Nova Reader - Subject

Inhibition of the PP2A activity by the histone chaperone ANP32B is long-range allosterically regulated by respiratory cytochrome c

2021-04-18T00:00

Repair of injured DNA relies on nucleosome dismantling by histone chaperones and de-phosphorylation events carried out by Protein Phosphatase 2A (PP2A). Typical histone chaperones are the Acidic leucine-rich Nuclear Phosphoprotein 32 family (ANP32) members, e.g. ANP32A, which is also a well-known PP2A inhibitor (a.k.a. I₁PP2A). Here we report the novel interaction between the endogenous family member B—so-called ANP32B—and endogenous cytochrome c in cells undergoing camptothecin-induced DNA damage. Soon after DNA lesions but prior to caspase cascade activation, the hemeprotein translocates to the nucleus to target the Low Complexity Acidic Region (LCAR) of ANP32B; in a similar way, our group recently reported that the hemeprotein targets the acidic domain of SET/Template Activating Factor-Iβ (SET/TAF-Iβ), which is another histone chaperone and PP2A inhibitor (a.k.a. I₂PP2A). The nucleosome assembly activity of ANP32B is indeed unaffected by cytochrome c binding. Like ANP32A, ANP32B inhibits PP2A activity and is thus herein referred to as I₃PP2A. Our data demonstrates that ANP32B-dependent inhibition of PP2A is regulated by respiratory cytochrome c, which induces long-distance allosteric changes in the structured N-terminal domain of ANP32B upon binding to the C-terminal LCAR. In agreement with the reported role of PP2A in the DNA damage response, we propose a model wherein cytochrome c is translocated from the mitochondria into the nucleus upon DNA damage to modulate PP2A activity via its interaction with ANP32B.

•

Respiratory cytochrome c interacts with ANP32B under DNA damage in the nucleus.

•

Cytochrome c binding to ANP32B LCAR restores ANP32B-mediated PP2A inhibition.

•

Cytochrome c emerges as a DNA Damage Response regulator.

Activation of NRF2 by APE1/REF1 is redox-dependent in Barrett's related esophageal adenocarcinoma cells

2021-04-19T00:00

Background

Chronic gastroesophageal reflux disease (GERD) is a major risk factor for the development of metaplastic Barrett's esophagus (BE) and its progression to esophageal adenocarcinoma (EAC). Uncontrolled accumulation of reactive oxygen species (ROS) in response to acidic bile salts (ABS) in reflux conditions can be lethal to cells. In this study, we investigated the role of APE1/REF1 in regulating nuclear erythroid factor-like 2 (NRF2), the master antioxidant transcription factor, in response to reflux conditions.

Results

We found that APE1 protein was critical for protecting against cellular ROS levels, oxidative DNA damage, double strand DNA breaks, and cell death in response to conditions that mimic reflux. Analysis of cell lines and de-identified tissues from patients with EAC demonstrated overexpression of both APE1 and NRF2 in EAC cells, as compared to non-neoplastic esophageal cells. Using reflux conditions, we detected concordant and prolonged increases of APE1 and NRF2 protein levels for several hours, following transient short exposure to ABS (20 min). NRF2 transcription activity, as measured by ARE luciferase reporter, and expression of its target genes (HO-1 and TRXND1) were similarly increased in response to ABS. Using genetic knockdown of APE1, we found that APE1 was required for the increase in NRF2 protein stability, nuclear localization, and transcription activation in EAC. Using knockdown of APE1 with reconstitution of wild-type and a redox-deficient mutant (C65A) of APE1, as well as pharmacologic APE1 redox inhibitor (E3330), we demonstrated that APE1 regulated NRF2 in a redox-dependent manner. Mechanistically, we found that APE1 is required for phosphorylation and inactivation of GSK-3β, an important player in the NRF2 degradation pathway.

Conclusion

APE1 redox function was required for ABS-induced activation of NRF2 by regulating phosphorylation and inactivation of GSK-3β. The APE1-NRF2 network played a critical role in protecting esophageal cells against ROS and promoting cell survival under oxidative reflux conditions.

•

APE1 protected esophageal neoplastic cells from acidic bile salts (ABS)-induced oxidative stress, DNA damage and cell death.

•

APE1 regulated oxidative stress through mediating the activity of master antioxidant transcription factor, NRF2.

•

APE1 was required to maintain NRF2 stability in the nucleus and suppressed NRF2 protein degradation, mainly through GSK-3β.

•

APE1 redox function was required for proper phosphorylation and inactivation of GSK-3β, and subsequent activation of NRF2.

AQP8 is a crucial H₂O₂ transporter in insulin-producing RINm5F cells

2021-04-01T00:00

Peroxiporins are distinct aquaporins (AQP) which, beside water, also facilitate the bidirectional transport of hydrogen peroxide (H₂O₂) across cellular membranes. H₂O₂ serves as the major reactive oxygen species that mediates essential cell signaling events. In pancreatic β-cells, H₂O₂ has been associated with the regulation of cell growth but in excess it leads to failure of insulin secretion, making it important for diabetes mellitus (DM) pathogenesis. In the present study, the role of aquaporin-8 (AQP8) as a peroxiporin was investigated in RINm5F cells. The role of AQP8 was studied in an insulin-producing cell model, on the basis of stable AQP8 overexpression (AQP8↑) and CRISPR/Cas9-mediated AQP8 knockdown (KD). A complete AQP8 knock-out was found to result in cell death, however we demonstrate that mild lentiviral re-expression through a Tet-On-regulated genetically modified AQP8 leads to cell survival, enabling functional characterization. Proliferation and insulin content were found to be increased in AQP8↑ cells underlining the importance of AQP8 in the regulation of H₂O₂ homeostasis in pancreatic β-cells. Colocalization analyses of V5-tagged AQP8 proteins based on confocal microscopic imaging revealed its membrane targeting to both the mitochondria and the plasma membrane, but not to the ER, the Golgi apparatus, insulin vesicles, or peroxisomes. By using the fluorescence H₂O₂ specific biosensor HyPer together with endogenous generation of H₂O₂ using d-amino acid oxidase, live cell imaging revealed enhanced H₂O₂ flux to the same subcellular regions in AQP8 overexpressing cells pointing to its importance in the development of type-1 DM. Moreover, the novel ultrasensitive H₂O₂ sensor HyPer7.2 clearly unveiled AQP8 as a H₂O₂ transporter in RINm5F cells. In summary, these studies establish that AQP8 is an important H₂O₂ pore in insulin-producing RINm5F cells involved in the transport of H₂O₂ through the mitochondria and cell membrane and may help to explain the H₂O₂ transport and toxicity in pancreatic β-cells.

The schematic pieces were provided by Smart Medical Art and adapted (https://smart.servier.com/image-set-download/). Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License.

•

AQP8 KO is lethal for insulin-producing RINm5F cells.

•

The peroxiporin AQP8 is localized in the plasma and mitochondrial membrane channeling H₂O₂ in RINm5F cells.

•

Tet-On regulated low AQP8 re-expression and APQ8 overexpression are feasible models to study H₂O₂ transport in β-cells.

•

Overexpression of AQP8 increases cell proliferation and cellular insulin content.

Decreased availability of nitric oxide and hydrogen sulfide is a hallmark of COVID-19

2021-05-08T00:00

Background

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is involved in a global outbreak affecting millions of people who manifest a variety of symptoms. Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 is increasingly associated with cardiovascular complications requiring hospitalizations; however, the mechanisms underlying these complications remain unknown. Nitric oxide (NO) and hydrogen sulfide (H₂S) are gasotransmitters that regulate key cardiovascular functions.

Methods

Blood samples were obtained from 68 COVID-19 patients and 33 controls and NO and H₂S metabolites were assessed. H₂S and NO levels were compared between cases and controls in the entire study population and subgroups based on race. The availability of gasotransmitters was examined based on severity and outcome of COVID-19 infection. The performance of H₂S and NO levels in predicting COVID-19 infection was also analyzed. Multivariable regression analysis was performed to identify the effects of traditional determinants of gasotransmitters on NO and H₂S levels in the patients with COVID-19 infection.

Results

Significantly reduced NO and H₂S levels were observed in both Caucasian and African American COVID-19 patients compared to healthy controls. COVID-19 patients who died had significantly higher NO and H₂S levels compared to COVID-19 patients who survived. Receiver-operating characteristic analysis of NO and H₂S metabolites in the study population showed free sulfide levels to be highly predictive of COVID-19 infection based on reduced availability. Traditional determinants of gasotransmitters, namely age, race, sex, diabetes, and hypertension had no effect on NO and H₂S levels in COVID-19 patients.

Conclusion

These observations provide the first insight into the role of NO and H₂S in COVID-19 infection, where their low availability may be a result of reduced synthesis secondary to endotheliitis, or increased consumption from scavenging of reactive oxygen species.

•

NO and H₂S availability is decreased in COVID-19 patients compared to healthy controls.

•

Decreased NO and H₂S availability in COVID-19 patients is independent of race, other demographics and comorbidities.

•

Decreased NO levels in COVID-19 patients parallel an increase in nitrotyrosine, an oxidative stress marker.

LRRc17 controls BMSC senescence via mitophagy and inhibits the therapeutic effect of BMSCs on ovariectomy-induced bone loss

2021-04-01T00:00

Senescence of bone marrow-derived mesenchymal stem cells (BMSCs) has been widely reported to be closely correlated with aging-related diseases, including osteoporosis (OP). Moreover, the beneficial functions of BMSCs decline with age, limiting their therapeutic efficacy in OP. In the present study, using RNA sequencing (RNA-Seq), we found that leucine-rich repeat containing 17 (LRRc17) expression in BMSCs was highly positively correlated with age. Therefore, we investigated whether LRRc17 knockdown could rejuvenate aged MSCs and increase their therapeutic efficacy in OP. Consistent with the RNA-Seq results, the protein expression of LRRc17 in senescent BMSCs was significantly increased, whereas LRRc17 knockdown inhibited cell apoptosis and reduced the expression of age-related proteins and G2 and S phase quiescence. Furthermore, LRRc17 knockdown shifted BMSCs from adipogenic to osteogenic differentiation, indicating the critical role of LRRc17 in BMSC senescence and differentiation. Additionally, similar to rapamycin (RAPA) treatment, LRRc17 knockdown activated mitophagy via inhibition of the mTOR/PI3K pathway, which consequently reduced mitochondrial dysfunction and inhibited BMSC senescence. However, the effects of LRRc17 knockdown were significantly blocked by the autophagy inhibitor hydroxychloroquine (HCQ), demonstrating that LRRc17 knockdown prevented BMSC senescence by activating mitophagy. In vivo, compared with untransfected aged mouse-derived BMSCs (O-BMSCs), O-BMSCs transfected with sh-LRRc17 showed effective amelioration of ovariectomy (OVX)-induced bone loss. Collectively, these results indicated that LRRc17 knockdown rejuvenated senescent BMSCs and thus enhanced their therapeutic efficacy in OP by activating autophagy.

Intestinal microbiota drives cholestasis-induced specific hepatic gene expression patterns

Intestinal microbiota regulates multiple host metabolic and immunological processes. Consequently, any difference in its qualitative and quantitative composition is susceptible to exert significant effects, in particular along the gut-liver axis. Indeed, recent findings suggest that such changes modulate the severity and the evolution of a wide spectrum of hepatobiliary disorders. However, the mechanisms linking intestinal microbiota and the pathogenesis of liver disease remain largely unknown. In this work, we investigated how a distinct composition of the intestinal microbiota, in comparison with germ-free conditions, may lead to different outcomes in an experimental model of acute cholestasis. Acute cholestasis was induced in germ-free (GF) and altered Schaedler’s flora (ASF) colonized mice by common bile duct ligation (BDL). Studies were performed 5 days after BDL and hepatic histology, gene expression, inflammation, lipids metabolism, and mitochondrial functioning were evaluated in normal and cholestatic mice. Differences in plasma concentration of bile acids (BA) were evaluated by UHPLC-HRMS. The absence of intestinal microbiota was associated with significant aggravation of hepatic bile infarcts after BDL. At baseline, we found the absence of gut microbiota induced altered expression of genes involved in the metabolism of fatty and amino acids. In contrast, acute cholestasis induced altered expression of genes associated with extracellular matrix, cell cycle, autophagy, activation of MAPK, inflammation, metabolism of lipids, and mitochondrial functioning pathways. Ductular reactions, cell proliferation, deposition of collagen 1 and autophagy were increased in the presence of microbiota after BDL whereas GF mice were more susceptible to hepatic inflammation as evidenced by increased gene expression levels of osteopontin, interleukin (IL)-1β and activation of the ERK/MAPK pathway as compared to ASF colonized mice. Additonally, we found that the presence of microbiota provided partial protection to the mitochondrial functioning and impairment in the fatty acid metabolism after BDL. The concentration of the majority of BA markedly increased after BDL in both groups without remarkable differences according to the hygiene status of the mice. In conclusion, acute cholestasis induced more severe liver injury in GF mice compared to mice with limited intestinal bacterial colonization. This protective effect was associated with different hepatic gene expression profiles mostly related to tissue repair, metabolic and immune functions. Our findings suggest that microbial-induced differences may impact the course of cholestasis and modulate liver injury, offering a background for novel therapies based on the modulation of the intestinal microbiota.

Development of gut microbiota and bifidobacterial communities of neonates in the first 6 weeks and their inheritance from mother

Microbiota especially Bifidobacterium play an important role in adjusting and maintaining homeostatic balance within the infant intestine. The aim of this study was to elucidate the relationship between maternal and infant gut microbiota and identify the Bifidobacterium species that may transfer from mother to infant over the first 42 days of the infant’s life. Nineteen mother-infant-pair fecal samples were collected and the diversity and composition of the total bacterial and Bifidobacterium communities were analyzed via 16S rDNA and bifidobacterial groEL gene high throughput sequencing. The results revealed that the relative abundance of Bifidobacterium was significantly higher in the infant gut while Parabacteroides, Blautia, Coprococcus, Lachnospira and Faecalibacterium were at lower relative abundance in 7-day and 42-day infant fecal samples compared to the maternal samples. The maternal gut has more B. pseudocatenulatum. In the infant group, B. breve and B. dentium relative abundance increased while B. animalis subsp. lactis decreased from days 7 to 42. Additionally, B. longum subsp. longum isolated from FGZ16 and FGZ35 may have transferred from mother to infant and colonized the infant gut. The results of the current study provide insight toward the infant gut microbiota composition and structure during the first 42 days and may help guide Bifidobacterium supplementation strategies in mothers and infants.

Sulforaphane exposure impairs contractility and mitochondrial function in three-dimensional engineered heart tissue

2021-03-31T00:00

Sulforaphane (SFN) is a phytochemical compound extracted from cruciferous plants, like broccoli or cauliflower. Its isothiocyanate group renders SFN reactive, thus allowing post-translational modification of cellular proteins to regulate their function with the potential for biological and therapeutic actions. SFN and stabilized variants recently received regulatory approval for clinical studies in humans for the treatment of neurological disorders and cancer. Potential unwanted side effects of SFN on heart function have not been investigated yet. The present study characterizes the impact of SFN on cardiomyocyte contractile function in cardiac preparations from neonatal rat, adult mouse and human induced-pluripotent stem cell-derived cardiomyocytes. This revealed a SFN-mediated negative inotropic effect, when administered either acutely or chronically, with an impairment of the Frank-Starling response to stretch activation. A direct effect of SFN on myofilament function was excluded in chemically permeabilized mouse trabeculae. However, SFN pretreatment increased lactate formation and enhanced the mitochondrial production of reactive oxygen species accompanied by a significant reduction in the mitochondrial membrane potential. Transmission electron microscopy revealed disturbed sarcomeric organization and inflated mitochondria with whorled membrane shape in response to SFN exposure. Interestingly, administration of the alternative energy source l-glutamine to the medium that bypasses the uptake route of pyruvate into the mitochondrial tricarboxylic acid cycle improved force development in SFN-treated EHTs, suggesting indeed mitochondrial dysfunction as a contributor of SFN-mediated contractile dysfunction. Taken together, the data from the present study suggest that SFN might impact negatively on cardiac contractility in patients with cardiovascular co-morbidities undergoing SFN supplementation therapy. Therefore, cardiac function should be monitored regularly to avoid the onset of cardiotoxic side effects.

Summary scheme of the functional effects of SFN exerted in cardiomyocytes: decline in force, elevation of diastolic tension and alteration of mitochondrial function and metabolism. MM: mitochondrial membrane potential; MPC: mitochondrial pyruvate carrier; mito: mitochondria; PC: pyruvate carboxylase; PDH: pyruvate dehydrogenase; Mitochondrium adapted from Servier Medical ART: SMART (smart.servier.com).

•

Sulforaphane has negative inotropic effects and increases diastolic tension.

•

Sulforaphane exposure increases lactate levels and mitochondrial ROS production and reduces mitochondrial membrane potential.

•

l-glutamine supplementation rescues the sulforaphane-mediated reduction in force development.

•

Sulforaphane plasma levels and cardiac function should be monitored to avoid unwanted cardiac side effects in patients.

Sustained IKKβ phosphorylation and NF-κB activation by superoxide-induced peroxynitrite-mediated nitrotyrosine modification of B56γ3 and PP2A inactivation

2020-12-18T00:00

Apart from its physiological role in inflammation and immunity, the nuclear factor-kappa B (NF-κB) protein complex has been implicated in tumorigenesis and its progression. Here, we provide evidence that a pro-oxidant milieu is an upstream effector of oncogenic NF-κB signaling. Through pharmacological or genetic inhibition of SOD1, we show that elevated intracellular superoxide (O₂•^-) mediates sustained IKK phosphorylation, and induces downstream degradation of IκBα, leading to the nuclear localization and transcriptional activation of NF-κB. Mechanistically, we show that such sustained NF-κB signaling is a function of protein phosphatase 2A (PP2A) inactivation brought about by the nitrative modification of its substrate-binding sub-unit B56γ. Importantly, the pro-oxidant driven NF-κB activation enhances the migratory and invasive potential of cancer cells. In summary, our work highlights the critical involvement of O₂•^--dependent peroxynitrite production in inhibiting PP2A-mediated dephosphorylation of IKK, thereby facilitating cancers to acquire an invasive phenotype. Given that NF-κB is a key player of chronic inflammation and carcinogenesis, our work unravels a novel synergistic node involving O₂•^--driven redox milieu and deregulated PP2A as a potential therapeutic target.

Cutaneous antimicrobial peptides: New “actors” in pollution related inflammatory conditions

2021-03-31T00:00

Ozone (O₃) exposure has been reported to contribute to various cutaneous inflammatory conditions, such as eczema, psoriasis, rush etc. via a redox-inflammatory pathway. O₃ is too reactive to penetrate cutaneous tissue; it interacts with lipids present in the outermost layer of skin, resulting in formation of oxidized molecules and hydrogen peroxide (H₂O₂). Interestingly, several inflammatory skin pathologies demonstrate altered levels of antimicrobial peptides (AMPs). These small, cationic peptides are found in various cells, including keratinocytes, eccrine gland cells, and seboctyes. Classically, AMPs function as antimicrobial agents. Recent studies indicate that AMPs also play roles in inflammation, angiogenesis, and wound healing. Since altered levels of AMPs have been detected in pollution-associated skin pathologies, we hypothesized that exposure to O₃ could affect the levels of AMPs in the skin. We examined levels of AMPs using qRT-PCR, Western blotting, and immunofluorescence in vitro (human keratinocytes), ex vivo (human skin explants), and in vivo (human volunteer subjects exposed to O₃) and observed increased levels of all the measured AMPs upon O₃ exposure. In addition, in vitro studies have confirmed the redox regulation of AMPs in keratinocytes. This novel finding suggests that targeting AMPs could be a possible defensive strategy to combat pollution-associated skin conditions.

•

AMPs (hBDs1-3, CAMP) increase in O₃ exposed human skin by a redox mechanism.

•

Transcriptional upregulation of AMPs in response to O₃ exposure is due to an altered redox status.

•

Pollution increase AMPs could be the connection between pollution exposure and the development/exacerbation of inflammatory skin conditions.

Skeletal muscle-specific Keap1 disruption modulates fatty acid utilization and enhances exercise capacity in female mice

2021-04-05T00:00

Skeletal muscle health is important for the prevention of various age-related diseases. The loss of skeletal muscle mass, which is known as sarcopenia, underlies physical disability, poor quality of life and chronic diseases in elderly people. The transcription factor NRF2 plays important roles in the regulation of the cellular defense against oxidative stress, as well as the metabolism and mitochondrial activity. To determine the contribution of skeletal muscle NRF2 to exercise capacity, we conducted skeletal muscle-specific inhibition of KEAP1, which is a negative regulator of NRF2, and examined the cell-autonomous and non-cell-autonomous effects of NRF2 pathway activation in skeletal muscles. We found that NRF2 activation in skeletal muscles increased slow oxidative muscle fiber type and improved exercise endurance capacity in female mice. We also observed that female mice with NRF2 pathway activation in their skeletal muscles exhibited enhanced exercise-induced mobilization and β-oxidation of fatty acids. These results indicate that NRF2 activation in skeletal muscles promotes communication with adipose tissues via humoral and/or neuronal signaling and facilitates the utilization of fatty acids as an energy source, resulting in increased mitochondrial activity and efficient energy production during exercise, which leads to improved exercise endurance.

•

Systemic Keap1 knockdown enhances exercise endurance capacity in mice.

•

Keap1 deficiency in skeletal muscle activates NRF2 pathway.

•

Keap1 deficiency in skeletal muscle enhances endurance capacity in female mice.

•

Keap1 deficiency in skeletal muscle promotes exercise-induced fatty acid utilization.

Therapeutic wavelengths of ultraviolet B radiation activate apoptotic, circadian rhythm, redox signalling and key canonical pathways in psoriatic epidermis

2021-03-10T00:00

Ultraviolet B radiation (UVB) exerts pleiotropic effects on human skin. DNA damage response and repair pathways are activated by UVB; if damage cannot be repaired, apoptosis ensues. Although cumulative UVB exposure predisposes to skin cancer, UVB phototherapy is widely used as an effective treatment for psoriasis. Previous studies defined the therapeutic action spectrum of UVB and showed that psoriasis is resistant to apoptosis. This study aimed to investigate early molecular responses within psoriasis plaques following irradiation with single equi-erythemogenic doses of clinically-effective (311 nm, narrow-band) compared to clinically-ineffective (290 nm) UVB. Forty-eight micro-dissected epidermal samples from 20 psoriatic patients were analyzed using microarrays. Our bioinformatic analysis compared gene expression between 311 nm irradiated, 290 nm irradiated and control psoriasis epidermis to specifically identify 311 nm UVB differentially expressed genes (DEGs) and their upstream regulatory pathways. Key DEGs and pathways were validated by immunohistochemical analysis.

There was a dynamic induction and repression of 311 nm UVB DEGs between 6 h and 18 h, only a limited number of DEGs maintained their designated expression status between time-points. Key disease and function pathways included apoptosis, cell death, cell migration and leucocyte chemotaxis. DNA damage response pathways, NRF2-mediated oxidative stress response and P53 signalling were key nodes, interconnecting apoptosis and cell cycle arrest. Interferon signalling, dendritic cell maturation, granulocyte adhesion and atherosclerotic pathways were also differentially regulated. Consistent with these findings, top transcriptional regulators of 311 nm UVB DEGs related to: a) apoptosis, DNA damage response and cell cycle control; b) innate/acquired immune regulation and inflammation; c) hypoxia/redox response and angiogenesis; d) circadian rhythmicity; f) EGR/AP1 signalling and keratinocyte differentiation; and g) mitochondrial biogenesis.

This research provides important insights into the molecular targets of 311 nm UVB, underscoring key roles for apoptosis and cell death. These and the other key pathways delineated may be central to the therapeutic effects of 311 nm in psoriasis.

Schematic representation of study to delineate pathways activated by therapeutic wavelengths of UVB in psoriasis.

Endogenous generation of nitro-fatty acid hybrids having dual nitrate ester (RONO₂) and nitroalkene (RNO₂) substituents

2021-02-24T00:00

Organic nitrate esters, long-recognized therapies for cardiovascular disorders, have not been detected biologically. We characterize in rat stomach unsaturated fatty acid nitration reactions that proceed by generation of nitro-nitrate intermediates (NO₂–ONO₂-FA) via oxygen and nitrite dependent reactions. NO₂–ONO₂-lipids represent ∼70% of all nitrated lipids in the stomach and they decay in vitro at neutral or basic pH by the loss of the nitrate ester group (-ONO₂) from the carbon backbone upon deprotonation of the α-carbon (pKa ∼7), yielding nitrate, nitrite, nitrosative species, and an electrophilic fatty acid nitroalkene product (NO₂-FA). Of note, NO₂-FA are anti-inflammatory and tissue-protective signaling mediators, which are undergoing Phase II trials for the treatment of kidney and pulmonary diseases. The decay of NO₂–ONO₂-FA occurs during intestinal transit and absorption, leading to the formation of NO₂-FA that were subsequently detected in circulating plasma triglycerides. These observations provide new insight into unsaturated fatty acid nitration mechanisms, identify nitro-nitrate ester-containing lipids as intermediates in the formation of both secondary nitrogen oxides and electrophilic fatty acid nitroalkenes, and expand the scope of endogenous products stemming from metabolic reactions of nitrogen oxides.

Lysosomal degradation ensures accurate chromosomal segregation to prevent chromosomal instability

Lysosomes, as primary degradative organelles, are the endpoint of different converging pathways, including macroautophagy. To date, lysosome degradative function has been mainly studied in interphase cells, while their role during mitosis remains controversial. Mitosis dictates the faithful transmission of genetic material among generations, and perturbations of mitotic division lead to chromosomal instability, a hallmark of cancer. Heretofore, correct mitotic progression relies on the orchestrated degradation of mitotic factors, which was mainly attributed to ubiquitin-triggered proteasome-dependent degradation. Here, we show that mitotic transition also relies on lysosome-dependent degradation, as impairment of lysosomes increases mitotic timing and leads to mitotic errors, thus promoting chromosomal instability. Furthermore, we identified several putative lysosomal targets in mitotic cells. Among them, WAPL, a cohesin regulatory protein, emerged as a novel SQSTM1-interacting protein for targeted lysosomal degradation. Finally, we characterized an atypical nuclear phenotype, the toroidal nucleus, as a novel biomarker for genotoxic screenings. Our results establish lysosome-dependent degradation as an essential event to prevent chromosomal instability.

Abbreviations: 3D: three-dimensional; APC/C: anaphase-promoting complex; ARL8B: ADP ribosylation factor like GTPase 8B; ATG: autophagy-related; BORC: BLOC-one-related complex; CDK: cyclin-dependent kinase; CENPE: centromere protein E; CIN: chromosomal instability; ConcA: concanamycin A; CQ: chloroquine; DAPI: 4,6-diamidino-2-penylinole; FTI: farnesyltransferase inhibitors; GFP: green fluorescent protein; H2B: histone 2B; KIF: kinesin family member; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; PDS5B: PDS5 cohesin associated factor B; SAC: spindle assembly checkpoint; PLEKHM2: pleckstrin homology and RUN domain containing M2; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; v-ATPase: vacuolar-type H⁺-translocating ATPase; WAPL: WAPL cohesion release factor.

The proppin Bcas3 and its interactor KinkyA localize to the early phagophore and regulate autophagy

To resolve the signaling mechanisms that mediate the starvation-induced processes of Dictyostelium sporulation and encystation, we performed insertional mutagenesis on cells harboring an mRFP-tagged spore gene. We isolated a mutant in kinkyA (knkA), a gene without known function, which formed fruiting bodies with a kinked stalk and lacking viable spores. Immunoprecipitation of lysates of KnkA-YFP-transformed knkA⁻ cells yielded a mammalian BCAS3 homolog as a KnkA interactor. bcas3⁻ phenocopied knkA⁻ and Bcas3 colocalized with KnkA to puncta. Bcas3 shares sequence similarity with proppins (beta-propellors that bind phosphoinositides). Mutation of 2 Bcas3 residues that are essential for PtdIns3P binding in proppins prevented Bcas3 binding to PtdIns3P as well as punctate Bcas3 and KnkA localization. KnkA puncta also colocalized with small but not large vesicles that contain the autophagy protein Atg8 and were contiguous with the endoplasmic reticulum. knkA⁻ and bcas3⁻ cells showed a pronounced decrease of RFP-GFP-Atg8 in neutral early autophagosomes, indicating that KnkA and Bcas3 are required for macroautophagy/autophagy. Knockouts in atg7, atg5 or atg9 substantiated this finding by showing similar sporulation defects as knkA⁻ and bcas3⁻. Defective Dictyostelium sporulation is evidently a useful diagnostic tool for the discovery of novel autophagy genes.

Abbreviations: Atg: Autophagy-related; BCAS3: BCAS3 microtubule associated cell migration factor; cAMP: 3ʹ,5ʹ-cyclic adenosine monophosphate; ER: endoplasmic reticulum; GFP: green fluorescent protein; PAS: phagophore assembly site; PRKA/PKA: protein kinase cAMP-dependent; Proppin: beta‐propellers that bind phosphoinositides; PtdIns3P: phosphatidylinositol 3-phosphate; REMI: restriction enzyme-mediated insertional mutagenesis; RFP: red fluorescent protein; RT-qPCR: reverse transcriptase - quantitative polymerase chain reaction; WIPI: WD repeat domain, phosphoinositide interacting; YFP: yellow fluorescent protein

Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5

Although the treatment of brain tumors by targeting kinase-regulated macroautophagy/autophagy, is under investigation, the precise mechanism underlying autophagy initiation and its significance in glioblastoma (GBM) remains to be defined. Here, we report that PAK1 (p21 [RAC1] activated kinase 1) is significantly upregulated and promotes GBM development. The Cancer Genome Atlas analysis suggests that the oncogenic role of PAK1 in GBM is mainly associated with autophagy. Subsequent experiments demonstrate that PAK1 indeed serves as a positive modulator for hypoxia-induced autophagy in GBM. Mechanistically, hypoxia induces ELP3-mediated PAK1 acetylation at K420, which suppresses the dimerization of PAK1 and enhances its activity, thereby leading to subsequent PAK1-mediated ATG5 (autophagy related 5) phosphorylation at the T101 residue. This event not only protects ATG5 from ubiquitination-dependent degradation but also increases the affinity between the ATG12–ATG5 complex and ATG16L1 (autophagy related 16 like 1). Consequently, ELP3-dependent PAK1 (K420) acetylation and PAK1-mediated ATG5 (T101) phosphorylation are required for hypoxia-induced autophagy and brain tumorigenesis by promoting autophagosome formation. Silencing PAK1 with shRNA or small molecule inhibitor FRAX597 potentially blocks autophagy and GBM growth. Furthermore, SIRT1-mediated PAK1-deacetylation at K420 hinders autophagy and GBM growth. Clinically, the levels of PAK1 (K420) acetylation significantly correlate with the expression of ATG5 (T101) phosphorylation in GBM patients. Together, this report uncovers that the acetylation modification and kinase activity of PAK1 plays an instrumental role in hypoxia-induced autophagy initiation and maintaining GBM growth. Therefore, PAK1 and its regulator in the autophagy pathway might represent potential therapeutic targets for GBM treatment.

Abbreviations: 3-MA: 3-methyladenine; Ac-CoA: acetyl coenzyme A; ATG5: autophagy related 5; ATG16L1, autophagy related 16 like 1; BafA₁: bafilomycin A₁; CDC42: cell division cycle 42; CGGA: Chinese Glioma Genome Atlas; CHX, cycloheximide; ELP3: elongator acetyltransferase complex subunit 3; GBM, glioblastoma; HBSS: Hanks balanced salts solution; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAP2K1: mitogen-activated protein kinase kinase 1; MAPK14, mitogen-activated protein kinase 14; PAK1: p21 (RAC1) activated kinase 1; PDK1: pyruvate dehydrogenase kinase 1; PGK1, phosphoglycerate kinase 1; PTMs: post-translational modifications; RAC1: Rac family small GTPase 1; SQSTM1: sequestosome 1; TCGA, The Cancer Genome Atlas.

TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium

Lysosomal Ca²⁺ contributes to macroautophagy/autophagy, an intracellular process for the degradation of cytoplasmic material and organelles in the lysosomes to protect cells against stress responses. TMBIM6 (transmembrane BAX inhibitor motif containing 6) is a Ca²⁺ channel-like protein known to regulate ER stress response and apoptosis. In this study, we examined the as yet unknown role of TMBIM6 in regulating lysosomal Ca²⁺ levels. The Ca²⁺ efflux from the ER through TMBIM6 was found to increase the resting lysosomal Ca²⁺ level, in which ITPR-independent regulation of Ca²⁺ status was observed. Further, TMBIM6 regulated the local release of Ca²⁺ through lysosomal MCOLN1/TRPML1 channels under nutrient starvation or MTOR inhibition. The local Ca²⁺ efflux through MCOLN1 channels was found to activate PPP3/calcineurin, triggering TFEB (transcription factor EB) nuclear translocation, autophagy induction, and lysosome biogenesis. Upon genetic inactivation of TMBIM6, lysosomal Ca²⁺ and the associated TFEB nuclear translocation were decreased. Furthermore, autophagy flux was significantly enhanced in the liver or kidney from starved Tmbim6^+/+ mice compared with that in the counter tmbim6^−/- mice. Together, our observations indicated that under stress conditions, TMBIM6 increases lysosomal Ca²⁺ release, leading to PPP3/calcineurin-mediated TFEB activation and subsequently enhanced autophagy. Thus, TMBIM6, an ER membrane protein, is suggested to be a lysosomal Ca²⁺ modulator that coordinates with autophagy to alleviate metabolism stress.Abbreviations: AVs: autophagic vacuoles; CEPIA: calcium-measuring organelle-entrapped protein indicator; ER: endoplasmic reticulum; GPN: glycyl-L-phenylalanine-beta-naphthylamide; ITPR/IP3R: inositol 1,4,5-trisphosphate receptor; LAMP1: lysosomal associated membrane protein 1; MCOLN/TRPML: mucolipin; MEF: mouse embryonic fibroblast; ML-SA1: mucolipin synthetic agonist 1; MTORC1: mechanistic target of rapamycin kinase complex 1; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TKO: triple knockout; TMBIM6/BI-1: transmembrane BAX inhibitor motif containing 6

Intestinal Cetobacterium and acetate modify glucose homeostasis via parasympathetic activation in zebrafish

The capability of carbohydrate utilization in fish is limited compared to mammals. It has scientific and practical significance to improve the ability of fish to use carbohydrates. The efficiency of dietary carbohydrate utilization varies among fish with different feeding habits, which are associated with differential intestinal microbiota. In this study, we found that zebrafish fed with omnivorous diet (OD) and herbivorous diet (HD) showed better glucose homeostasis compared with carnivorous diet (CD) fed counterpart and the differential glucose utilization efficiency was attributable to the intestinal microbiota. The commensal bacterium Cetobacterium somerae, an acetate producer, was enriched in OD and HD groups, and administration of C. somerae in both adult zebrafish and gnotobiotic larval zebrafish models resulted in improved glucose homeostasis and increased insulin expression, supporting a causative role of C. somerae enrichment in glucose homeostasis in fish. The enrichment of C. somerae was constantly associated with higher acetate levels, and dietary supplementation of acetate promotes glucose utilization in zebrafish, suggesting a contribution of acetate in the function of C. somerae. Furthermore, we found that the beneficial effect of both acetate and C. somerae on glucose homeostasis was mediated through parasympathetic activation. Overall, this work highlights the existence of a C. somerae-brain axis in the regulation of glucose homeostasis in fish and suggests a role of acetate in mediating the axis function. Our results suggest potential strategies for improvement of fish carbohydrate utilization.

Coxiella effector protein CvpF subverts RAB26-dependent autophagy to promote vacuole biogenesis and virulence

Coxiella burnetii, the etiological agent of the zoonosis Q fever, replicates inside host cells within a large vacuole displaying autolysosomal characteristics. The development of this compartment is mediated by bacterial effectors, which interfere with a number of host membrane trafficking pathways. By screening a Coxiella transposon mutant library, we observed that transposon insertions in cbu0626 led to intracellular replication and vacuole biogenesis defects. Here, we demonstrate that CBU0626 is a novel member of the Coxiella vacuolar protein (Cvp) family of effector proteins, which is translocated by the Dot/Icm secretion system and localizes to vesicles with autolysosomal features as well as Coxiella-containing vacuoles (CCVs). We thus renamed this effector CvpF for Coxiella vacuolar protein F. CvpF specifically interacts with the host small GTPase RAB26, leading to the recruitment of the autophagosomal marker MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) to CCVs. Importantly, cvpF::Tn mutants were highly attenuated compared to wild-type bacteria in the SCID mouse model of infection, highlighting the importance of CvpF for Coxiella virulence. These results suggest that CvpF manipulates endosomal trafficking and macroautophagy/autophagy induction for optimal C. burnetii vacuole biogenesis.

Abbreviations: ACCM: acidified citrate cystein medium; AP: adaptor related protein complex; CCV: Coxiella-containing vacuole; Cvp: Coxiella vacuolar protein; GDI: guanosine nucleotide dissociation inhibitor; GDF: GDI dissociation factor; GEF: guanine exchange factor; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTORC1: mechanistic target of rapamycin kinase MTOR complex 1; PBS: phosphate-buffered saline; PMA: phorbol myristate acetate; SQSTM1/p62: sequestosome 1; WT: wild-type.

Prediction of the spread of Corona-virus carrying droplets in a bus - A computational based artificial intelligence approach

2021-02-09T00:00

Public transport has been identified as high risk as the corona-virus carrying droplets generated by the infected passengers could be distributed to other passengers. Therefore, predicting the patterns of droplet spreading in public transport environment is of primary importance. This paper puts forward a novel computational and artificial intelligence (AI) framework for fast prediction of the spread of droplets produced by a sneezing passenger in a bus. The formation of droplets of salvia is numerically modelled using a volume of fluid methodology applied to the mouth and lips of an infected person during the sneezing process. This is followed by a large eddy simulation of the resultant two phase flow in the vicinity of the person while the effects of droplet evaporation and ventilation in the bus are considered. The results are subsequently fed to an AI tool that employs deep learning to predict the distribution of droplets in the entire volume of the bus. This combined framework is two orders of magnitude faster than the pure computational approach. It is shown that the droplets with diameters less than 250 micrometers are most responsible for the transmission of the virus, as they can travel the entire length of the bus.

Macrophage migration inhibitory factor (MIF) enhances hypochlorous acid production in phagocytic neutrophils

2021-03-30T00:00

Background

Macrophage migration inhibitory factor (MIF) is an important immuno-regulatory cytokine and is elevated in inflammatory conditions. Neutrophils are the first immune cells to migrate to sites of infection and inflammation, where they generate, among other mediators, the potent oxidant hypochlorous acid (HOCl). Here, we investigated the impact of MIF on HOCl production in neutrophils in response to phagocytic stimuli.

Methods

Production of HOCl during phagocytosis of zymosan was determined using the specific fluorescent probe R19-S in combination with flow cytometry and live cell microscopy. The rate of phagocytosis was monitored using fluorescently-labeled zymosan. Alternatively, HOCl production was assessed during phagocytosis of Pseudomonas aeruginosa by measuring the oxidation of bacterial glutathione to the HOCl-specific product glutathione sulfonamide. Formation of neutrophil extracellular traps (NETs), an oxidant-dependent process, was quantified using a SYTOX Green plate assay.

Results

Exposure of human neutrophils to MIF doubled the proportion of neutrophils producing HOCl during early stages of zymosan phagocytosis, and the concentration of HOCl produced was greater. During phagocytosis of P. aeruginosa, a greater fraction of bacterial glutathione was oxidized to glutathione sulfonamide in MIF-treated compared to control neutrophils. The ability of MIF to increase neutrophil HOCl production was independent of the rate of phagocytosis and could be blocked by the MIF inhibitor 4-IPP. Neutrophils pre-treated with MIF produced more NETs than control cells in response to PMA.

Conclusion

Our results suggest a role for MIF in potentiating HOCl production in neutrophils in response to phagocytic stimuli. We propose that this newly discovered activity of MIF contributes to its role in mediating the inflammatory response and enhances host defence.

•

MIF augments phagosomal HOCl production.

•

This results in increased oxidation of bacterial glutathione.

•

MIF increases superoxide production in response to soluble stimuli.

•

This results in increased NET formation.

Gut bacteriophage dynamics during fecal microbial transplantation in subjects with metabolic syndrome

Metabolic Syndrome (MetS) is a growing public health concern worldwide. Individuals with MetS have an increased risk for cardiovascular (CV) disease and type 2 diabetes (T2D). These diseases – in part preventable with the treatment of MetS – increase the chances of premature death and pose a great economic burden to health systems. A healthy gut microbiota is associated with a reduction in MetS, T2D, and CV disease. Treatment of MetS with fecal microbiota transplantation (FMT) can be effective, however, its success rate is intermediate and difficult to predict. Because bacteriophages significantly affect the microbiota membership and function, the aim of this pilot study was to explore the dynamics of the gut bacteriophage community after FMT in MetS subjects. We performed a longitudinal study of stool bacteriophages from healthy donors and MetS subjects before and after FMT treatment. Subjects were assigned to either a control group (self-stool transplant, n = 3) or a treatment group (healthy-donor-stool transplant; n-recipients = 6, n-donors = 5). Stool samples were collected over an 18-week period and bacteriophage-like particles were purified and sequenced. We found that FMT from healthy donors significantly alters the gut bacteriophage community. Subjects with better clinical outcome clustered closer to the heathy donor group, suggesting that throughout the treatment, their bacteriophage community was more similar to healthy donors. Finally, we identified bacteriophage groups that could explain these differences and we examined their prevalence in individuals from a larger cohort of MetS FMT trial.

Trial information- http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=2705; NTR 2705

The Impact of Migration on the Gut Metagenome of South Asian Canadians

South Asian (SA) Canadian immigrants have a higher risk of developing certain immune-mediated inflammatory diseases compared to non-migrant SAs. We sought to investigate the effect of migration on the gut metagenome and to identify microbiological associations between migration and conditions that may influence the development of immune-mediated inflammatory diseases. Metagenomic analysis of 58 first-generation (GEN1) SA immigrants and 38 unrelated Canadian born children-of-immigrants (GEN2) determined that the time lived in Canada was associated with continued changes in gut microbial communities. Migration of GEN1 to Canada early in life results in a gut community with similarities to GEN2 SA Canadians and non-SA North Americans. Conversely, GEN1 immigrants who arrived recently to Canada exhibited pronounced differences from GEN2, while displaying microbial similarities to a non-migrating SA cohort. Multivariate analysis identified that community composition was primarily influenced by high abundance taxa. Prevotella copri dominated in GEN1 and non-migrant SAs. Clostridia and functionally related Bacteroidia spp. replaced P. copri dominance over generations in Canada. Mutually exclusive Dialister species occurred at differing relative abundances over time and generations in Canada. This shift in species composition is accompanied by a change in genes associated with carbohydrate utilization and short-chain fatty acid production. Total energy derived from carbohydrates compared to protein consumption was significantly higher for GEN1 recent immigrants, which may influence the functional requirements of the gut community. This study demonstrates the associations between migration and the gut microbiome, which may be further associated with the altered risk of immune-mediated inflammatory diseases observed for SA Canadians.

NOX4 promotes ferroptosis of astrocytes by oxidative stress-induced lipid peroxidation via the impairment of mitochondrial metabolism in Alzheimer's diseases

2021-03-19T00:00

Oxidative stress has been implicated in the pathogenesis of Alzheimer's disease (AD). Mitochondrial dysfunction is linked to oxidative stress and reactive oxygen species (ROS) in neurotoxicity during AD. Impaired mitochondrial metabolism has been associated with mitochondrial dysfunction in brain damage of AD. While the role of NADPH oxidase 4 (NOX4), a major source of ROS, has been identified in brain damage, the mechanism by which NOX4 regulates ferroptosis of astrocytes in AD remains unclear. Here, we show that the protein levels of NOX4 were significantly elevated in impaired astrocytes of cerebral cortex from patients with AD and APP/PS1 double-transgenic mouse model of AD. The levels of 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), a marker of oxidative stress-induced lipid peroxidation, were significantly also elevated in impaired astrocytes of patients with AD and mouse AD. We demonstrate that the over-expression of NOX4 significantly increases the impairment of mitochondrial metabolism by inhibition of mitochondrial respiration and ATP production via the reduction of five protein complexes in the mitochondrial ETC in human astrocytes. Moreover, the elevation of NOX4 induces oxidative stress by mitochondrial ROS (mtROS) production, mitochondrial fragmentation, and inhibition of cellular antioxidant process in human astrocytes. Furthermore, the elevation of NOX4 increased ferroptosis-dependent cytotoxicity by the activation of oxidative stress-induced lipid peroxidation in human astrocytes. These results suggest that NOX4 promotes ferroptosis of astrocytes by oxidative stress-induced lipid peroxidation via the impairment of mitochondrial metabolism in AD.

•

The levels of NOX4 were elevated in impaired astrocytes of human and mouse AD.

•

The levels of 4-HNE and MDA were elevated in impaired astrocytes of human and mouse AD.

•

NOX4 induces oxidative stress by the impairment of mitochondria in human astrocytes.

•

NOX4 promotes ferroptosis by oxidative stress in human astrocytes.

Dexmedetomidine ameliorates endotoxin-induced acute lung injury in vivo and in vitro by preserving mitochondrial dynamic equilibrium through the HIF-1a/HO-1 signaling pathway

2021-03-21T00:00

Increasing lines of evidence identified that dexmedetomidine (DEX) exerted protective effects against sepsis-stimulated acute lung injury via anti-inflammation, anti-oxidation and anti-apoptosis. However, the mechanisms remain unclear. Herein, we investigated whether DEX afforded lung protection by regulating the process of mitochondrial dynamics through the HIF-1a/HO-1 pathway in vivo and in vitro. Using C57BL/6J mice exposed to lipopolysaccharide, it was initially observed that preemptive administration of DEX (50μg/kg) alleviated lung pathologic injury, reduced oxidative stress indices (OSI), improved mitochondrial dysfunction, upregulated the expression of HIF-1α and HO-1, accompanied by shifting the dynamic course of mitochondria into fusion. Moreover, HO-1-knockout mice or HO-1 siRNA transfected NR8383 cells were pretreated with HIF-1α stabilizer DMOG and DEX to validate the effect of HIF-1a/HO-1 pathway on DEX-mediated mitochondrial dynamics in a model of endotoxin-induced lung injury. We found that pretreatment with DEX and DMOG distinctly relieved lung injury, decreased the levels of mitochondrial ROS and mtDNA, reduced OSI, increased nuclear accumulation of HIF-1a and HO-1 protein in wild type mice but not HO-1 KO mice. Similar observations were recapitulated in NC siRNA transfected NR8383 cells after LPS stimulation but not HO-1 siRNA transfected cells. Concertedly, DEX reversed the impaired mitochondrial morphology in LPS stimulated-wild type mice or NC siRNA transfected NR8383 cells, upregulated the expression of mitochondrial fusion protein, while downregulated the expression of fission protein in HIF-1a/HO-1 dependent pathway. Altogether, our data both in vivo and in vitro certified that DEX treatment ameliorated endotoxin-induced acute lung injury by preserving the dynamic equilibrium of mitochondrial fusion/fission through the regulation of HIF-1a/HO-1 signaling pathway.

Quiescin sulfhydryl oxidase 1 promotes sorafenib-induced ferroptosis in hepatocellular carcinoma by driving EGFR endosomal trafficking and inhibiting NRF2 activation

2021-03-13T00:00

Sorafenib is a first-line molecular-target drug for advanced hepatocellular carcinoma (HCC), but its clinical effects are still limited. In this study we identify Quiescin sulfhydryl oxidase 1 (QSOX1) acting as a cellular pro-oxidant, specifically in the context of sorafenib treatment of HCC. QSOX1 disrupts redox homoeostasis and sensitizes HCC cells to oxidative stress by inhibiting activation of the master antioxidant transcription factor NRF2. A negative correlation between QSOX1 and NRF2 expression was validated in tumor tissues from 151 HCC patients. Mechanistically, QSOX1 restrains EGF-induced EGFR activation by promoting ubiquitination-mediated degradation of EGFR and accelerating its intracellular endosomal trafficking, leading to suppression of NRF2 activity. Additionally, QSOX1 potentiates sorafenib-induced ferroptosis by suppressing NRF2 in vitro and in vivo. In conclusion, the data presented identify QSOX1 as a novel candidate target for sorafenib-based combination therapeutic strategies in HCC or other EGFR-dependent tumor types.

•

QSOX1 impairs the antioxidant capacity of HCC cells by inhibiting NRF2 activation.

•

QSOX1 inhibits NRF2 activation by accelerating EGFR signaling termination.

•

QSOX1 promotes sorafenib-induced ferroptosis in HCC.

•

QSOX1 is a potential biomarker for adjuvant sorafenib treatment in HCC patients.

Htd2 deficiency-associated suppression of α-lipoic acid production provokes mitochondrial dysfunction and insulin resistance in adipocytes

2021-03-19T00:00

Mitochondria harbor a unique fatty acid synthesis pathway (mtFAS) with mysterious functions gaining increasing interest, while its involvement in metabolic regulation is essentially unknown. Here we show that 3-Hydroxyacyl-ACP dehydratase (HTD2), a key enzyme in mtFAS pathway was primarily downregulated in adipocytes of mice under metabolic disorders, accompanied by decreased de novo production of lipoic acid, which is the byproduct of mtFAS pathway. Knockdown of Htd2 in 3T3-L1 preadipocytes or differentiated 3T3-L1 mature adipocytes impaired mitochondrial function via suppression of complex I activity, resulting in enhanced oxidative stress and impaired insulin sensitivity, which were all attenuated by supplement of lipoic acid. Moreover, lipidomic study revealed limited lipid alterations in mtFAS deficient cells which primarily presenting accumulation of triglycerides, attributed to mitochondrial dysfunction. Collectively, the present study highlighted the pivotal role of mtFAS pathway in regulating mitochondrial function and adipocytes insulin sensitivity, demonstrating supportive evidence for lipoic acid being potential effective nutrient for improving insulin resistance and related metabolic disorders.

•

3-Hydroxyacyl-ACP dehydratase is decreased in adipocytes under diabetic condition.

•

Deficient of 3-Hydroxyacyl-ACP dehydratase (HTD2) triggers mitochondrial dysfunction.

•

Deficient of HTD2 promotes insulin resistance in adipocytes.

•

Supplement of lipoic acid ameliorates deleterious effects of HTD2 deficiency.

Cohousing-mediated microbiota transfer from milk bioactive components-dosed mice ameliorate colitis by remodeling colonic mucus barrier and lamina propria macrophages

Human milk oligosaccharides (HMOs) and milk fat globule membrane (MFGM) are highly abundant in breast milk, and have been shown to exhibit potent immunomodulatory effects. Yet, their role in the gut microbiota modulation in relation to colitis remains understudied. Since the mixtures of fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS) perfectly mimic the properties and functions of HMOs, the combination of MFGM, FOS, and GOS (CMFG) has therefore been developed and used in this study. Here, CMFG were pre-fed to mice for three weeks to investigate its preventive effect on dextran sodium sulfate (DSS) induced colitis. Moreover, CMFG-treated and vehicle-treated mice were cohoused to further elucidate the preventive role of the gut microbiota transfer in colitis. At the end of the study, 16S rDNA gene amplicon sequencing, short-chain fatty acids (SCFAs) profiling, transcriptome sequencing, histological analysis, immunofluorescence staining and flow cytometry analysis were conducted. Our results showed that CMFG pre-supplementation alleviated DSS-induced colitis as evidenced by decreased disease activity index (DAI) score, reduced body weight loss, increased colon length and mucin secretion, and ameliorated intestinal damage. Moreover, CMFG reduced macrophages in the colon, resulting in decreased levels of IL-1β, IL-6, IL-8, TNF-α, and MPO in the colon and circulation. Furthermore, CMFG altered the gut microbiota composition and promoted SCFAs production in DSS-induced colitis. Markedly, the cohousing study revealed that transfer of gut microbiota from CMFG-treated mice largely improved the DSS-induced colitis as evidenced by reduced intestinal damage and decreased macrophages infiltration in the colon. Moreover, transfer of the gut microbiota from CMFG-treated mice protected against DSS-induced gut microbiota dysbiosis and promotes SCFAs production, which showed to be associated with colitis amelioration. Collectively, these findings demonstrate the beneficial role of CMFG in the gastrointestinal diseases, and further provide evidence for the rational design of effective prophylactic functional diets in both animals and humans.

Expansion and persistence of antibiotic-specific resistance genes following antibiotic treatment

Oral antibiotics are commonly prescribed to non-hospitalized adults. However, antibiotic-induced changes in the human gut microbiome are often investigated in cohorts with preexisting health conditions and/or concomitant medication, leaving the effects of antibiotics not completely understood. We used a combination of omic approaches to comprehensively assess the effects of antibiotics on the gut microbiota and particularly the gut resistome of a small cohort of healthy adults. We observed that 3 to 19 species per individual proliferated during antibiotic treatment and Gram-negative species expanded significantly in relative abundance. While the overall relative abundance of antibiotic resistance gene homologs did not significantly change, antibiotic-specific gene homologs with presumed resistance toward the administered antibiotics were common in proliferating species and significantly increased in relative abundance. Virome sequencing and plasmid analysis showed an expansion of antibiotic-specific resistance gene homologs even 3 months after antibiotic administration, while paired-end read analysis suggested their dissemination among different species. These results suggest that antibiotic treatment can lead to a persistent expansion of antibiotic resistance genes in the human gut microbiota and provide further data in support of good antibiotic stewardship.

Abbreviation: ARG – Antibiotic resistance gene homolog; AsRG – Antibiotic-specific resistance gene homolog; AZY – Azithromycin; CFX – Cefuroxime; CIP – Ciprofloxacin; DOX – Doxycycline; FDR – False discovery rate; GRiD – Growth rate index value; HGT – Horizontal gene transfer; NMDS – Non-metric multidimensional scaling; qPCR – Quantitative polymerase chain reaction; RPM – Reads per million mapped reads; TA – Transcriptional activity; TE – Transposable element; TPM – Transcripts per million mapped reads

Opa1 relies on cristae preservation and ATP synthase to curtail reactive oxygen species accumulation in mitochondria

2021-03-19T00:00

Reactive oxygen species (ROS) are a common product of active mitochondrial respiration carried in mitochondrial cristae, but whether cristae shape influences ROS levels is unclear. Here we report that the mitochondrial fusion and cristae shape protein Opa1 requires mitochondrial ATP synthase oligomers to reduce ROS accumulation. In cells fueled with galactose to force ATP production by mitochondria, cristae are enlarged, ATP synthase oligomers destabilized, and ROS accumulate. Opa1 prevents both cristae remodeling and ROS generation, without impinging on levels of mitochondrial antioxidant defense enzymes that are unaffected by Opa1 overexpression. Genetic and pharmacologic experiments indicate that Opa1 requires ATP synthase oligomerization and activity to reduce ROS levels upon a blockage of the electron transport chain. Our results indicate that the converging effect of Opa1 and mitochondrial ATP synthase on mitochondrial ultrastructure regulate ROS abundance to sustain cell viability.

•

Mitochondrial ROS levels mirror changes in cristae shape.

•

F₁F_O-ATP synthase oligomerization and reversal link mitochondrial ultrastructure to ROS generation.

•

Disrupted F₁F_O-ATP synthase dimerization abolishes Opa1 ability to curtail mitochondrial ROS accumulation.

The intestinal microbiota and metabolites in patients with anorexia nervosa

Brain-gut microbiota interactions are intensively studied in connection with various neurological and psychiatric diseases. While anorexia nervosa (AN) pathophysiology is not entirely clear, it is presumably linked to microbiome dysbiosis. We aimed to elucidate the gut microbiota contribution in AN disease pathophysiology. We analyzed the composition and diversity of the gut microbiome of patients with AN (bacteriome and mycobiome) from stool samples before and after renourishment, and compared them to healthy controls. Further, levels of assorted neurotransmitters and short-chain fatty acids (SCFA) were analyzed in stool samples by MS and NMR, respectively. Biochemical, anthropometric, and psychometric profiles were assessed. The bacterial alpha-diversity parameter analyses revealed only increased Chao 1 index in patients with AN before the realimentation, reflecting their interindividual variation. Subsequently, core microbiota depletion signs were observed in patients with AN. Overrepresented OTUs (operation taxonomic units) in patients with AN taxonomically belonged to Alistipes, Clostridiales, Christensenellaceae, and Ruminococcaceae. Underrepresented OTUs in patients with AN were Faecalibacterium, Agathobacter, Bacteroides, Blautia, and Lachnospira. Patients exhibited greater interindividual variation in the gut bacteriome, as well as in metagenome content compared to controls, suggesting altered bacteriome functions. Patients had decreased levels of serotonin, GABA, dopamine, butyrate, and acetate in their stool samples compared to controls. Mycobiome analysis did not reveal significant differences in alpha diversity and fungal profile composition between patients with AN and healthy controls, nor any correlation of the fungal composition with the bacterial profile. Our results show the changed profile of the gut microbiome and its metabolites in patients with severe AN. Although therapeutic partial renourishment led to increased body mass index and improved psychometric parameters, SCFA, and neurotransmitter profiles, as well as microbial community compositions, did not change substantially during the hospitalization period, which can be potentially caused by only partial weight recovery.

Bacteroides uniformis combined with fiber amplifies metabolic and immune benefits in obese mice

Gut microbiota represents a therapeutic target for obesity. We hypothesize that B. uniformis CECT 7771 combined with wheat bran extract (WBE), its preferred carbon source, may exert superior anti-obesity effects. We performed a 17-week intervention in diet-induced obese mice receiving either B. uniformis, WBE, or their combination to identify interactions and independent actions on metabolism and immunity. B. uniformis combined with WBE was the most effective intervention, curbing weight gain and adiposity, while exerting more modest effects separately. The combination restored insulin-dependent metabolic routes in fat and liver, although the bacterium was the primary driver for improving whole-body glucose disposal. Moreover, B. uniformis-combined with WBE caused the highest increases in butyrate and restored the proportion of induced intraepithelial lymphocytes and type-3 innate lymphoid cells in the intestinal epithelium. Thus, strengthening the first line of immune defense against unhealthy diets and associated dysbiosis in the intestine. This intervention also attenuated the altered IL22 signaling and liver inflammation. Our study shows opportunities for employing B. uniformis, combined with WBE, to aid in the treatment of obesity.

Methionine restriction alleviates age-associated cognitive decline via fibroblast growth factor 21

2021-03-11T00:00

Methionine restriction (MR) extends lifespan and delays the onset of aging-associated pathologies. However, the effect of MR on age-related cognitive decline remains unclear. Here, we find that a 3-month MR ameliorates working memory, short-term memory, and spatial memory in 15-month-old and 18-month-old mice by preserving synaptic ultrastructure, increasing mitochondrial biogenesis, and reducing the brain MDA level in aged mice hippocampi. Transcriptome data suggest that the receptor of fibroblast growth factor 21 (FGF21)-related gene expressions were altered in the hippocampi of MR-treated aged mice. MR increased FGF21 expression in serum, liver, and brain. Integrative modelling reveals strong correlations among behavioral performance, MR altered nervous structure-related genes, and circulating FGF21 levels. Recombinant FGF21 treatment balanced the cellular redox status, prevented mitochondrial structure damages, and upregulated antioxidant enzymes HO-1 and NQO1 expression by transcriptional activation of Nrf2 in SH-SY5Y cells. Moreover, knockdown of Fgf21 by i.v. injection of adeno-associated virus abolished the neuroprotective effects of MR in aged mice. In conclusion, the MR exhibited the protective effects against age-related behavioral disorders, which could be partly explained by activating circulating FGF21 and promoting mitochondrial biogenesis, and consequently suppressing the neuroinflammation and oxidative damages. These results demonstrate that FGF21 can be used as a potential nutritional factor in dietary restriction-based strategies for improving cognition associated with neurodegeneration disorders.

•

MR suppresses age-associated cognitive impairment.

•

MR improves synapse ultrastructure and mitochondrial biogenesis in the hippocampus.

•

FGF21 is required for the beneficial effects of MR.

•

FGF21 activates Nrf2 signaling and alleviates neuroinflammation and oxidative stress.

bFGF alleviates diabetes-associated endothelial impairment by downregulating inflammation via S-nitrosylation pathway

2021-02-20T00:00

Protein S-nitrosylation is a reversible protein modification implicated in both physiological and pathophysiological regulation of protein function. However, the relationship between dysregulated S-nitrosylation homeostasis and diabetic vascular complications remains incompletely understood. Here, we demonstrate that basic fibroblast growth factor (bFGF) is a key regulatory link between S-nitrosylation homeostasis and inflammation, and alleviated endothelial dysfunction and angiogenic defects in diabetes. Subjecting human umbilical vein endothelial cells (HUVECs) to hyperglycemia and hyperlipidemia significantly decreased endogenous S-nitrosylated proteins, including S-nitrosylation of inhibitor kappa B kinase β (IKKβ^C179) and transcription factor p65 (p65^C38), which was alleviated by bFGF co-treatment. Pretreatment with carboxy-PTIO (c-PTIO), a nitric oxide scavenger, abolished bFGF-mediated S-nitrosylation increase and endothelial protection. Meanwhile, nitrosylation-resistant IKKβ^C179S and p65^C38S mutants exacerbated endothelial dysfunction in db/db mice, and in cultured HUVECs subjected to hyperglycemia and hyperlipidemia. Mechanistically, bFGF-mediated increase of S-nitrosylated IKKβ and p65 was attributed to synergistic effects of increased endothelial nitric oxide synthase (eNOS) and thioredoxin (Trx) activity. Taken together, the endothelial protective effect of bFGF under hyperglycemia and hyperlipidemia can be partially attributed to its role in suppressing inflammation via the S-nitrosylation pathway.

Far Upstream Binding Protein 1 (FUBP1) participates in translational regulation of Nrf2 protein under oxidative stress

2021-02-23T00:00

Oxidative stress is ubiquitously involved in disease etiology or progression. While the damaging effects have been well characterized, how cells deal with oxidative stress for prevention or removal of damage remains to be fully elucidated. Works from our laboratory have revealed de novo Nrf2 protein translation when cells are encountering low to mild levels of oxidative stress. Nrf2 encodes a transcription factor controlling a myriad of genes important for antioxidation, detoxification, wound repair and tissue remodeling. Here we report a role of FUBP1 in regulating de novo Nrf2 protein translation. An increase of FUBP1 binding to Nrf2 5′UTR due to H₂O₂ treatment has been found by LC-MS/MS, Far Western blot and ribonucleoprotein immunoprecipitation assays. Blocking FUBP1 expression using siRNA abolished H₂O₂ from inducing Nrf2 protein elevation or Nrf2 5′UTR activity. While no nuclear to cytoplasmic translocation was detected, cytosolic redistribution to the ribosomal fractions was observed due to oxidant treatment. The presence of FUBP1 in 40/43S ribosomal fractions confirm its involvement in translation initiation of Nrf2 protein. When tested by co-immunoprecipitation with eIF4E, eIF2a, eIF3η and eIF1, only eIF3η was found to gain physical interaction with FUBP1 due to H₂O₂ treatment. Our data support a role of FUBP1 for promoting the attachment of 40S ribosomal subunit to Nrf2 mRNA and formation of 43S pre-initiation complex for translation initiation of Nrf2 protein under oxidative stress.

The lncRNA Malat1 regulates microvascular function after myocardial infarction in mice via miR-26b-5p/Mfn1 axis-mediated mitochondrial dynamics

2021-02-22T00:00

Rationale

Myocardial infarction (MI) is a leading cause of cardiovascular mortality globally. The improvement of microvascular function is critical for cardiac repair after MI. Evidence now points to long non-coding RNAs (lncRNAs) as key regulators of cardiac remodelling processes. The lncRNA Malat1 is involved in the development and progression of multiple cardiac diseases. Studies have shown that Malat1 is closely related to the regulation of endothelial cell regeneration. However, the potential molecular mechanisms of Malat1 in repairing cardiac microvascular dysfunction after MI remain unreported.

Methods and results

The present study found that Malat1 is upregulated in the border zone of infarction in mouse hearts, as well as in isolated cardiac microvascular endothelial cells (CMECs). Targeted knockdown of Malat1 in endothelial cells exacerbated oxidative stress, attenuated angiogenesis and microvascular perfusion, and as a result decreased cardiac function in MI mice. Further studies showed that silencing Malat1 obviously inhibited CMEC proliferation, migration and tube formation, which was at least in part attributed to disturbed mitochondrial dynamics and activation of the mitochondrial apoptosis pathway. Moreover, bioinformatic analyses, luciferase assays and pull-down assays indicated that Malat1 acted as a competing endogenous RNA (ceRNA) for miR-26b-5p and formed a signalling axis with Mfn1 to regulate mitochondrial dynamics and endothelial functions. Overexpression of Mfn1 markedly reversed the microvascular dysfunction and CMEC injuries that were aggravated by silencing Malat1 via inhibition of excessive mitochondrial fragments and mitochondria-dependent apoptosis.

Conclusions

The present study elucidated the functions and mechanisms of Malat1 in cardiac microcirculation repair after MI. The underlying mechanisms of the effects of Malat1 could be attributed to its blocking effects on miR-26b-5p/Mfn1 pathway-mediated mitochondrial dynamics and apoptosis.

Taurine rescues mitochondria-related metabolic impairments in the patient-derived induced pluripotent stem cells and epithelial-mesenchymal transition in the retinal pigment epithelium

2021-02-28T00:00

Mitochondria participate in various metabolic pathways, and their dysregulation results in multiple disorders, including aging-related diseases. However, the metabolic changes and mechanisms of mitochondrial disorders are not fully understood. Here, we found that induced pluripotent stem cells (iPSCs) from a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) showed attenuated proliferation and survival when glycolysis was inhibited. These deficits were rescued by taurine administration. Metabolomic analyses showed that the ratio of the reduced (GSH) to oxidized glutathione (GSSG) was decreased; whereas the levels of cysteine, a substrate of GSH, and oxidative stress markers were upregulated in MELAS iPSCs. Taurine normalized these changes, suggesting that MELAS iPSCs were affected by the oxidative stress and taurine reduced its influence. We also analyzed the retinal pigment epithelium (RPE) differentiated from MELAS iPSCs by using a three-dimensional culture system and found that it showed epithelial mesenchymal transition (EMT), which was suppressed by taurine. Therefore, mitochondrial dysfunction caused metabolic changes, accumulation of oxidative stress that depleted GSH, and EMT in the RPE that could be involved in retinal pathogenesis. Because all these phenomena were sensitive to taurine treatment, we conclude that administration of taurine may be a potential new therapeutic approach for mitochondria-related retinal diseases.

•

iPS cell lines were derived from a MELAS patient with the mtDNA A3243G mutation.

•

Decreased proliferation and survival of MELAS iPSCs were rescued by taurine.

•

Reduction in GSH/GSSG ratio in MELAS iPSCs was suppressed by taurine.

•

EMT in MELAS iPSC-derived retinal pigment epithelium was suppressed by taurine.

•

Oxidative stress markers in MELAS iPSCs and RPE were suppressed by taurine.

MKL1 cooperates with p38MAPK to promote vascular senescence, inflammation, and abdominal aortic aneurysm

2021-02-20T00:00

Abdominal aortic aneurysm (AAA) is a catastrophic disease with little effective therapy. Myocardin related transcription factor A (MRTFA, MKL1) is a multifaceted transcription factor, regulating diverse biological processes. However, a detailed understanding of the mechanistic role of MKL1 in AAA has yet to be elucidated. In this study, we showed induced MKL1 expression in thoracic and abdominal aneurysmal tissues, respectively in both mice and humans. MKL1 global knockout mice displayed reduced AAA formation and aortic rupture compared with wild-type mice. Both gene deletion and pharmacological inhibition of MKL1 markedly protected mice from aortic dissection, an early event in Angiotensin II (Ang II)-induced AAA formation. Loss of MKL1 was accompanied by reduced senescence/proinflammation in the vessel wall and cultured vascular smooth muscle cells (VSMCs). Mechanistically, a deficiency in MKL1 abolished AAA-induced p38 mitogen activated protein kinase (p38MAPK) activity. Similar to MKL1, loss of MAPK14 (p38α), the dominant isoform of p38MAPK family in VSMCs suppressed Ang II-induced AAA formation, vascular inflammation, and senescence marker expression. These results reveal a molecular pathway of AAA formation involving MKL1/p38MAPK stimulation and a VSMC senescent/proinflammatory phenotype. These data support targeting MKL1/p38MAPK pathway as a potential effective treatment for AAA.

•

MKL1 expression is induced in both thoracic and abdominal aneurysmal tissues.

•

Genetic ablation and pharmacological inhibition of MKL1 protect mice from aortic dissection and AAA induced by Ang II.

•

Depletion of MKL1 in mice suppresses Ang II-induced vascular inflammation and senescence.

•

Depletion of MKL1 blunts the activation of p38MAPK and STAT3 pathways.

•

Loss of MAPK14 in VSMCs suppresses Ang II-induced AAA formation, vascular inflammation, and senescence marker expression.

Divergent trajectories of cellular bioenergetics, intermediary metabolism and systemic redox status in survivors and non-survivors of critical illness

2021-02-20T00:00

Background

Numerous pathologies result in multiple-organ failure, which is thought to be a direct consequence of compromised cellular bioenergetic status. Neither the nature of this phenotype nor its relevance to survival are well understood, limiting the efficacy of modern life-support.

Methods

To explore the hypothesis that survival from critical illness relates to changes in cellular bioenergetics, we combined assessment of mitochondrial respiration with metabolomic, lipidomic and redox profiling in skeletal muscle and blood, at multiple timepoints, in 21 critically ill patients and 12 reference patients.

Results

We demonstrate an end-organ cellular phenotype in critical illness, characterized by preserved total energetic capacity, greater coupling efficiency and selectively lower capacity for complex I and fatty acid oxidation (FAO)-supported respiration in skeletal muscle, compared to health. In survivors, complex I capacity at 48 h was 27% lower than in non-survivors (p = 0.01), but tended to increase by day 7, with no such recovery observed in non-survivors. By day 7, survivors’ FAO enzyme activity was double that of non-survivors (p = 0.048), in whom plasma triacylglycerol accumulated. Increases in both cellular oxidative stress and reductive drive were evident in early critical illness compared to health. Initially, non-survivors demonstrated greater plasma total antioxidant capacity but ultimately higher lipid peroxidation compared to survivors. These alterations were mirrored by greater levels of circulating total free thiol and nitrosated species, consistent with greater reductive stress and vascular inflammation, in non-survivors compared to survivors. In contrast, no clear differences in systemic inflammatory markers were observed between the two groups.

Conclusion

Critical illness is associated with rapid, specific and coordinated alterations in the cellular respiratory machinery, intermediary metabolism and redox response, with different trajectories in survivors and non-survivors. Unravelling the cellular and molecular foundation of human resilience may enable the development of more effective life-support strategies.

mtROS-mediated Akt/AMPK/mTOR pathway was involved in Copper-induced autophagy and it attenuates Copper-induced apoptosis in RAW264.7 mouse monocytes

2021-03-01T00:00

Copper (Cu) is a trace element necessary in animals as well as human beings. However, excessive Cu is toxic to immunocytes, but the precise mechanism is largely unclear so far. This work was conducted aiming to examine the Cu-mediated autophagy mechanism together with its role in Cu toxicology in RAW264.7 cells. Here, we demonstrated that CuSO₄ reduced the cell viability depending on its dose. CuSO₄ could obviously increase autophagy in RAW264.7 cells. According to the obtained results, CuSO₄ induced autophagy through Akt/AMPK/mTOR pathway which characterized by down regulation of p-Akt (Ser473)/Akt, p-mTOR/mTOR, p-ULK1(Ser757)/ULK1 and subsequent up-regulation of p-AMPKα/AMPKα and p-ULK1(Ser555)/ULK1. Furthermore, CuSO₄ significantly induced the production of mitochondrial reactive oxygen species (mtROS). In addition, CuSO₄-mediated apoptosis and autophagy might be suppressed through suppressing mtROS generation by exposure to Mito-TEMPO. Intriguingly, autophagy promotion with rapamycin could decrease the apoptosis and the inhibition of autophagy with knock down Atg5 could enhance the apoptosis induced by CuSO₄. Moreover, our results suggested that mtROS is the original cause in CuSO₄-induced apoptosis and autophagy. Additionally, CuSO₄ induced autophagy through mtROS-dependent Akt/AMPK/mTOR signalling pathwayin RAW264.7 cells. Moreover, autophagy activation might potentially generate a protection mechanism for improving CuSO₄-induced RAW264.7 cell apoptosis.

•

mtROS is the original cause in CuSO₄-induced apoptosis and autophagy.

•

CuSO₄ induced autophagy through mtROS-dependent Akt/AMPK/mTOR signalling pathway.

•

Autophagy attenuates CuSO₄-induced RAW264.7 cell apoptosis.

Progression from Monoclonal gammopathy of undetermined significance of the immunoglobulin M class (IgM-MGUS) to Waldenstrom Macroglobulinemia is associated with an alteration in lipid metabolism

2021-03-04T00:00

The molecular events that modulate the progression of monoclonal gammopathy of undetermined significance of the immunoglobulin M class (IgM-MGUS) to Waldenstrom Macroglobulinemia (WM) are mostly unknown. We implemented comparative proteomics and metabolomics analyses on patient serum samples to identify differentially expressed molecules crucial to the progression from IgM-MGUS to WM. Our data identified altered lipid metabolism as a discriminating factor between MGUS, WM, and matched normal controls. Levels of many fatty acids, including polyunsaturated fatty acids and dicarboxylic acids, were significantly downregulated in WM sera when compared to MGUS. These reductions were associated with diminished 15-LOX and PPAR protein expression and increased 5-LOX and GPX4 expression in WM versus MGUS patients’ samples. Furthermore, WM serum samples showed increased lipid peroxidation compared to MGUS. Treatment with IL-6 or TNFα, upstream regulators of differentially expressed proteins between MGUS and WM, increased lipid absorption and lipid peroxidation in WM cell lines. Knock-down of 15-LOX expression increased WM cell survival, an effect accompanied by increased 5-LOX and GPX4 expression. In summary, our data show that reduced fatty acid and lipid metabolite levels in the serum of the WM patients are associated with increased lipid peroxidation and that downregulation of 15-LOX increases the survival of WM cells. These data are highly significant in identifying the biomarkers of disease progression and designing targeted therapeutic intervention.

Endogenous SO₂-dependent Smad3 redox modification controls vascular remodeling

2021-02-18T00:00

Sulfur dioxide (SO₂) has emerged as a physiological relevant signaling molecule that plays a prominent role in regulating vascular functions. However, molecular mechanisms whereby SO₂ influences its upper-stream targets have been elusive. Here we show that SO₂ may mediate conversion of hydrogen peroxide (H₂O₂) to a more potent oxidant, peroxymonosulfite, providing a pathway for activation of H₂O₂ to convert the thiol group of protein cysteine residues to a sulfenic acid group, aka cysteine sulfenylation. By using site-centric chemoproteomics, we quantified >1000 sulfenylation events in vascular smooth muscle cells in response to exogenous SO₂. Notably, ~42% of these sulfenylated cysteines are dynamically regulated by SO₂, among which is cysteine-64 of Smad3 (Mothers against decapentaplegic homolog 3), a key transcriptional modulator of transforming growth factor β signaling. Sulfenylation of Smad3 at cysteine-64 inhibits its DNA binding activity, while mutation of this site attenuates the protective effects of SO₂ on angiotensin II-induced vascular remodeling and hypertension. Taken together, our findings highlight the important role of SO₂ in vascular pathophysiology through a redox-dependent mechanism.

•

Vascular smooth muscle cell-derived endogenous SO₂ regulates vascular remodeling and hypertension in vivo.

•

SO₂ may facilitate H₂O₂-mediated protein cysteine oxidation.

•

Chemoproteomics reveals Smad3^C64 as a target of SO₂-dependent sulfenylation.

•

Smad3^C64 is required for SO₂-dependent regulation of vascular functions in vivo.

Identification of proteins and cellular pathways targeted by 2-nitroimidazole hypoxic cytotoxins

2021-02-21T00:00

Tumour hypoxia negatively impacts therapy outcomes and continues to be a major unsolved clinical problem. Nitroimidazoles are hypoxia selective compounds that become entrapped in hypoxic cells by forming drug-protein adducts. They are widely used as hypoxia diagnostics and have also shown promise as hypoxia-directed therapeutics. However, little is known about the protein targets of nitroimidazoles and the resulting effects of their modification on cancer cells. Here, we report the synthesis and applications of azidoazomycin arabinofuranoside (N₃-AZA), a novel click-chemistry compatible 2-nitroimidazole, designed to facilitate (a) the LC-MS/MS-based proteomic analysis of 2-nitroimidazole targeted proteins in FaDu head and neck cancer cells, and (b) rapid and efficient labelling of hypoxic cells and tissues. Bioinformatic analysis revealed that many of the 62 target proteins we identified participate in key canonical pathways including glycolysis and HIF1A signaling that play critical roles in the cellular response to hypoxia. Critical cellular proteins such as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the detoxification enzyme glutathione S-transferase P (GSTP1) appeared as top hits, and N₃-AZA adduct formation significantly reduced their enzymatic activities only under hypoxia. Therefore, GAPDH, GSTP1 and other proteins reported here may represent candidate targets to further enhance the potential for nitroimidazole-based cancer therapeutics.

•

Azidoazomycin arabinofuranoside (N₃-AZA) is a novel hypoxia-targeting compound designed to undergo click chemistry.

•

We report 62 protein targets of N₃-AZA; many of which are critical for cellular response to hypoxia, such as GAPDH and GST.

•

Binding of N₃-AZA reduced GAPDH and GST enzyme activity, indicating that nitroimidazoles can disrupt key cellular pathways.

•

Fluorescent imaging with N₃-AZA click chemistry is highly efficient for mapping cellular and tissue hypoxia.

•

No relationship was observed between tumour size and hypoxia.

Dietary vitamin K is remodeled by gut microbiota and influences community composition

Vitamins have well-established roles in bacterial metabolism. Menaquinones (MKn, n = prenyl units in sidechain) are bacterially produced forms of vitamin K produced by the gut microbiota and consumed in the diet. Little is known about the influence of dietary vitamin K quinones on gut microbial composition and MKn production. Here, male and female C57BL6 mice were fed a vitamin K deficient diet or vitamin K sufficient diets containing phylloquinone (PK, plant-based vitamin K form), MK4, and/or MK9. DNA was extracted from cecal contents and 16S sequencing conducted to assess microbial composition. Cecal microbial community composition was significantly different in vitamin K deficient female mice compared to females on vitamin K sufficient diets (all p < .007). Parallel trends were seen in male mice, but were not statistically significant (all p > .05 but <0.1). Next, stable isotope-labeled vitamin K quinones were supplemented to male and female C57BL6 mice (²H₇PK, ¹³C₁₁MK4, ²H₇MK7, ²H₇MK9) and to an in vitro fermentation model inoculated with human stool (²H₇PK, ²H₇MK4, ²H₇MK9, or vitamin K precursor ²H₈-menadione). Vitamin K quinones in feces and culture aliquots were measured using LC-MS. In vivo, supplemented vitamin K quinones were remodeled to other MKn (²H₇- or ¹³C₆-labeled MK4, MK10, MK11, and MK12), but in vitro only the precursor ²H₈-menadione was remodeled to ²H₇MK4, ²H₇MK9, ²H₇MK10, and ²H₇MK11. These results suggest that dietary vitamin K deficiency alters the gut microbial community composition. Further studies are needed to determine if menadione generated by host metabolism may serve as an intermediate in dietary vitamin K remodeling in vivo.

Links between gut microbiome composition and fatty liver disease in a large population sample

Fatty liver disease is the most common liver disease in the world. Its connection with the gut microbiome has been known for at least 80 y, but this association remains mostly unstudied in the general population because of underdiagnosis and small sample sizes. To address this knowledge gap, we studied the link between the Fatty Liver Index (FLI), a well-established proxy for fatty liver disease, and gut microbiome composition in a representative, ethnically homogeneous population sample of 6,269 Finnish participants. We based our models on biometric covariates and gut microbiome compositions from shallow metagenome sequencing. Our classification models could discriminate between individuals with a high FLI (≥60, indicates likely liver steatosis) and low FLI (<60) in internal cross-region validation, consisting of 30% of the data not used in model training, with an average AUC of 0.75 and AUPRC of 0.56 (baseline at 0.30). In addition to age and sex, our models included differences in 11 microbial groups from class Clostridia, mostly belonging to orders Lachnospirales and Oscillospirales. Our models were also predictive of the high FLI group in a different Finnish cohort, consisting of 258 participants, with an average AUC of 0.77 and AUPRC of 0.51 (baseline at 0.21). Pathway analysis of representative genomes of the positively FLI-associated taxa in (NCBI) Clostridium subclusters IV and XIVa indicated the presence of, e.g., ethanol fermentation pathways. These results support several findings from smaller case–control studies, such as the role of endogenous ethanol producers in the development of the fatty liver.

Association between antibiotics and gut microbiome dysbiosis in children: systematic review and meta-analysis

Antibiotics in childhood have been linked with diseases including asthma, juvenile arthritis, type 1 diabetes, Crohn’s disease and mental illness. The underlying mechanisms are thought related to dysbiosis of the gut microbiome. We conducted a systematic review of the association between antibiotics and disruption of the pediatric gut microbiome. Searches used MEDLINE, EMBASE and Web of Science. Eligible studies: association between antibiotics and gut microbiome dysbiosis; children 0–18 years; molecular techniques of assessment; outcomes of microbiome richness, diversity or composition. Quality assessed by Newcastle–Ottawa Scale or Cochrane Risk of Bias Tool. Meta-analysis where possible. A total of 4,668 publications identified: 12 in final analysis (5 randomized controlled trials (RCTs), 5 cohort studies, 2 cross-sectional studies). Microbiome richness was measured in 3 studies, species diversity in 6, and species composition in 10. Quality of evidence was good or fair. 5 studies found a significant reduction in diversity and 3 a significant reduction in richness. Macrolide exposure was associated with reduced richness for twice as long as penicillin. Significant reductions were seen in Bifidobacteria (5 studies) and Lactobacillus (2 studies), and significant increases in Proteobacteria such as E. coli (4 studies). A meta-analysis of RCTs of the effect of macrolide (azithromycin) exposure on the gut microbiome found a significant reduction in alpha-diversity (Shannon index: mean difference −0.86 (95% CI −1.59, −0.13). Antibiotic exposure was associated with reduced microbiome diversity and richness, and with changes in bacterial abundance. The potential for dysbiosis in the microbiome should be taken into account when prescribing antibiotics for children.

Systematic review registration number: CRD42018094188

Gut microbiota shape the inflammatory response in mice with an epithelial defect

Intestinal epithelial cell endoplasmic reticulum (ER) stress has been implicated in intestinal inflammation. It remains unclear whether ER stress is an initiator of or a response to inflammation. Winnie mice, carrying a Muc2 gene mutation resulting in intestinal goblet cell ER stress, develop spontaneous colitis with a depleted mucus barrier and increased bacterial translocation. This study aims to determine whether the microbiota was required for the development of Winnie colitis, and whether protein misfolding itself can initiate inflammation directly in absence of the microbiota. To assess the role of microbiota in driving Winnie colitis, WT and Winnie mice on the same background were rederived into the germ-free facility and housed in the Trexler-type soft-sided isolators. The colitis phenotype of these mice was assessed and compared to WT and Winnie mice housed within a specific pathogen-free facility. We found that Winnie colitis was substantially reduced but not abolished under germ-free conditions. Expression of inflammatory cytokine genes was reduced but several chemokines remained elevated in absence of microbiota. Concomitantly, ER stress was also diminished, although mucin misfolding persisted. RNA-Seq revealed that Winnie differentiated colon organoids have decreased expression of the negative regulators of the inflammatory response compared to WT. This data along with the increase in Mip2a chemokine expression, suggests that the epithelial cells in the Winnie mice are more responsive to stimuli. Moreover, the data demonstrate that intestinal epithelial intrinsic protein misfolding can prime an inflammatory response without initiating the unfolded protein response in the absence of the microbiota. However, the microbiota is necessary for the amplification of colitis in Winnie mice. Genetic predisposition to mucin misfolding in secretory cells initiates mild inflammatory signals. However, the inflammatory signal sets a forward-feeding cycle establishing progressive inflammation in the presence of microbiota.

Abbreviations: Endoplasmic Reticulum: ER; Mucin-2: Muc-2; GF: Germ-Free; Inflammatory Bowel Disease: IBD

Intestinal and systemic inflammation induced by symptomatic and asymptomatic enterotoxigenic E. coli infection and impact on intestinal colonization and ETEC specific immune responses in an experimental human challenge model

Recent studies have gained a better appreciation of the potential impacts of enteric infections beyond symptomatic diarrhea. It is recognized that infections by several enteropathogens could be associated with growth deficits in children and intestinal and systemic inflammation may play an important underlying role. With enterotoxigenic E. coli (ETEC) being one of the leading causes of diarrhea among children in the developing world and important contributor to stunting, a better understanding of the impact of ETEC infection beyond diarrhea is timely and greatly needed. To address this, we evaluated if ETEC infection induces intestinal and systemic inflammation and its impact on colonization and immune responses to ETEC vaccine-specific antigens in a dose descending experimental human challenge model using ETEC strain H10407. This study demonstrates that the concentrations of myeloperoxidase (MPO) in stool and intestinal fatty acid-binding protein (an indicator of compromised intestinal epithelial integrity) in serum, significantly increased following ETEC infection in both diarrhea and asymptomatic cases and the magnitudes and kinetics of MPO are dose and clinical outcome dependent. Cytokines IL-17A and IFN-γ were significantly increased in serum post-ETEC challenge. In addition, higher pre-challenge concentrations of cytokines IL-10 and GM-CSF were associated with protection from ETEC diarrhea. Interestingly, higher MPO concentrations were associated with higher intestinal colonization of ETEC and lower seroconversions of colonization factor I antigen, but the reverse was noted for seroconversions to heat-labile toxin B-subunit. Together this study has important implications for understanding the acute and long-term negative health outcomes associated with ETEC infection.

Sirt3-mediated mitophagy regulates AGEs-induced BMSCs senescence and senile osteoporosis

2021-02-24T00:00

Senile osteoporosis (SOP) is widely regarded as one of the typical aging-related diseases due to a decrease in bone mass and the destruction in microarchitecture. The inhibition of mitophagy can promote bone marrow mesenchymal stem cells (BMSCs) senescence, and increasing studies have shown that interventions targeting BMSCs senescence can ameliorate osteoporosis, exhibiting their potential for use as therapeutic strategies. Sirtuin-3 (Sirt3) is an essential mitochondria metabolic regulatory enzyme that plays an important role in mitochondrial homeostasis, but its role in bone homeostasis remains largely unknown. This study seeks to investigate whether advanced glycation end products (AGEs) accumulation aggravated BMSCs senescence and SOP, and explored the mechanisms underlying these effects. We observed that AGEs significantly aggravated BMSCs senescence, as well as promoted mitochondrial dysfunction and inhibited mitophagy in a concentration-dependent manner. In addition, this effect could be further strengthened by Sirt3 silencing. Importantly, we identified that the reduction of Sirt3 expression and the mitophagy were vital mechanisms in AGEs-induced BMSCs senescence. Furthermore, overexpression of Sirt3 by intravenously injection with recombinant adeno-associated virus 9 carrying Sirt3 plasmids (rAAV-Sirt3) significantly alleviated BMSCs senescence and the formation of SOP in SAMP6. In conclusion, our data demonstrated that Sirt3 protects against AGEs-induced BMSCs senescence and SOP. Targeting Sirt3 to improve mitophagy may represent a potential therapeutic strategy for attenuating AGEs-associated SOP.

Binding of the human antioxidation protein α₁-microglobulin (A1M) to heparin and heparan sulfate. Mapping of binding site, molecular and functional characterization, and co-localization in vivo and in vitro

2021-02-10T00:00

Heparin and heparan sulfate (HS) are linear sulfated disaccharide polymers. Heparin is found mainly in mast cells, while heparan sulfate is found in connective tissue, extracellular matrix and on cell membranes in most tissues. α₁-microglobulin (A1M) is a ubiquitous protein with thiol-dependent antioxidant properties, protecting cells and matrix against oxidative damage due to its reductase activities and radical- and heme-binding properties. In this work, it was shown that A1M binds to heparin and HS and can be purified from human plasma by heparin affinity chromatography and size exclusion chromatography. The binding strength is inversely dependent of salt concentration and proportional to the degree of sulfation of heparin and HS. Potential heparin binding sites, located on the outside of the barrel-shaped A1M molecule, were determined using hydrogen deuterium exchange mass spectrometry (HDX-MS). Immunostaining of endothelial cells revealed pericellular co-localization of A1M and HS and the staining of A1M was almost completely abolished after treatment with heparinase. A1M and HS were also found to be co-localized in vivo in the lungs, aorta, kidneys and skin of mice. The redox-active thiol group of A1M was unaffected by the binding to HS, and the cell protection and heme-binding abilities of A1M were slightly affected. The discovery of the binding of A1M to heparin and HS provides new insights into the biological role of A1M and represents the basis for a novel method for purification of A1M from plasma.

Elevated levels of oxidized nucleosides in individuals with the JAK2V617F mutation from a general population study

2021-02-13T00:00

It is unknown if the somatic mutations in chronic myeloproliferative neoplasms (MPNs), JAK2V617F and Calreticulin, are associated with oxidative stress, or impaired mitochondrial defense against reactive oxygen species. In the Danish General Suburban Population Study (GESUS), including 116 JAK2V617F-mutated, 8 CALR-mutated, and 3310 mutation-negative participants without overt MPN, and in a study of 39 patients with myelofibrosis, the most advances type of MPNs, and 179 matched controls, we compared the urinary concentration of oxidized nucleosides – 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) – as markers of oxidative stress. In GESUS, we performed Mendelian randomization analyses, using the Ala16Val single nucleotide polymorphism in the superoxide dismutase2 (SOD2) gene. In the multivariate analyses in GESUS, the 8-oxodG and 8-oxoGuo concentration were 13% (95%CI: 6–21%, p < 0.001) and 6% (95%CI: 0.4–11%, p = 0.035) higher in mutation-positive than in mutation-negative participants, respectively. Each SOD2 T allele was associated with an odds ratio of being mutation-positive of 1.69 (95%CI: 1.12–2.55, p = 0.013) through 8-oxodG. The 8-oxodG and 8-oxoGuo concentrations were 77% (95%CI: 49–110%, p < 0.001) and 105% (95%CI: 80–133%, p < 0.001) higher in myelofibrosis patients than in controls, respectively. In conclusion, an impaired mitochondrial antioxidative defense, that is causatively associated with markers of oxidative stress, may contribute to the development of mutations associated with MPNs.

Formation of protein cross-links by singlet oxygen-mediated disulfide oxidation

2021-01-23T00:00

Cross-links formed within and between proteins are a major cause of protein dysfunction, and are postulated to drive the accumulation of protein aggregates in some human pathologies. Cross-links can be formed from multiple residues and can be reversible (usually sulfur-sulfur bonds) or irreversible (typically carbon-carbon or carbon-heteroatom bonds). Disulfides formed from oxidation of two Cys residues are widespread, with these formed both deliberately, via enzymatic reactions, or as a result of unintended oxidation reactions. We have recently demonstrated that new protein-glutathione mixed disulfides can be formed through oxidation of a protein disulfide to a thiosulfinate, and subsequent reaction of this species with glutathione. Here we investigate whether similar reactions occur between an oxidized protein disulfide, and a Cys residues on a second protein, to give novel protein cross-links. Singlet oxygen (¹O₂)-mediated oxidation of multiple proteins (α-lactalbumin, lysozyme, beta-2-microglobulin, C-reactive protein), and subsequent incubation with the Cys-containing protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH), generates inter-protein cross-links as detected by SDS-PAGE, immunoblotting and mass spectrometry (MS). The cross-link yield is dependent on the ¹O₂ concentration, the presence of the original protein disulfide bond, and the free Cys on GAPDH. MS with ¹⁸O-labeling has allowed identification of the residues involved in some cases (e.g. Cys25 from the Cys25-Cys80 disulfide in beta-2-microglobulin, with Cys149 or Cys244 of GAPDH). The formation of these cross-links results in a loss of GAPDH enzymatic activity. These data provide ‘proof-of-concept’ for a novel mechanism of protein cross-link formation which may help rationalize the accumulation of cross-linked proteins in multiple human pathologies.

•

Disulfide bonds (DSBs) are critical to protein structure and function.

•

DSBs are rapidly oxidized by singlet oxygen and other oxidants to reactive intermediates.

•

These intermediates react with Cys-containing proteins to give new protein-protein cross-links.

•

This novel disulfide cross-linking pathway affects the functional activity of the proteins.

•

These cross-links can be diminished by reductants, but this does not repair the DSB damage.

Reductively modified albumin attenuates DSS-Induced mouse colitis through rebalancing systemic redox state

2021-02-05T00:00

Albumin (Alb) is the most abundant plasma protein with multiple biological functions, including antioxidative property through its thiol activity. Given that inflammatory bowel disease is associated with a decreased level of Alb and an increased level of Alb oxidation, we asked whether Alb could have a therapeutic effect on colitis. Here we tested this possibility. Bovine serum albumin (BSA) was reductively modified with dithiothreitol (DTT) and administrated via gavage or intraperitoneal injection. Dextran sulfate sodium (DSS)-induced mice colitis was associated with massive oxidative stress, as indicated by the elevated sulfenic acid formation in blood, colon tissues, and feces. Treatment of mice with the reductively modified albumin (r-Alb) attenuated the oxidative stress and reduced local inflammation and tissue injury. These effects of r-Alb were only partially achieved by unmodified Alb and wholly lost after blocking the –SH groups with maleimide. In cultured colon epithelial cells, r-Alb prevented DSS- and H₂O₂-induced ROS elevation and barrier dysfunction, preceded by inhibition of sulfenic acid formation and P38 activation. Further analysis revealed that Alb was susceptible to H₂O₂-induced oxidation, and it detoxified H₂O₂ in a –SH group-dependent way. Moreover, Alb reacted with GSH/GSSG via thiol-disulfide exchange and reciprocally regulated the availability of –SH groups. Collectively, our study shows that r-Alb effectively attenuates DSS colitis via –SH group-mediated antioxidative action. Given that the oxidative stress underlies many life-threatening diseases, r-Alb, functioning as a potent antioxidant, could have a wide range of applications.

•

Albumin, the major thiol antioxidant in body, is decreased and oxidized in colitis.

•

Reductive modification of albumin increased its thiol-antioxidative activity.

•

Modified albumin ameliorated DSS colitis and improved systemic redox state.

•

Modified albumin detoxified H₂O₂ and prevented H₂O₂-induced cell injury.

•

Modified albumin interacted with GSH/GSSG/H₂O₂ via sulfhydryl group.

Effects of colon-targeted vitamins on the composition and metabolic activity of the human gut microbiome– a pilot study

An increasing body of evidence has shown that gut microbiota imbalances are linked to diseases. Currently, the possibility of regulating gut microbiota to reverse these perturbations by developing novel therapeutic and preventive strategies is being extensively investigated. The modulatory effect of vitamins on the gut microbiome and related host health benefits remain largely unclear. We investigated the effects of colon-delivered vitamins A, B2, C, D, and E on the gut microbiota using a human clinical study and batch fermentation experiments, in combination with cell models for the assessment of barrier and immune functions. Vitamins C, B2, and D may modulate the human gut microbiome in terms of metabolic activity and bacterial composition. The most distinct effect was that of vitamin C, which significantly increased microbial alpha diversity and fecal short-chain fatty acids compared to the placebo. The remaining vitamins tested showed similar effects on microbial diversity, composition, and/or metabolic activity in vitro, but in varying degrees. Here, we showed that vitamins may modulate the human gut microbiome. Follow-up studies investigating targeted delivery of vitamins to the colon may help clarify the clinical significance of this novel concept for treating and preventing dysbiotic microbiota-related human diseases. Trial registration: ClinicalTrials.gov, NCT03668964. Registered 13 September 2018 – Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT03668964.

The gut virome in Irritable Bowel Syndrome differs from that of controls

Irritable Bowel Syndrome (IBS), the most common gastrointestinal disorder, is diagnosed solely on symptoms. Potentially diagnostic alterations in the bacterial component of the gut microbiome (the bacteriome) are associated with IBS, but despite the known role of the virome (particularly bacteriophages), in shaping the gut bacteriome, few studies have investigated the virome in IBS. We performed metagenomic sequencing of fecal Virus-Like Particles (VLPs) from 55 patients with IBS and 51 control individuals. We detected significantly lower alpha diversity of viral clusters comprising both known and novel viruses (viral ‘dark matter’) in IBS and a significant difference in beta diversity compared to controls, but not between IBS symptom subtypes. The three most abundant bacteriophage clusters belonged to the Siphoviridae, Myoviridae, and Podoviridae families (Order Caudovirales). A core virome (defined as a cluster present in at least 50% of samples) of 5 and 12 viral clusters was identified in IBS and control subjects, respectively. We also identified a subset of viral clusters that showed differential abundance between IBS and controls. The virome did not co-vary significantly with the bacteriome, with IBS clinical subtype, or with Bile Acid Malabsorption status. However, differences in the virome could be related back to the bacteriome as analysis of CRISPR spacers indicated that the virome alterations were at least partially related to the alterations in the bacteriome. We found no evidence for a shift from lytic to lysogenic replication of core viral clusters, a phenomenon reported for the gut virome of patients with Inflammatory Bowel Disease. Collectively, our data show alterations in the virome of patients with IBS, regardless of clinical subtype, which may facilitate development of new microbiome-based therapeutics.

Tempol ameliorates polycystic ovary syndrome through attenuating intestinal oxidative stress and modulating of gut microbiota composition-serum metabolites interaction

2021-02-03T00:00

Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder, which is often accompanied by oxidative stress. Tempol, a superoxide dismutase mimetic, protects against several diseases caused by oxidative stress. However, the effect of tempol on PCOS has not been investigated. The present study demonstrated the alleviation of ovarian dysfunction and glucose tolerance in dehydroepiandrosterone (DHEA)-induced PCOS rats treated with tempol. Tempol significantly reduced the intestinal oxidative stress in PCOS rats without affecting the ovarian redox rate. The 16S rDNA sequencing of the intestinal microbiome and non-targeted metabolomics analysis indicated significant differences in gut microbiota composition and serum metabolite profiles between the control and PCOS rats, and most of these differences were reduced after tempol intervention. Tempol alters the gut microbiome by increasing the abundance of genus Ruminococcus_1 and by decreasing the abundance of Ruminococcus_2, Staphylococcus, Ideonella, and Corynebnacterium genera. Tempol also attenuates the reduction of serum bile acid and stachyose levels in PCOS rats, and the serum stachyose level was significantly correlated with the abundance of 15 genera, particularly Ruminococcus_1 and Ruminococcus_2. Moreover, stachyose administration improved ovarian dysfunction in PCOS rats. Thus, our data indicate that tempol ameliorates PCOS phenotype by reducing intestinal oxidative stress, restoring gut dysbiosis, and modulating the interaction between gut microbiota and host metabolite. Therefore, tempol intervention is a potential therapeutic approach for PCOS.

•

Tempol improved ovarian dysfunction and glucose tolerance in polycystic ovary syndrome rats.

•

Tempol ameliorates intestinal oxidative stress and gut microbiota dysbiosis.

•

The protective effect of tempol is associated alternations in serum bile acid and stachyose levels.

•

Stachyose administration improved ovarian dysfunction in polycystic ovary syndrome rats.

Ethnic variability associating gut and oral microbiome with obesity in children

Obesity is a growing worldwide problem that generally starts in the early years of life and affects minorities more often than Whites. Thus, there is an urgency to determine factors that can be used as targets as indicators of obesity. In this study, we attempt to generate a profile of gut and oral microbial clades predictive of disease status in African American (AA) and European American (EA) children. 16S rDNA sequencing of the gut and saliva microbial profiles were correlated with salivary amylase, socioeconomic factors (e.g., education and family income), and obesity in both ethnic populations. Gut and oral microbial diversity between AA and EA children showed significant differences in alpha-, beta-, and taxa-level diversity. While gut microbial diversity between obese and non-obese was not evident in EA children, the abundance of gut Klebsiella and Magasphaera was associated with obesity in AA children. In contrast, an abundance of oral Aggregatibacter and Eikenella in obese EA children was observed. These observations suggest an ethnicity-specific association with gut and oral microbial profiles. Socioeconomic factors influenced microbiota in obesity, which were ethnicity dependent, suggesting that specific approaches to confront obesity are required for both populations.

Endothelial specific deletion of HMGB1 increases blood pressure and retards ischemia recovery through eNOS and ROS pathway in mice^☆

2021-02-05T00:00

Recent studies demonstrated HMGB1, an extracellular inflammation molecule, played an important role on endothelial cells. This study aimed to define the role and related mechanism of HMGB1 in endothelial cells. Endothelial-specific deletion of HMGB1(HMGB1ECKO) was generated and Akt/eNOS signaling, reactive oxygen species (ROS) production, endothelium dependent relaxation (EDR), and angiogenesis were determined in vitro and in vivo. Decreased activation of Akt/eNOS signaling, sprouting, and proliferation, and increased ROS production were evidenced in endothelial cells derived from HMGB1ECKO mice as compared with wild type controls. Decreased EDR and retarded blood flow recovery after hind limb ischemia were also demonstrated in HMGB1ECKO mice. Both impaired EDR and angiogenesis could be partly rescued by superoxide dismutase in HMGB1ECKO mice. In conclusion, intracellular HMGB1 might be a key regulator of endothelial Akt/eNOS pathway and ROS production, thus plays an important role in EDR regulation and angiogenesis.

Roux-en-Y gastric bypass surgery in Zucker rats induces bacterial and systemic metabolic changes independent of caloric restriction-induced weight loss

Mechanisms of Roux-en-Y gastric bypass (RYGB) surgery are not fully understood. This study aimed to investigate weight loss-independent bacterial and metabolic changes, as well as the absorption of bacterial metabolites and bile acids through the hepatic portal system following RYGB surgery. Three groups of obese Zucker (fa/fa) rats were included: RYGB (n = 11), sham surgery and body weight matched with RYGB (Sham-BWM, n = 5), and sham surgery fed ad libitum (Sham-obese, n = 5). Urine and feces were collected at multiple time points, with portal vein and peripheral blood obtained at the end of the study. Metabolic phenotyping approaches and 16S rRNA gene sequencing were used to determine the biochemical and bacterial composition of the samples, respectively. RYGB surgery-induced distinct metabolic and bacterial disturbances, which were independent of weight loss through caloric restriction. RYGB resulted in lower absorption of phenylalanine and choline, and higher urinary concentrations of host-bacterial co-metabolites (e.g., phenylacetylglycine, indoxyl sulfate), together with higher fecal trimethylamine, suggesting enhanced bacterial aromatic amino acid and choline metabolism. Short chain fatty acids (SCFAs) were lower in feces and portal vein blood from RYGB group compared to Sham-BWM, accompanied with lower abundances of Lactobacillaceae, and Ruminococcaceae known to contain SCFA producers, indicating reduced bacterial fiber fermentation. Fecal γ-amino butyric acid (GABA) was found in higher concentrations in RYGB than that in Sham groups and could play a role in the metabolic benefits associated with RYGB surgery. While no significant difference in urinary BA excretion, RYGB lowered both portal vein and circulating BA compared to Sham groups. These findings provide a valuable resource for how dynamic, multi-systems changes impact on overall metabolic health, and may provide potential therapeutic targets for developing downstream non-surgical treatment for metabolic disease.

The rectal mucosal but not fecal microbiota detects subclinical ulcerative colitis

Ulcerative colitis (UC), a subtype of inflammatory bowel disease, is characterized by repetitive remission and relapse. Gut microbiome is critically involved in pathogenesis of UC. The shifts in microbiome profile during disease remission remain under-investigated. Recent studies revealed that UC pathogenesis is likely to originate in the mucosal barrier. Therefore, we investigated the effectiveness of mucosal tissue microbiomes to differentiate patients with subclinical UC from healthy individuals. The microbiomes of cecal and rectal biopsies and feces were characterized from 13 healthy individuals and 45 patients with subclinical UC. Total genomic DNA was extracted from the samples, and their microbial communities determined using next-generation sequencing. We found that changes in relative abundance of subclinical UC were marked by a decrease in Proteobacteria and an increase in Bacteroidetes phyla in microbiome derived from rectal tissues but not cecal tissue nor feces. Only in the microbiome of rectal tissue had significantly higher community richness and evenness in subclinical UC patients than controls. Twenty-seven operational taxonomic units were enriched in subclinical UC cohort with majority of the taxa from the Firmicutes phylum. Inference of putative microbial functional pathways from rectal biopsy microbiome suggested a differential increase in interleukin-17 signaling and T-helper cell differentiation pathways. Rectal biopsy tissue was suggested to be more suitable than fecal samples for microbiome assays to distinguish patients with subclinical UC from healthy adults. Assessment of the rectal biopsy microbiome may offer clinical insight into UC disease progression and predict relapse of the diseases.

Involvement of the microbiota-gut-brain axis in chronic restraint stress: disturbances of the kynurenine metabolic pathway in both the gut and brain

Emerging evidence suggests that the gut microbiota may interact with the host brain and play pivotal roles in the pathogenesis of neuropsychiatric disorders. However, the mechanism underlying reciprocal interactions along the microbiota-gut-brain axis in depression remains unclear. In this study, a murine model of chronic restraint stress (CRS) was established to investigate the metabolic signaling of tryptophan (Trp) neurotransmission at the intestinal and central levels in depression. The results showed that CRS mice displayed depression- and anxiety-like behaviors. Additionally, kynurenine (Kyn) and its metabolites, an important Trp metabolic pathway, were strongly activated in the brain. Intriguingly, the Kyn toxic signaling was exacerbated in the gut, especially in the colon. Indoleamine 2,3-dioxygenase (IDO), a rate-limiting enzyme responsible for Kyn metabolic pathway initiation, was significantly upregulated in the brain and gut in CRS mice compared with control mice, promoting transfer of Trp metabolic pathway to Kyn signaling. Additionally, administration of IDO inhibitor, 1-methyl-tryptophan (1-MT), partially rescued CRS-induced depression- and anxiety-like changes. Moreover, the enhanced intestinal permeability mediated by CRS allowed toxic metabolites to “leak” into the bloodstream. The microbiome profiles of CRS mice displayed obviously altered taxonomic composition and negative correlations were observed between Enterorhabdus, Parabacteroides and Kyn levels in the brain. Reciprocal crosstalk between the brain and gut was further validated by citalopram treatment, IDO inhibitor and microbiota intervention, which counteracted depression-like behavior, Kyn metabolic signaling and microbiota composition in CRS mice. Meanwhile, Parabacteroides treatment affected Trp metabolism in mouse hippocampus, manifesting as elevated concentration of 5-HT as well as ratio of 5-HT to Trp. These results suggest that long-term stress disrupts Kyn metabolism and endocrine function along the gut-brain axis, accompanied by the disrupted homeostasis of certain microbiota, which collectively contribute to the development of depression-like behavior.

Early life environmental exposures have a minor impact on the gut ecosystem following a natural birth

A growing body of evidence suggests that the environment is an important source of colonizing bacteria for the gastrointestinal tract of C-section delivered infants, who undergo multiple birth-related interventions; however, the extent to which environmental microbes impact vaginally delivered infants remains unclear. Here we investigated the impact of rural and urban environmental exposures on microbial establishment and immunity in vaginally delivered mice. We simulated rural and urban home environments by adding soil types to cages from breeding to weaning. Our aims were to determine the impact of rural and urban soil exposures on the gut microbiome in young mice and to understand whether these changes persisted into adulthood. Host immune cytokines and microbial short-chain fatty acids were quantified to understand the impact on immunity. We found that early-life soil exposure had a minor effect on the richness of the neonatal gut microbiota contributing 5% and 9% variation in the bacterial community structure between mice during early-life and adulthood, respectively. Exposure to urban soil increased Clostridiaceae and propionic acid which persisted into adulthood. While soil exposure had a limited effect on the gut taxa, systemic cytokine and chemokine profiles were altered in adulthood. The findings presented here show that unlike in C-section deliveries previously reported, environmental exposures following a natural birth have a limited impact on the gut microbial taxa but potentially play an important role in immune-mediated disease susceptibility later in life.

Dietary betaine prevents obesity through gut microbiota-drived microRNA-378a family

Betaine is a natural compound present in commonly consumed foods and may have a potential role in the regulation of glucose and lipids metabolism. However, the underlying molecular mechanism of its action remains largely unknown. Here, we show that supplementation with betaine contributes to improved high-fat diet (HFD)-induced gut microbiota dysbiosis and increases anti-obesity strains such as Akkermansia muciniphila, Lactobacillus, and Bifidobacterium. In mice lacking gut microbiota, the functional role of betaine in preventing HFD-induced obesity, metabolic syndrome, and inactivation of brown adipose tissues are significantly reduced. Akkermansia muciniphila is an important regulator of betaine in improving microbiome ecology and increasing strains that produce short-chain fatty acids (SCFAs). Increasing two main members of SCFAs including acetate and butyrate can significantly regulate the levels of DNA methylation at host miR-378a promoter, thus preventing the development of obesity and glucose intolerance. However, these beneficial effects are partially abolished by Yin yang (YY1), a common target gene of the miR-378a family. Taken together, our findings demonstrate that betaine can improve obesity and associated MS via the gut microbiota-derived miR-378a/YY1 regulatory axis, and reveal a novel mechanism by which gut microbiota improve host health.

Human galectin-1 and galectin-3 promote Tropheryma whipplei infection

Tropheryma whipplei, is an actinobacterium that causes different infections in humans, including Whipple’s disease. The bacterium infects and replicates in macrophages, leading to a Th2-biased immune response. Previous studies have shown that T. whipplei harbors complex surface glycoproteins with evidence of sialylation. However, the exact contribution of these glycoproteins for infection and survival remains obscure. To address this, we characterized the bacterial glycoprofile and evaluated the involvement of human β-galactoside-binding lectins, Galectin-1 (Gal-1) and Galectin-3 (Gal-3) which are highly expressed by macrophages as receptors for bacterial glycans.

Tropheryma whipplei glycoproteins harbor different sugars including glucose, mannose, fucose, β-galactose and sialic acid. Mass spectrometry identification revealed that these glycoproteins were membrane- and virulence-associated glycoproteins. Most of these glycoproteins are highly sialylated and N-glycosylated while some of them are rich in poly-N-acetyllactosamine (Poly-LAcNAc) and bind Gal-1 and Gal-3. In vitro, T. whipplei modulates the expression and cellular distribution of Gal-1 and Gal-3. Although both galectins promote T. whipplei infection by enhancing bacterial cell entry, only Gal-3 is required for optimal bacterial uptake. Finally, we found that serum levels of Gal-1 and Gal-3 were altered in patients with T. whipplei infections as compared to healthy individuals, suggesting that galectins are also involved in vivo.

Among T. whipplei membrane-associated proteins, poly-LacNAc rich-glycoproteins promote infection through interaction with galectins. T. whipplei modulates the expression of Gal-1 and Gal-3 both in vitro and in vivo. Drugs interfering with galectin–glycan interactions may provide new avenues for the treatment and diagnosis of T. whipplei infections.

B. adolescentis ameliorates chronic colitis by regulating Treg/Th2 response and gut microbiota remodeling

Inflammatory bowel disease (IBD) is defined as an immune dysregulation disease with poor prognosis. Various therapies based on gut microbe modulation have been proposed. In this study, we aim to explore the therapeutic effect of B. adolescentis on IBD, as well as the immune and microecology mechanism of B. adolescentis in IBD. The fecal level of B. adolescentis was decreased in the IBD patients compared with the normal people in our cohort and the GMrepo database. To further clarify the role of B. adolescentis in IBD, we induced chronic colitis with three cycles of dextran sulfate sodium (DSS). We found B. adolescentis gavage exhibited protective effects as evidenced by the significantly decreased diarrhea score, spleen weight, and increased colon length. Accordingly, the cumulative histological grading was decreased in the B. adolescentis administration group. In addition, tight junction protein and mucin family were enhanced after B. adolescentis treatment. Furthermore, distinct effects were found with decreased pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β, IL-18, IL-22, IL-9 and increased anti-inflammatory cytokines IL-10, IL-4, IL-5. Importantly, the colon lamina propria in the B. adolescentis group consisted of more Treg and Th2 cells, which inhibited extreme gut inflammation. Additionally, 16srRNA sequencing showed an evident increase in the B:F ratio in the B. adolescentis group. In particular, B. adolescentis application inhibited the excessive growth of Akkermansia and Escherichia-Shigella in genus level. In conclusion, B. adolescentis refined the DSS-induced chronic colitis by stimulating protective Treg/Th2 response and gut microbiota remodeling. B. adolescentis regularly treatment might improve the therapeutic effects for inflammatory bowel disease.

Helicobacter hepaticus is required for immune targeting of bacterial heat shock protein 60 and fatal colitis in mice

Gut microbiota and the immune system are in constant exchange shaping both host immunity and microbial communities. Here, improper immune regulation can cause inflammatory bowel disease (IBD) and colitis. Antibody therapies blocking signaling through the CD40–CD40L axis showed promising results as these molecules are deregulated in certain IBD patients. To better understand the mechanism, we used transgenic DC-LMP1/CD40 animals with a constitutive CD40-signal in CD11c⁺ cells, causing a lack of intestinal CD103⁺ dendritic cells (DCs) and failure to induce regulatory T (iTreg) cells. These mice rapidly develop spontaneous fatal colitis, accompanied by dysbiosis and increased inflammatory IL-17⁺IFN-γ⁺ Th17/Th1 and IFN-γ ⁺ Th1 cells. In the present study, we analyzed the impact of the microbiota on disease development and detected elevated IgA- and IgG-levels in sera from DC-LMP1/CD40 animals. Their serum antibodies specifically bound intestinal bacteria, and by proteome analysis, we identified a 60 kDa chaperonin GroEL (Hsp60) from Helicobacter hepaticus (Hh) as the main specific antigen targeted in the absence of iTregs. When re-derived to a different Hh-free specific-pathogen-free (SPF) microbiota, mice showed few signs of disease, normal microbiota, and no fatality. Upon recolonization of mice with Hh, the disease developed rapidly. Thus, the present work identifies GroEL/Hsp60 as a major Hh-antigen and its role in disease onset, progression, and outcome in this colitis model. Our results highlight the importance of CD103⁺ DC- and iTreg-mediated immune tolerance to specific pathobionts to maintain healthy intestinal balance.

YjbH mediates the oxidative stress response and infection by regulating SpxA1 and the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) in Listeria monocytogenes

The foodborne pathogen Listeria monocytogenes relies on its ability to fine-tune the expression of virulence factors and stress regulators in response to rapidly changing environments. Here, we reveal that YjbH, a putative thioredoxin family oxidoreductase, plays a pivotal role in bacterial adaption to oxidative stress and host infection. YjbH directly interacts with SpxA1, an ArsC family oxidative stress response regulator, and the deletion of YjbH compromised the oxidative stress tolerance of L. monocytogenes. Also, YjbH is required for the bacterial spread in host cells and proliferation in mouse organs, thereby contributing to virulence. Transcriptomic analysis of strains treated with Cd²⁺ revealed that most virulence genes and phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) genes were significantly downregulated in the absence of YjbH. However, YjbH inhibits PrfA expression when bacteria were grown in the media, suggesting that YjbH participates in regulating the virulence genes via a complicated regulatory network involving PrfA and PTS. Collectively, these findings provide a valuable model for clarifying the roles of thioredoxins from foodborne pathogens regarding improving survival in the external environment and, more importantly, successfully establishing infection within the host.

Elevated gut microbiome abundance of Christensenellaceae, Porphyromonadaceae and Rikenellaceae is associated with reduced visceral adipose tissue and healthier metabolic profile in Italian elderly

Aging is accompanied by physiological changes affecting body composition and functionality, including accumulation of fat mass at the expense of muscle mass, with effects upon morbidity and quality of life. The gut microbiome has recently emerged as a key environmental modifier of human health that can modulate healthy aging and possibly longevity. However, its associations with adiposity in old age are still poorly understood. Here we profiled the gut microbiota in a well-characterized cohort of 201 Italian elderly subjects from the NU-AGE study, by 16S rRNA amplicon sequencing. We then tested for association with body composition from dual-energy X-ray absorptiometry (DXA), with a focus on visceral and subcutaneous adipose tissue. Dietary patterns, serum metabolome and other health-related parameters were also assessed. This study identified distinct compositional structures of the elderly gut microbiota associated with DXA parameters, diet, metabolic profiles and cardio-metabolic risk factors.

The mitochondrial redistribution of eNOS is involved in lipopolysaccharide induced inflammasome activation during acute lung injury

2021-01-26T00:00

Acute lung injury (ALI) is a devastating clinical syndrome with no effective therapies. Inflammasome activation has been reported to play a critical role in the initiation and progression of ALI. The molecular mechanisms involved in regulating the activation of inflammasome in ALI remains unresolved, although increases in mitochondrial derived reactive oxygen species (mito-ROS) are involved. Our previous work has shown that the mitochondrial redistribution of uncoupled eNOS impairs mitochondrial bioenergetics and increases mito-ROS generation. Thus, the focus of our study was to determine if lipopolysaccharide (LPS)-mediated inflammasome activation involves the mitochondrial redistribution of uncoupled eNOS. Our data show that the increase in mito-ROS involved in LPS-mediated inflammasome activation is associated with the disruption of mitochondrial bioenergetics in human lung microvascular endothelial cells (HLMVEC) and the mitochondrial redistribution of eNOS. These effects are dependent on RhoA-ROCK signaling and are mediated via increased phosphorylation of eNOS at Threonine (T)-495. A derivative of the mitochondrial targeted Szeto‐Schiller peptide (SSP) attached to the antioxidant Tiron (T-SSP), significantly attenuated LPS-mediated mito-ROS generation and inflammasome activation in HLMVEC. Further, T-SSP attenuated mitochondrial superoxide production in a mouse model of sepsis induced ALI. This in turn significantly reduced the inflammatory response and attenuated lung injury. Thus, our findings show that the mitochondrial redistribution of uncoupled eNOS is intimately involved in the activation of the inflammatory response in ALI and implicate attenuating mito-ROS as a therapeutic strategy in humans.

•

The mitochondrial redistribution of uncoupled eNOS is involved in LPS-mediated activation of the NLRP3 inflammasome.

•

Nitration-mediated activation of RhoA is involved in the activation of NF-κb signaling during sepsis.

•

Blocking RhoA nitration attenuates the increase in mitochondrial ROS required for NLRP3 inflammasome activation.

•

A mitochondrial targeted antioxidant reduces NLRP3 inflammasome activation and sepsis mediated lung injury.

Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions

2021-02-01T00:00

Background

The adaptation to hypoxia is mainly controlled by the HIF transcription factors. Increased expression/activity of HIF-1α correlates with poor prognosis in cancer patients. PARP-1 inhibitors are used in the clinic to treat BRCAness breast/ovarian cancer and have been shown to regulate the hypoxic response; therefore, their use could be expanded.

Methods

In this work by integrating molecular/cell biology approaches, genome-wide ChIP-seq, and patient samples, we elucidate the extent to which PARP-1 exerts control over HIF-1-regulated genes.

Results

In human melanoma, PARP-1 and HIF-1α expression are strongly associated. In response to a hypoxic challenge poly(ADP-ribose) (PAR) is synthesized, HIF-1α is post-transcriptionally modified (PTM) and stabilized by PARylation at specific K/R residues located at its C-terminus. Using an unbiased ChIP-seq approach we demonstrate that PARP-1 dictates hypoxia-dependent HIF-recruitment to chromatin in a range of HIF-regulated genes while analysis of HIF-binding motifs (RCGTG) reveals a restriction on the recognition of hypoxia responsive elements in the absence of PARP-1. Consequently, the cells are poorly adapted to hypoxia, showing a reduced fitness during hypoxic induction.

Conclusions

These data characterize the fine-tuning regulation by PARP-1/PARylation of HIF activation and suggest that PARP inhibitors might have therapeutic potential against cancer types displaying HIF-1α over-activation.

Citrulline supplementation attenuates the development of non-alcoholic steatohepatitis in female mice through mechanisms involving intestinal arginase

2021-01-26T00:00

Non-alcoholic fatty liver disease (NAFLD) is by now the most prevalent liver disease worldwide. The non-proteogenic amino acid l-citrulline (L-Cit) has been shown to protect mice from the development of NAFLD. Here, we aimed to further assess if L-Cit also attenuates the progression of a pre-existing diet-induced NAFLD and to determine molecular mechanisms involved. Female C57BL/6J mice were either fed a liquid fat-, fructose- and cholesterol-rich diet (FFC) or control diet (C) for 8 weeks to induce early stages of NASH followed by 5 more weeks with either FFC-feeding +/- 2.5 g L-Cit/kg bw or C-feeding. In addition, female C57BL/6J mice were either pair-fed a FFC +/- 2.5 g L-Cit/kg bw +/- 0.01 g/kg bw i.p. N(ω)-hydroxy-nor-l-arginine (NOHA) or C diet for 8 weeks.

The protective effects of supplementing L-Cit on the progression of a pre-existing NAFLD were associated with an attenuation of 1) the increased translocation of bacterial endotoxin and 2) the loss of tight junction proteins as well as 3) arginase activity in small intestinal tissue, while no marked changes in intestinal microbiota composition were prevalent in small intestine. Treatment of mice with the arginase inhibitor NOHA abolished the protective effects of L-Cit on diet-induced NAFLD. Our results suggest that the protective effects of L-Cit on the development and progression of NAFLD are related to alterations of intestinal arginase activity and intestinal permeability.

•

l-citrulline diminished progression of non-alcoholic fatty liver disease (NAFLD).

•

l-citrulline protects from fructose-induced small intestinal barrier dysfunction.

•

NASH development is associated with a loss of arginase activity in small intestine.

•

l-citrulline improves intestinal arginase activity in diet-induced NAFLD.

•

Arginase inhibitor attenuates effects of l-citrulline on NAFLD development.

Increased survivability of coronavirus and H1N1 influenza virus under electrostatic aerosol-to-hydrosol sampling

2021-02-12T00:00

Airborne virus susceptibility is an underlying cause of severe respiratory diseases, raising pandemic alerts worldwide. Following the first reports of the novel severe acute respiratory syndrome coronavirus-2 in 2019 and its rapid spread worldwide and the outbreak of a new highly variable strain of influenza A virus (H1N1) in 2009, developing quick, accurate monitoring and diagnostic approaches for emerging infections is considered critical. Efficient air sampling of coronaviruses and the H1N1 virus allows swift, real-time identification, triggering early adjuvant interventions. Electrostatic precipitation is an efficient method for sampling bio-aerosols as hydrosols; however, sampling conditions critically impact this method. Corona discharge ionizes surrounding air, generating reactive oxygen species (ROS), which may impair virus structural components, leading to RNA and/or protein damage and preventing virus detection. Herein, ascorbic acid (AA) dissolved in phosphate-buffered saline (PBS) was used as the sampling solution of an electrostatic sampler to counteract virus particle impairment, increasing virus survivability throughout sampling. The findings of this study indicate that the use of PBS+AA is effective in reducing the ROS damage of viral RNA by 95%, viral protein by 45% and virus yield by 60%.

Modulation of hypochlorous acid (HOCl) induced damage to vascular smooth muscle cells by thiocyanate and selenium analogues

2021-01-21T00:00

The production of hypochlorous acid (HOCl) by myeloperoxidase (MPO) plays a key role in immune defense, but also induces host tissue damage, particularly in chronic inflammatory pathologies, including atherosclerosis. This has sparked interest in the development of therapeutic approaches that decrease HOCl formation during chronic inflammation, including the use of alternative MPO substrates. Thiocyanate (SCN⁻) supplementation decreases HOCl production by favouring formation of hypothiocyanous acid (HOSCN), which is more selectively toxic to bacterial cells. Selenium-containing compounds are also attractive therapeutic agents as they react rapidly with HOCl and can be catalytically recycled. In this study, we examined the ability of SCN⁻, selenocyanate (SeCN⁻) and selenomethionine (SeMet) to modulate HOCl-induced damage to human coronary artery smooth muscle cells (HCASMC), which are critical to both normal vessel function and lesion formation in atherosclerosis. Addition of SCN⁻ prevented HOCl-induced cell death, altered the pattern and extent of intracellular thiol oxidation, and decreased perturbations to calcium homeostasis and pro-inflammatory signaling. Protection was also observed with SeCN⁻ and SeMet, though SeMet was less effective than SeCN⁻ and SCN⁻. Amelioration of damage was detected with sub-stoichiometric ratios of the added compound to HOCl. The effects of SCN⁻ are consistent with conversion of HOCl to HOSCN. Whilst SeCN⁻ prevented HOCl-induced damage to a similar extent to SCN⁻, the resulting product hyposelenocyanous acid (HOSeCN), was more toxic to HCASMC than HOSCN. These results provide support for the use of SCN⁻ and/or selenium analogues as scavengers, to decrease HOCl-induced cellular damage and HOCl production at inflammatory sites in atherosclerosis and other pathologies.

•

HOCl induces extensive smooth muscle cell death and irreversible thiol oxidation.

•

Addition of SCN⁻ decreases the extent of HOCl-induced cell damage.

•

SeCN⁻ has similar protective effects to SCN⁻ towards HOCl-induced cell damage.

•

HOSeCN is less toxic than HOCl but more damaging than HOSCN.

•

SeMet modulates HOCl-induced damage but less effectively than SCN⁻ or SeCN⁻.

Apolipoprotein E deficiency induces a progressive increase in tissue iron contents with age in mice

2021-01-16T00:00

Association of both iron/hepcidin and apolipoprotein E (ApoE) with development of Alzheimer disease (AD) and atherosclerosis led us to hypothesize that ApoE might be required for body iron homeostasis. Here, we demonstrated that ApoE knock-out (KO) induced a progressive accumulation of iron with age in the liver and spleen of mice. Subsequent investigations showed that the increased iron in the liver and spleen was due to phosphorylated extracellular regulated protein kinases (pERK) mediated up-regulation of transferrin receptor 1 (TfR1), and nuclear factor erythroid 2-related factor-2 (Nrf2)-dependent down-regulation of ferroportin 1. Furthermore, replenishment of ApoE could partially reverse the iron-related phenotype in ApoE KO mice. The findings imply that ApoE may be essential for body iron homeostasis and also suggest that clinical late-onset diseases with unexplained iron abnormality may partly be related to deficiency or reduced expression of ApoE.

•

Apolipoprotein E deficiency induces a progressive increase in tissue iron contents with age in mice.

•

ApoE−/− induced a progressive accumulation of iron with age in the liver and spleen of mice.

•

The increased iron was due to upregulation of TfR1 and downregulation of Fpn1.

•

Replenishment of ApoE could partially reverse the iron-related phenotype in ApoE KO mice.

•

ApoE may be essential for body iron homeostasis.

Gene variations in Autism Spectrum Disorder are associated with alternation of gut microbiota, metabolites and cytokines

The genetic variations and dysbiosis of gut microbiota are associated with ASD. However, the role of the microbiota in the etiology of ASD in terms of host genetic susceptibility remains unclear. This study aims to systematically explore the interplay between host genetic variation and gut microbiota in ASD children. Whole-exon sequencing was applied to 26 ASD children and 26 matched controls to identify the single nucleotide variations (SNVs) in ASD. Our previous study revealed alteration in gut microbiota and disorder of metabolism activity in ASD for this cohort. Systematic bioinformatic analyses were further performed to identify associations between SNVs and gut microbiota, as well as their metabolites. The ASD SNVs were significantly enriched in genes associated with innate immune response, protein glycosylation process, and retrograde axonal transport. These SNVs were also correlated with the microbiome composition and a broad aspect of microbial functions, especially metabolism. Additionally, the abundance of metabolites involved in the metabolic network of neurotransmitters was inferred to be causally related to specific SNVs and microbes. Furthermore, our data suggested that the interaction of host genetics and gut microbes may play a crucial role in the immune and metabolism homeostasis of ASD. This study may provide valuable clues to investigate the interaction of host genetic variations and gut microbiota in the pathogenesis of ASD.

Cystathionine γ-lyase promotes estrogen-stimulated uterine artery blood flow via glutathione homeostasis

2020-12-08T00:00

During pregnancy, estrogen (E₂) stimulates uterine artery blood flow (UBF) by enhancing nitric oxide (NO)-dependent vasodilation. Cystathionine γ-lyase (CSE) promotes vascular NO signaling by producing hydrogen sulfide (H₂S) and by maintaining the ratio of reduced-to-oxidized intracellular glutathione (GSH/GSSG) through l-cysteine production. Because redox homeostasis can influence NO signaling, we hypothesized that CSE mediates E₂ stimulation of UBF by modulating local intracellular cysteine metabolism and GSH/GSSG levels to promote redox homeostasis. Using non-pregnant ovariectomized WT and CSE-null (CSE KO) mice, we performed micro-ultrasound of mouse uterine and renal arteries to assess changes in blood flow upon exogenous E₂ stimulation. We quantified serum and uterine artery NO metabolites (NO_x), serum amino acids, and uterine and renal artery GSH/GSSG. WT and CSE KO mice exhibited similar baseline uterine and renal blood flow. Unlike WT, CSE KO mice did not exhibit expected E₂ stimulation of UBF. Renal blood flow was E₂-insensitive for both genotypes. While serum and uterine artery NO_x were similar between genotypes at baseline, E₂ decreased NO_x in CSE KO serum. Cysteine was also lower in CSE KO serum, while citrulline and homocysteine levels were elevated. E₂ and CSE deletion additively decreased GSH/GSSG in uterine arteries. In contrast, renal artery GSH/GSSG was insensitive to E₂ or CSE deletion. Together, these findings suggest that CSE maintenance of uterine artery GSH/GSSG facilitates nitrergic signaling in uterine arteries and is required for normal E₂ stimulation of UBF. These data have implications for pregnancy pathophysiology and the selective hormone responses of specific vascular beds.

•

CSE-null mice exhibit abnormal estrogen augmentation of uterine artery blood flow.

•

Estrogen lowers uterine artery nitric oxide metabolites in CSE null mice.

•

CSE loss and estrogen additively impair uterine artery glutathione homeostasis.

•

Neither CSE loss nor estrogen influences renal artery blood flow or glutathione.

Investigation of a monoclonal antibody against enterotoxigenic Escherichia coli, expressed as secretory IgA1 and IgA2 in plants

Passive immunization with antibodies is a promising approach against enterotoxigenic Escherichia coli diarrhea, a prevalent disease in LMICs. The objective of this study was to investigate expression of a monoclonal anti-ETEC CfaE secretory IgA antibody in N. benthamiana plants, with a view to facilitating access to ETEC passive immunotherapy. SIgA1 and SIgA2 forms of mAb 68–81 were produced by co-expressing the light and engineered heavy chains with J chain and secretory component in N. benthamiana. Antibody expression and assembly were compared with CHO-derived antibodies by SDS-PAGE, western blotting, size-exclusion chromatography and LC-MS peptide mapping. N-linked glycosylation was assessed by rapid fluorescence/mass spectrometry and LC-ESI-MS. Susceptibility to gastric digestion was assessed in an in vitro model. Antibody function was compared for antigen binding, a Caco-2 cell-based ETEC adhesion assay, an ETEC hemagglutination inhibition assay and a murine in vivo challenge study. SIgA1 assembly appeared superior to SIgA2 in plants. Both sub-classes exhibited resistance to degradation by simulated gastric fluid, comparable to CHO-produced 68–61 SIgA1. The plant expressed SIgAs had more homogeneous N-glycosylation than CHO-derived SIgAs, but no alteration of in vitro functional activity was observed, including antibodies expressed in a plant line engineered for mammalian-like N glycosylation. The plant-derived SIgA2 mAb demonstrated protection against diarrhea in a murine infection model. Although antibody yield and purification need to be optimized, anti-ETEC SIgA antibodies produced in a low-cost plant platform are functionally equivalent to CHO antibodies, and provide promise for passive immunotherapy in LMICs.

Cell-penetrating, antioxidant SELENOT mimetic protects dopaminergic neurons and ameliorates motor dysfunction in Parkinson's disease animal models

2020-12-28T00:00

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor dysfunction for which there is an unmet need for better treatment options. Although oxidative stress is a common feature of neurodegenerative diseases, notably PD, there is currently no efficient therapeutic strategy able to tackle this multi-target pathophysiological process. Based on our previous observations of the potent antioxidant and neuroprotective activity of SELENOT, a vital thioredoxin-like selenoprotein, we designed the small peptide PSELT from its redox active site to evaluate its antioxidant properties in vivo, and its potential polyfunctional activity in PD models. PSELT protects neurotoxin-treated dopaminergic neurons against oxidative stress and cell death, and their fibers against neurotoxic degeneration. PSELT is cell-permeable and acts in multiple subcellular compartments of dopaminergic neurons that are vulnerable to oxidative stress. In rodent models of PD, this protective activity prevented neurodegeneration, restored phosphorylated tyrosine hydroxylase levels, and led to improved motor skills. Transcriptomic analysis revealed that gene regulation by PSELT after MPP⁺ treatment negatively correlates with that occurring in PD, and positively correlates with that occurring after resveratrol treatment. Mechanistically, a major impact of PSELT is via nuclear stimulation of the transcription factor EZH2, leading to neuroprotection. Overall, these findings demonstrate the potential of PSELT as a therapeutic candidate for treatment of PD, targeting oxidative stress at multiple intracellular levels.

Compensatory intestinal immunoglobulin response after vancomycin treatment in humans

Intestinal immunoglobulins (Ig) are abundantly secreted antibodies that bind bacteria and bacterial components in the gut. This binding is considered to accelerate bacterial transit time and prevent the interaction of potentially immunogenic compounds with intestinal immune cells. Ig secretion is regulated by alterations in gut microbiome composition, an event rarely mapped in an intervention setting in humans. Here, we determined the intestinal and systemic Ig response to a major intervention in gut microbiome composition. Healthy humans and humans with metabolic syndrome received oral vancomycin 500 mg four times per day for 7 days. Coinciding with a vancomycin-induced increase in Gram-negative bacteria, fecal levels of the immunogenic bacterial components lipopolysaccharide (LPS) and flagellin drastically increased. Intestinal antibodies (IgA and IgM) significantly increased, whereas peripheral antibodies (IgG, IgA, and IgM) were mostly unaffected by vancomycin treatment. Bacterial cell sorting followed by 16S rRNA sequencing revealed that the majority of Gram-negative bacteria, including opportunistic pathogens, were IgA-coated after the intervention. We suggest that the intestinal Ig response after vancomycin treatment prevents the intrusion of pathogens and bacterial components into systemic sites.

Characterization of fructooligosaccharide metabolism and fructooligosaccharide-degrading enzymes in human commensal butyrate producers

Butyrate produced by gut microbiota has multiple beneficial effects on host health, and oligosaccharides derived from host diets and glycans originating from host mucus are major sources of its production. A significant reduction of butyrate-producing bacteria has been reported in patients with inflammatory bowel diseases and colorectal cancers. Although gut butyrate levels are important for host health, oligosaccharide metabolic properties in butyrate producers are poorly characterized. We studied the metabolic properties of fructooligosaccharides (FOSs) and other prebiotic oligosaccharides (i.e. raffinose and xylooligosaccharides; XOSs) in gut butyrate producers. 1-Kestose (kestose) and nystose, FOSs with degrees of polymerization of 3 and 4, respectively, were also included. Fourteen species of butyrate producers were divided into four groups based on their oligosaccharide metabolic properties, which are group A (two species) metabolizing all oligosaccharides tested, group F (four species) metabolizing FOSs but not raffinose and XOSs, group XR (four species) metabolizing XOSs and/or raffinose but not FOSs, and group N (four species) metabolizing none of the oligosaccharides tested. Species assigned to groups A and XR are rich glycoside hydrolase (GH) holders, whereas those in groups F and N are the opposite. In total, 17 enzymes assigned to GH32 were observed in nine of the 14 butyrate producers tested, and species that metabolized FOSs had at least one active GH32 enzyme. The GH32 enzymes were divided into four clusters by phylogenetic analysis. Heterologous gene expression analysis revealed that the GH32 enzymes in each cluster had similar FOS degradation properties within clusters, which may be linked to the conservation/substitution of amino acids to bind with substrates in GH32 enzymes. This study provides important knowledge to understand the impact of FOS supplementation on the activation of gut butyrate producers.

Abbreviations: SCFA, short chain fatty acid; FOS, fructooligosaccharide; XOS, xylooligosaccharide; CAZy, Carbohydrate Active Enzymes; CBM, carbohydrate-binding module; PUL, polysaccharide utilization locus; S6PH sucrose-6-phosphate hydrolase.

Ex vivo fecal fermentation of human ileal fluid collected after raspberry consumption modifies (poly)phenolics and modulates genoprotective effects in colonic epithelial cells

2021-01-12T00:00

Diets rich in fruit and vegetables are associated with a decreased incidence of colorectal cancer (CRC) due, in part, to the bioactive (poly)phenolic components and their microbiota-mediated metabolites. This study investigated how such compounds, derived from ingested raspberries in the gastrointestinal tract, may exert protective effects by reducing DNA damage. Ileal fluids collected pre- and post-consumption of 300 g of raspberries by ileostomists (n = 11) were subjected to 24 h ex vivo fermentation with fecal inoculum to simulate interaction with colonic microbiota. The impact of fermentation on (poly)phenolics in ileal fluid was determined and the bioactivity of ileal fluids pre- and post fermentation investigated. (Poly)phenolic compounds including sanguiin H-6, sanguiin H-10 and cyanidin-3-O-sophoroside decreased significantly during fermentation while, in contrast, microbial catabolites, including 3-(3′-hydroxyphenyl)propanoic acid, 3-hydroxybenzoic acid and benzoic acid increased significantly. The post-raspberry ileal fermentate from 9 of the 11 ileostomates significantly decreased DNA damage (~30%) in the CCD 841 CoN normal cell line using an oxidative challenge COMET assay. The raspberry ileal fermentates also modulated gene expression of the nuclear factor 2–antioxidant responsive element (Nrf2-ARE) pathway involved in oxidative stress cytoprotection, namely Nrf2, NAD(P)H dehydrogenase, quinone-1 and heme oxygenase-1. Four of the phenolic catabolites were assessed individually, each significantly reducing DNA damage from an oxidative challenge over a physiologically relevant 10–100 μM range. They also induced a differential pattern of expression of key genes in the Nrf2-ARE pathway in CCD 841 CoN cells. The study indicates that the colon-available raspberry (poly)phenols and their microbial-derived catabolites may play a role in protection against CRC in vivo.

•

Health effects of dietary (poly)phenols linked to interactions within the GI tract.

•

Ileostomy-based bioavailability studies allow effective interrogation of the GI tract.

•

Fecal fermentation of raspberry-enriched ileal fluid, increases phenolic content.

•

Raspberry ileal fluid fermentates & phenolic acids reduce DNA damage in colonocytes.

•

Cytoprotective Nrf2-ARE pathway modulated by ileal fluid fermentates & phenolic acids.

Long non-coding RNA Meg3 deficiency impairs glucose homeostasis and insulin signaling by inducing cellular senescence of hepatic endothelium in obesity

2021-01-19T00:00

Obesity-induced insulin resistance is a risk factor for diabetes and cardiovascular disease. However, the mechanisms underlying endothelial senescence in obesity, and how it impacts obesity-induced insulin resistance remain incompletely understood. In this study, transcriptome analysis revealed that the long non-coding RNA (lncRNA) Maternally expressed gene 3 (Meg3) is one of the top differentially expressed lncRNAs in the vascular endothelium in diet-induced obese mice. Meg3 knockdown induces cellular senescence of endothelial cells characterized by increased senescence-associated β–galactosidase activity, increased levels of endogenous superoxide, impaired mitochondrial structure and function, and impaired autophagy. Moreover, Meg3 knockdown causes cellular senescence of hepatic endothelium in diet-induced obese mice. Furthermore, Meg3 expression is elevated in human nonalcoholic fatty livers and nonalcoholic steatohepatitis livers, which positively correlates with the expression of CDKN2A encoding p16, an important hallmark of cellular senescence. Meg3 knockdown potentiates obesity-induced insulin resistance and impairs glucose homeostasis. Insulin signaling is reduced by Meg3 knockdown in the liver and, to a lesser extent, in the skeletal muscle, but not in the visceral fat of obese mice. We found that the attenuation of cellular senescence of hepatic endothelium by ablating p53 expression in vascular endothelium can restore impaired glucose homeostasis and insulin signaling in obesity. In conclusion, our data demonstrate that cellular senescence of hepatic endothelium promotes obesity-induced insulin resistance, which is tightly regulated by the expression of Meg3. Our results suggest that manipulation of Meg3 expression may represent a novel approach to managing obesity-associated hepatic endothelial senescence and insulin resistance.

•LncRNA Meg3 is a top differentially expressed lncRNA in the vascular endothelium in obese mice.

•Meg3 knockdown causes cellular senescence of HUVECs and of hepatic endothelium in obese mice.

•Meg3 expression is elevated in human NAFLD and NASH Nlivers, and correlates with CDKN2A expression -a senescent marker.

•Meg3 knockdown impairs glucose homeostasis and insulin signaling in obese mice.

•Attenuation of hepatic endothelial senescence improves glucose homeostasis and insulin signaling in obese mice.

Trem2 mediated Syk-dependent ROS amplification is essential for osteoclastogenesis in periodontitis microenvironment

2020-12-28T00:00

Periodontitis is the sixth most prevalent diseases around the globe, which is closely related to many systemic diseases and affects general health. As the leading cause of tooth loss, periodontitis is characterized by irreversible alveolar bone loss and activated osteoclastogenic process, which might be closely related to the activated intracellular reactive oxygen species (ROS) in osteoclasts. Here, we demonstrated triggering receptor expressed on myeloid cells 2 (Trem2) as a key regulator of osteoclastogenesis with the regulation of intracellular ROS signals in periodontitis. In the present study, the expression of Trem2 was significantly upregulated in human alveolar bones diagnosed with chronic periodontitis, as assessed by RNA-seq. In the mice model of periodontitis, the alveolar bone resorption was impeded in the presence of the conditional knockout of Trem2 in osteoclasts. Furthermore, we identified Trem2/DAP12/Syk-dependent cascade as a vital intracellular signaling for the amplification of reactive oxygen species (ROS) signals in osteoclastogenesis, while the accumulation of soluble Aβ₄₂ oligomers (Aβo) in periodontitis microenvironment further strengthened the signals and enhanced osteoclastogenesis through direct interactions with Trem2. Collectively, Trem2 mediated ROS signal amplification cascade was crucial in the process of osteoclastogenesis in periodontitis, suggesting the potential of Trem2 as a target for the prevention and treatment of bone destruction in periodontitis.

Noninvasive monitoring of fibre fermentation in healthy volunteers by analyzing breath volatile metabolites: lessons from the FiberTAG intervention study

The fermentation of dietary fibre (DF) leads to the production of bioactive metabolites, the most volatile ones being excreted in the breath. The aim of this study was to analyze the profile of exhaled breath volatile metabolites (BVM) and gastrointestinal symptoms in healthy volunteers after a single ingestion of maltodextrin (placebo) versus chitin-glucan (CG), an insoluble DF previously shown to be fermented into short-chain fatty acids (SCFA) by the human microbiota in vitro. Maltodextrin (4.5 g at day 0) or CG (4.5 g at day 2) were added to a standardized breakfast in fasting healthy volunteers (n = 15). BVM were measured using selected ion flow tube mass spectrometry (SIFT-MS) throughout the day. A single ingestion of 4.5 g CG did not induce significant gastrointestinal discomfort. Untargeted metabolomics analysis of breath highlighted that 13 MS-fragments (among 408 obtained from ionizations of breath) discriminated CG versus maltodextrin acute intake in the posprandial state. The targeted analysis revealed that CG increased exhaled butyrate and 5 other BVM – including the microbial metabolites 2,3-butanedione and 3-hydroxybutanone – with a peak observed 6 h after CG intake. Correlation analyses with fecal microbiota (Illumina 16S rRNA sequencing) spotlighted Mitsuokella as a potential genus responsible for the presence of butyric acid, triethylamine and 3-hydroxybutanone in the breath. In conclusion, measuring BMV in the breath reveals the microbial signature of the fermentation of DF after a single ingestion. This protocol allows to analyze the time-course of released bioactive metabolites that could be proposed as new biomarkers of DF fermentation, potentially linked to their biological properties.

Trial registration: Clinical Trials NCT03494491. Registered 11 April 2018 – Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT03494491

NADPH oxidase 4 (Nox4) deletion accelerates liver regeneration in mice

2020-12-23T00:00

Liver is a unique organ in displaying a reparative and regenerative response after acute/chronic damage or partial hepatectomy, when all the cell types must proliferate to re-establish the liver mass. The NADPH oxidase NOX4 mediates Transforming Growth Factor-beta (TGF-β) actions, including apoptosis in hepatocytes and activation of stellate cells to myofibroblasts. Aim of this work was to analyze the impact of NOX4 in liver regeneration by using two mouse models where Nox4 was deleted: 1) general deletion of Nox4 (NOX4−/−) and 2) hepatocyte-specific deletion of Nox4 (NOX4hepKO). Liver regeneration was analyzed after 2/3 partial hepatectomy (PH). Results indicated an earlier recovery of the liver-to-body weight ratio in both NOX4−/− and NOX4hepKO mice and an increased survival, when compared to corresponding WT mice. The regenerative hepatocellular fat accumulation and the parenchyma organization recovered faster in NOX4 deleted livers. Hepatocyte proliferation, analyzed by Ki67 and phospho-Histone3 immunohistochemistry, was accelerated and increased in NOX4 deleted mice, coincident with an earlier and increased Myc expression. Primary hepatocytes isolated from NOX4 deleted mice showed higher proliferative capacity and increased expression of Myc and different cyclins in response to serum. Transcriptomic analysis through RNA-seq revealed significant changes after PH in NOX4−/− mice and support a relevant role for Myc in a node of regulation of proliferation-related genes. Interestingly, RNA-seq also revealed changes in the expression of genes related to activation of the TGF-β pathway. In fact, levels of active TGF-β1, phosphorylation of Smads and levels of its target p21 were lower at 24 h in NOX4 deleted mice. Nox4 did not appear to be essential for the termination of liver regeneration in vivo, neither for the in vitro hepatocyte response to TGF-β1 in terms of growth inhibition, which suggest its potential as therapeutic target to improve liver regeneration, without adverse effects.

•

General or hepatocyte-specific Nox4 deletion accelerates mice liver regeneration.

•

Increased hepatocyte proliferation is observed, coincident with higher Myc expression.

•

RNA-seq analysis reveals a role for Myc in a node of regulation of gene expression.

•

Transcriptional and functional attenuation of the TGF-β1 pathway is observed in vivo.

•

In vitro, Nox4 deleted hepatocytes maintain the growth inhibitory response to TGF-β1.

Heat shock protein 22 modulates NRF1/TFAM-dependent mitochondrial biogenesis and DRP1-sparked mitochondrial apoptosis through AMPK-PGC1α signaling pathway to alleviate the early brain injury of subarachnoid hemorrhage in rats

2021-01-06T00:00

Mitochondrial dysfunction has been widely accepted as a detrimental factor in subarachnoid hemorrhage (SAH)-induced early brain injury (EBI), which is eminently related to poor neurologic function outcome. Previous studies have revealed that enhancement of heat shock protein 22 (hsp22) under conditions of stress is a friendly mediator of mitochondrial homeostasis, oxidative stress and apoptosis, thus accelerating neurological recovery. However, no study has confirmed whether hsp22 attenuates mitochondrial stress and apoptosis in the setting of SAH-induced EBI. Our results indicated that endogenous hsp22, p-AMPK/AMPK, PGC1α, TFAM, Nrf1 and Drp1 were significantly upregulated in cortical neurons in response to SAH, accompanied by neurologic impairment, brain edema, neuronal degeneration, lower level of mtDNA and ATP, mitochondria-cytosol translocation of cytochrome c, oxidative injury and caspase 3-involved mitochondrial apoptosis. However, exogenous hsp22 maintained neurological function, reduced brain edema, improved oxidative stress and mitochondrial apoptosis, these effects were highly dependent on PGC1α-related mitochondrial biogenesis/fission, as evidenced by co-application of PGC1α siRNA. Furthermore, we demonstrated that blockade of AMPK with dorsomorphin also compromised the neuroprotective actions of hsp22, along with the alterations of PGC1α and its associated pathway molecules. These data revealed that hsp22 exerted neuroprotective effects by salvaging mitochondrial function in an AMPK-PGC1α dependent manner, which modulates TFAM/Nrf1-induced mitochondrial biogenesis with positive feedback and DRP1-triggered mitochondrial apoptosis with negative feedback, further reducing oxidative stress and brain injury. Boosting the biogenesis and repressing excessive fission of mitochondria by hsp22 may be an efficient treatment to relieve SAH-elicited EBI.

The schematic diagram demonstrating that Hsp22 modulates mitochondrial biogenesis and fission through AMPK-PGC1α signaling pathway to alleviate the early brain injury after SAH in rats. SAH subarachnoid hemorrhage, Hsp22 heat shock protein 22, AMPK Adenosine 5'monophosphate-activated protein kinase, PGC1α peroxisome proliferative activated receptor γ (PPARγ) coactivator 1α, Drp1 dynamin-related protein 1, TFAM mitochondrial transcription factor A, Nrf1 nuclear respiratory factor 1, UCP2 uncoupling protein 2, ROS reactive oxygen species, 8-OHdG 8-hydroxyguanine, MDA malondialdehyde, PCO protein carbonyl, Bcl-2 B-cell lymphoma-2, Bax Bcl-2 associated X protein, siRNA small interfering ribonucleic acid.

•

Hsp22 is notably upregulated in neurons at 24 h after SAH.

•

Hsp22 boosts the NRF1/TFAM-dependent mitochondrial biogenesis.

•

Hsp22 represses DRP1-sparked mitochondrial apoptosis.

•

AMPK-PGC1α pathway is involved in hsp22-mediated neuroprotection after SAH.

•

Modulation of mitochondrial biogenesis and fission may be efficient for treating SAH.

Enriched metabolites that potentially promote age-associated diseases in subjects with an elderly-type gut microbiota

We previously investigated the gut microbiota of 453 healthy Japanese subjects aged 0 to 104 years and found that the composition of the gut microbiota could be classified into some age-related clusters. In this study, we compared fecal metabolites between age-matched and age-mismatched elderly subjects to examine the roles of the gut microbiota in the health of the elderly. Fecal metabolites in 16 elderly subjects who fell into an age-matched cluster (elderly-type gut microbiota group, E-GM) and another 16 elderly subjects who fell into an age-mismatched cluster (adult-type gut microbiota group, A-GM) were measured by CE-TOF-MS. A total of eight metabolites were significantly different between the groups: cholic acid and taurocholic acid were enriched in the A-GM group, whereas choline, trimethylamine (TMA), N8-acetylspermidine, propionic acid, 2-hydroxy-4-methylvaleric acid, and 5-methylcytosine were enriched in the E-GM group. Some metabolites (choline, TMA, N8-acetylspermidine) elevated in the E-GM group were metabolites or precursors reported as risk factors for age-associated diseases such as arteriosclerosis and colorectal cancer. The abundance of some species belongs to Proteobacteria, which were known as TMA-producing bacteria, was increased in the E-GM group and correlated with fecal TMA levels. In vitro assays showed that these elderly-type fecal metabolites suppressed the expression of genes related to tight junctions in normal colonic epithelial cells and induced the expression of inflammatory cytokines in colon cancer cells. These findings suggest that metabolites produced by the aged gut microbiota could contribute to intestinal and systemic homeostasis and could be targeted for preventing aging-associated diseases.

The program of renal fibrogenesis is controlled by microRNAs regulating oxidative metabolism

2020-12-28T00:00

Excessive accumulation of extracellular matrix (ECM) is the hallmark of fibrotic diseases. In the kidney, it is the final common pathway of prevalent diseases, leading to chronic renal failure. While cytokines such as TGF-β play a fundamental role in myofibroblast transformation, recent work has shown that mitochondrial dysfunction and defective fatty acid oxidation (FAO), which compromise the main source of energy for renal tubular epithelial cells, have been proposed to be fundamental contributors to the development and progression of kidney fibrosis. MicroRNAs (miRNAs), which regulate gene expression post-transcriptionally, have been reported to control renal fibrogenesis. To identify miRNAs involved in the metabolic derangement of renal fibrosis, we performed a miRNA array screen in the mouse model of unilateral ureteral obstruction (UUO). MiR-150-5p and miR-495-3p were selected for their link to human pathology, their role in mitochondrial metabolism and their targeting of the fatty acid shuttling enzyme CPT1A. We found a 2- and 4-fold upregulation of miR-150-5p and miR-495-5p, respectively, in both the UUO and the folic acid induced nephropathy (FAN) models, while TGF-β1 upregulated their expressions in the human renal tubular epithelial cell line HKC-8. These miRNAs synergized with TGF-β regarding its pro-fibrotic effect by enhancing the fibrosis-associated markers Acta2, Col1α1 and Fn1. Bioenergetics studies showed a reduction of FAO-associated oxygen consumption rate (OCR) in HKC-8 cells in the presence of both miRNAs. Consistently, expression levels of their mitochondrial-related target genes CPT1A, PGC1α and the mitochondrial transcription factor A (TFAM), were reduced by half in renal epithelial cells exposed to these miRNAs. By contrast, we did not detect changes in mitochondrial mass and transmembrane potential (ΔѰm) or mitochondrial superoxide radical anion production. Our data support that miR-150 and miR-495 may contribute to renal fibrogenesis by aggravating the metabolic failure critically involved in tubular epithelial cells, ultimately leading to fibrosis.

MiR-150-5p and miR-495-3p exert pro-fibrotic effects by synergizing with TGF-β in the fibrotic response and contributing to the mitochondrial impairment associated to renal fibrosis.

•

MiR-150-5p and miR-495-3p were upregulated both in the UUO and FAN models.

•

MiR-150-5p and miR-495-3p synergized with TGF-β profibrotic effects and reduced FAO-associated OCR in renal epithelial cells.

•

MiR-150-5p and miR-495-3p did not alter mitochondrial transmembrane potential and superoxide radical anion production.

•

MiRNAs 150-5p and 495-3p are contributors to the metabolic impairment leading to renal fibrosis.

Galanin promotes autophagy and alleviates apoptosis in the hypertrophied heart through FoxO1 pathway

2021-01-16T00:00

Autophagy and apoptosis are powerful regulators of multiple facets of cellular metabolism and homeostasis. Here, we uncover that galanin, a pleiotropic peptide, regulates cardiac autophagy and deactivates apoptotic cell death through the Forkhead box protein O1 (FoxO1) pathway. In hypertrophied heart, galanin promotes autophagy and metabolic shift from fatty acid (FA) to glucose oxidation and preserves mitochondrial integrity. In cardiomyoblasts, galanin triggers autophagosome formation and alleviates hypertrophy, apoptotic cell death, and mitochondrial stress. Mechanistically, galanin dictates cell autophagic and anti-apoptotic phenotypes through FoxO1 pathway. Together, these findings uncover a previously unknown role for galanin in the regulation of cardiac autophagy and provide new insights into the molecular mechanisms supporting cell survival in the hypertrophic reprogramming of the heart.

•

Galanin dictates autophagic phenotype in cardiomyoblasts.

•

Galanin suppresses myocardial apoptosis and mitochondrial oxidative stress in hypertrophic remodeling.

•

Galanin promotes metabolic shift from fatty acid to glucose oxidation in the hypertrophied hearts.

•

Galanin regulates cardiac autophagy and apoptosis through FoxO1 pathway.

Microbial and metabolic features associated with outcome of infliximab therapy in pediatric Crohn’s disease

Gut microbial dysbiosis and altered metabonomics have been implicated in the pathogenesis of Crohn’s disease (CD). The aim of our study was to characterize the gut microbiome structure and metabolic activities in pediatric CD patients with different clinical outcomes after infliximab (IFX) therapy. Fecal samples were collected from 20 healthy children and 29 newly diagnosed pediatric CD patients. 16S rRNA/ITS2 gene sequencing and targeted metabolomics analysis were applied to profile the gut bacterial microbiome, mycobiome, and metabolome, respectively. Pediatric CD patients exhibited lower relative abundances of short-chain fatty acids (SCFAs)-producing bacteria including Faecalibacterium, Clostridium clusters IV and XIVb, Roseburia, and Ruminococcus, which were correlated with reduced fecal levels of SCFAs. Decreased unconjugated bile acids (BAs) pool size and a lower unconjugated/conjugated BAs ratio were associated with reduced relative abundances of Bifidobacterium and Clostridium clusters IV and XIVb which contain bile salt hydrolases (BSH) genes. IFX treatment enriched the BSH-producing bacteria in CD subjects, which may explain a decreased level of conjugated BAs and an increase in unconjugated BAs as well as the unconjugated/conjugated BAs ratio. Furthermore, a sustained response (SR) of IFX therapy was associated with higher abundances of Methylobacterium, Sphingomonas, Staphylococcus, and Streptococcus, and higher fecal concentrations of amino acids, including L-aspartic acid, linoleic acid, and L-lactic acid at baseline. Our study suggests that the effects of IFX might be partially mediated by enriching bacteria taxa that producing SCFAs and BSH thereby inhibiting inflammation and restoring the BA metabolism. Some fecal bacteria and metabolites may be predictive of outcomes of IFX therapy for pediatric CD patients.

Salmonella effector SpvB aggravates dysregulation of systemic iron metabolism via modulating the hepcidin−ferroportin axis

Iron withholding, an essential component of nutritional immunity, plays a fundamental role in host resistance to Salmonella infection. Our previous study showed that SpvB, an important pSLT-encoded cytotoxic effector, facilitated Salmonella pathogenesis within macrophages via perturbing cellular iron metabolism. However, the underlying mechanisms of SpvB in Salmonella-relevant disorders of systemic iron metabolism have not yet been identified. Here, we demonstrated that SpvB facilitated Salmonella to scavenge iron from the host by modulating the hepcidin–ferroportin axis, a key regulator of systemic iron metabolism. We observed that SpvB enhanced hepatic hepcidin synthesis in a STAT3-dependent manner, but not the BMP/SMAD pathway. This subsequently resulted in a reduction of the unique cellular iron exporter ferroportin, which facilitated hypoferremia and hepatic iron accumulation and ultimately countered the limitation of iron availability, thereby improving the chances of Salmonella survival and replication. Moreover, SpvB promoted the production of proinflammatory molecules associated with the infiltration of inflammatory cells via highly upregulating TREM-1 signaling. Our data supported a role of TREM-1 in SpvB-related dysregulation of host iron metabolism and suggested that targeting TREM-1 might provide a potential therapeutic strategy to prevent or alleviate Salmonella pathogenesis.

Reactive oxygen species induce Cys106-mediated anti-parallel HMGB1 dimerization that protects against DNA damage

2021-01-07T00:00

Oxidative stress can induce covalent disulfide bond formation between protein-protein thiol groups and generate hydroxyl free radicals that damage DNA. HMGB1 is a DNA chaperone and damage-associated molecular pattern molecule. As a redox-sensitive protein, HMGB1 contains three cysteine residues: Cys23, Cys45, and Cys106. In this study, we focused on the relationship between HMGB1 dimerization and DNA stabilization under oxidative stress conditions. HMGB1 dimerization was positively modulated by CuCl₂ and H₂O₂. Mutation of the Cys106 residue blocked dimer formation. Treatment of HEK293T cells with CuCl₂ and H₂O₂ enhanced the oxidative self-dimerization of HMGB1, whereas this dimerization was inhibited in mutant HMGB1^C106A cells. Furthermore, we performed a bimolecular fluorescence complementation assay to visualize Cys106 oxidation-induced HMGB1 dimerization in live cells exposed to oxidative stress and were able to reproduce the dimerization effect of HMGB1 in fluorescence resonance energy transfer analysis. Interestingly, dimerized HMGB1 bound to DNA with higher affinity than monomeric HMGB1. Dimerized HMGB1 protected DNA from damage due to hydroxyl free radicals and prevented cell death. In conclusion, dimerized HMGB1 may play a regulatory role in DNA stabilization under oxidative stress.

•

Accumulation of excessive ROS induces DNA damage, causing cell death.

•

HMGB1 dimerizes in the presence of excessive ROS and binds DNA with high affinity.

•

Binding of dimerized HMGB1 (Di-HMGB1) protects DNA from ROS action.

•

We prepared an in vitro HMGB1 dimerization due to excessive ROS.

•

Di-HMGB1 protected against DNA damage induced by radiation exposure.

High air flow-rate electrostatic sampler for the rapid monitoring of airborne coronavirus and influenza viruses

2021-01-23T00:00

Capturing virus aerosols in a small volume of liquid is essential when monitoring airborne viruses. As such, aerosol-to-hydrosol enrichment is required to produce a detectable viral sample for real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays. To meet this requirement, the efficient and non-destructive collection of airborne virus particles is needed, while the incoming air flow rate should be sufficiently high to quickly collect a large number of virus particles. To achieve this, we introduced a high air flow-rate electrostatic sampler (HAFES) that collected virus aerosols (human coronavirus 229E, influenza A virus subtypes H1N1 and H3N2, and bacteriophage MS2) in a continuously flowing liquid. Viral collection efficiency was evaluated using aerosol particle counts, while viral recovery rates were assessed using real-time qRT-PCR and plaque assays. An air sampling period of 20 min was sufficient to produce a sample suitable for use in real-time qRT-PCR in a viral epidemic scenario.

Hypochlorite-induced aggregation of fibrinogen underlies a novel antioxidant role in blood plasma

2020-12-30T00:00

Fibrinogen, a major constituent of blood plasma, is highly susceptible to reaction with biological oxidants. It has been proposed that fibrinogen plays a role in antioxidant defence, but oxidation of fibrinogen is also known to disrupt normal blood clotting and is implicated in the pathology of atherosclerosis. In the present study, we show that the biological oxidant hypochlorite promotes the formation of soluble high molecular weight fibrinogen assemblies ≥40 × 10⁶ Da, that do not accumulate when fibrinogen is induced to aggregate by other stresses such as heating or hydroxyl-mediated damage in vitro. Hypochlorite-modified fibrinogen is stable at 37 °C as assessed by precipitation assays, and has reduced susceptibility to iron-induced (hydroxyl-mediated) precipitation compared to native fibrinogen. In contrast to hypochlorite-modified albumin, which is known to be immunostimulatory, hypochlorite-modified fibrinogen does not induce RAW 264.7 (macrophage-like) cells or EOC 13.31 (microglia-like) cells to produce reactive oxygen species or induce cell death. Furthermore, depletion of fibrinogen from human blood plasma increases the immunostimulatory property of blood plasma after it is supplemented with hypochlorite in situ. We propose that reaction of hypochlorite with fibrinogen in blood plasma potentially reduces the accumulation of other hypochlorite-modified species such as immunostimulatory hypochlorite-modified albumin. The latter represent a novel role for fibrinogen in blood plasma antioxidant defence.

•

Plasma proteins have distinct responses to reaction with hypochlorite.

•

Hypochlorite-modified fibrinogen is prone to aggregation.

•

Hypochlorite-modified albumin is resistant to aggregation.

•

Hypochlorite-modified albumin induces ROS production and is cytotoxic.

•

Hypochlorite-modified fibrinogen does not induce ROS production or cell death.

Melatonin protects against environmental stress-induced fetal growth restriction via suppressing ROS-mediated GCN2/ATF4/BNIP3-dependent mitophagy in placental trophoblasts

2021-01-06T00:00

Gestational exposure to environmental stress induces fetal growth restriction (FGR), and thereby increasing the risk of infant death and chronic noncommunicable diseases in adults. However, the mechanism by which environmental stress induces FGR remains unclear. Based on case-control study, we found that the reduced level of melatonin (MT), a major secretory product from the pineal gland, was observed in placentae of FGR. This work was to investigate the protective effect of MT on environmental stress-caused FGR and its mechanisms. We used cadmium (Cd) as an environmental stressor to stimulate pregnant mice and thereby establishing a FGR model. The data showed that maternal Cd exposure lowered the P4 concentration in maternal sera, placentae and amniotic fluid, and caused FGR. Correspondingly, the expression of CYP11A1, a critical P4 synthase, was markedly downregulated in Cd-treated placentae. Simultaneously, Cd triggered BNIP3-dependent mitophagy in placental trophoblasts, as determined by the degradation of mitochondrial proteins, including HSP60 and COX IV, and the accumulation of puncta representing co-localization of TOM20 with LC3B or BNIP3 with LC3B. Based on our case-control study, we also found that activated BNIP3-dependent mitophagy and P4 synthesis inhibition occurred in SGA placentae. Most importantly, BNIP3 siRNA reversed Cd-induced P4 synthesis suppression in human placental trophoblasts. It is noteworthy that MT alleviated Cd-caused P4 synthesis suppression and FGR via antagonizing BNIP3-dependent mitophagy in placental trophoblasts. Further results confirmed that MT attenuated Cd-triggered BNIP3-dependent mitophagy via blocking GCN2/ATF4 signaling. Amusingly, Cd triggered oxidative stress and then activating GCN2/ATF4 signaling in placental trophoblasts. As expected, MT obviously suppressed Cd-caused reactive oxygen species (ROS) release. In the present study, we propose a neoteric mechanism by which MT protects against environmental stress-impaired P4 synthesis and fetal growth via suppressing ROS-mediated GCN2/ATF4/BNIP3-dependent mitophagy in placental trophoblasts. As above, MT is a potential therapeutic agent antagonizing environmental stress-induced developmental toxicity.

•

Melatonin protects against Cd-induced fetal growth restriction.

•

Melatonin attenuates Cd-induced placental P4 synthesis inhibition by mitophagy.

•

Melatonin suppresses Cd-triggered placental mitophagy via blocking GCN2/ATF4.

•

Melatonin blocks Cd-activated placental GCN2/ATF4 signaling via repressing ROS.

•

Activated mitophagy and reduced P4 synthesis occur in SGA placentae.

Localized increases in CEPT1 and ATGL elevate plasmalogen phosphatidylcholines in HDLs contributing to atheroprotective lipid profiles in hyperglycemic GCK-MODY

2021-01-06T00:00

Glucokinase-maturity onset diabetes of the young (GCK-MODY) represents a rare genetic disorder due to mutation in the glucokinase (GCK) gene. The low incidence of vascular complications in GCK-MODY makes it a natural paradigm for interrogating molecular mechanisms promoting vascular health under prolonged hyperglycemia. Clinical rate of misdiagnosis has remained high, and a reliable serum lipid biomarker that precedes genetic screening can facilitate correct diagnosis and treatment. Herein, we comprehensively quantitated 565 serum lipids from 25 classes in 105 subjects (42 nondiabetic controls, 30 GC K-MODY patients, 33 drug-naïve, and newly-onset T2D patients). At false-discovery rate (FDR) < 0.05, several phosphatidylcholines (PCs) and plasmalogen PCs were specifically increased in GCK-MODY, while triacylglycerols (TAGs) and diacylglycerols (DAGs) were reduced. Correlation matrices between lipids uncovered coregulation between plasmalogen PCs (PCps) and glycerolipid precursors was distinctly enhanced in GCK-MODY compared to T2D. Strengthened positive correlations between serum PCps and circulating HDLs was specifically observed in hyperglycemic subjects (i.e. T2D and GCK-MODY) compared to normglycemic controls, suggesting that HDL-PCps may elicit distinct physiological effects under hyperglycemia. Amongst GCK-MODY patients, individuals harboring variants of GCK mutations with elevated PCps also exhibited higher HDLs. Isolated HDLs displayed localized increases (p < 0.05) in very-long-chain PUFA-PCs and PCps in GCK-MODY. Protein analyses revealed elevated levels of HDL-resident ATGL (P = 0.003) and CEPT1 (P < 0.0001), which mediate critical steps of PCps production along the TAG-DAG-PC axis, in GCK-MODY relative to T2D. A panel of four lipids differentiated GCK-MODY from T2D with AUC of 0.950 (95% CI 0.903–9.997). This study provides the first evidence that enhanced recruitment of CEPT1 and ATGL onto HDLs essentially underlie the atheroprotective profiles associated with GCK-MODY. Resultant increases in the production of HDL-PCps and PUFA-PCs provides an active, circulating form of protection towards the vasculature of GCK-MODY, thereby lowering the incidence of vascular complications despite chronic exposure to hyperglycemia since birth.

Chalcone suppresses tumor growth through NOX4-IRE1α sulfonation-RIDD-miR-23b axis

2021-01-06T00:00

Chalcone is a polyphenolic compound found abundantly in natural plant components. They have been acclaimed as potential antitumor compounds in multiple tumor cells. However, not much attention has been paid to elucidate its antitumor mechanism of action. Here, chalcone was demonstrated to trigger endoplasmic reticulum (ER) stress-induced apoptosis through sulfonation of IRE1α by ER-localized NADPH oxidase 4 (NOX4). IRE1α-sulfonation at a cysteine residue was shown to induce “regulated IRE1α-dependent decay” (RIDD) of mRNA rather than specific splicing of XBP1. The IRE1α sulfonation-induced RIDD degraded miR-23b, enhancing the expression of NOX4. The expression of NOX4 was also upregulated in breast, and prostate cancer tissue. In chalcone-administered mice in vivo, tumor growth was regressed by the consistent mechanisms “NOX4-IRE1α sulfonation-RIDD”. Similarly, NOX4 activation and IRE1α sulfonation were also highly increased under severe ER stress conditions. Together, these findings suggest chalcone as a lead anticancer compound where it acts through NOX4-IRE1α-RIDD-miR-23b axis providing a promising vision of chalcone derivatives’ anticancer mechanism.

OGG1 co-inhibition antagonizes the tumor-inhibitory effects of targeting MTH1

2021-01-02T00:00

Cancer cells develop protective adaptations against oxidative DNA damage, providing a strong rationale for targeting DNA repair proteins. There has been a high degree of recent interest in inhibiting the mammalian Nudix pyrophosphatase MutT Homolog 1 (MTH1). MTH1 degrades 8-oxo-dGTP, thus limiting its incorporation into genomic DNA. MTH1 inhibition has variously been shown to induce genomic 8-oxo-dG elevation, genotoxic strand breaks in p53-functional cells, and tumor-inhibitory outcomes. Genomically incorporated 8-oxo-dG is excised by the base excision repair enzyme, 8-oxo-dG glycosylase 1 (OGG1). Thus, OGG1 inhibitors have been developed with the idea that their combination with MTH1 inhibitors will have anti-tumor effects by increasing genomic oxidative DNA damage. However, contradictory to this idea, we found that human lung adenocarcinoma with low OGG1 and MTH1 were robustly represented in patient datasets. Furthermore, OGG1 co-depletion mitigated the extent of DNA strand breaks and cellular senescence in MTH1-depleted p53-wildtype lung adenocarcinoma cells. Similarly, shMTH1-transduced cells were less sensitive to the OGG1 inhibitor, SU0268, than shGFP-transduced counterparts. Although the dual OGG1/MTH1 inhibitor, SU0383, induced greater cytotoxicity than equivalent combined or single doses of its parent scaffold MTH1 and OGG1 inhibitors, IACS-4759 and SU0268, this effect was only observed at the highest concentration assessed. Collectively, using both genetic depletion as well as small molecule inhibitors, our findings suggest that OGG1/MTH1 co-inhibition is unlikely to yield significant tumor-suppressive benefit. Instead such co-inhibition may exert tumor-protective effects by preventing base excision repair-induced DNA nicks and p53 induction, thus potentially conferring a survival advantage to the treated tumors.

•

Low MTH1/low OGG1 tumors are robustly represented in patient lung adenocarcinoma datasets but low MTH1/high OGG1 are not.

•

Co-depletion of OGG1 in lung adenocarcinoma cells mitigates shMTH1-induced DNA strand breaks and p53-induced senescence.

•

p53-null tumor cells have lower OGG1 vs. wt p53 counterparts and are more resistant to MTH1 loss-induced anti-tumor effects.

•

Pharmacologic co-inhibition of OGG1 and MTH1 does not enhance cytotoxicity over the respective single inhibitors.

The protective effects of fibroblast growth factor 10 against hepatic ischemia-reperfusion injury in mice

2021-01-07T00:00

Hepatic ischemia-reperfusion injury (IRI) is a major complication of liver surgery and transplantation. IRI leads to hepatic parenchymal cell death, resulting in liver failure, and lacks effective therapeutic approaches. Fibroblast growth factor 10 (FGF10) is a paracrine factor which is well-characterized with respect to its pro-proliferative effects during embryonic liver development and liver regeneration, but its role in hepatic IRI remains unknown. In this study, we investigated the role of FGF10 in liver IRI and identified signaling pathways regulated by FGF10. In a mouse model of warm liver IRI, FGF10 was highly expressed during the reperfusion phase. In vitro experiments demonstrated that FGF10 was primarily secreted by hepatic stellate cells and acted on hepatocytes. The role of FGF10 in liver IRI was further examined using adeno-associated virus-mediated gene silencing and overexpression. Overexpression of FGF10 alleviated liver dysfunction, reduced necrosis and inflammation, and protected hepatocytes from apoptosis in the early acute injury phase of IRI. Furthermore, in the late phase of IRI, FGF10 overexpression also promoted hepatocyte proliferation. Meanwhile, gene silencing of FGF10 had the opposite effect. Further studies revealed that overexpression of FGF10 activated nuclear factor-erythroid 2-related factor 2 (NRF2) and decreased oxidative stress, mainly through activation of the phosphatidylinositol-3-kinase/AKT pathway, and the protective effects of FGF10 overexpression were largely abrogated in NRF2 knockout mice. These results demonstrate the protective effects of FGF10 in liver IRI, and reveal the important role of NRF2 in FGF10-mediated hepatic protection during IRI.

•

FGF10 is markedly upregulated in the early phase of liver IRI.

•

FGF10 overexpression exerts great potential in ameliorating hepatic IRI.

•

FGF10 knockdown significantly aggravates hepatic IRI.

•

FGF10 overexpression activates PI3K/AKT-NRF2 signaling and thus ameliorates hepatic IRI.

•

NRF2 knockout abrogates the protective effects of FGF10 overexpression during liver IRI.

Long-term effects of antimicrobial drugs on the composition of the human gut microbiota

Introduction

Antimicrobial drugs are known to have effects on the human gut microbiota. We studied the long-term temporal relationship between several antimicrobial drug groups and the composition of the human gut microbiota determined in feces samples.

Methods

Feces samples were obtained from a community-dwelling cohort of middle-aged and elderly individuals (Rotterdam Study). Bacterial DNA was isolated and sequenced using V3/V4 16 S ribosomal RNA sequencing (Illumina MiSeq). The time between the last prescription of several antimicrobial drug groups and the day of sampling was categorized into 0–12, 12–24, 24–48 and >48 months. The effects of the antimicrobial drug groups on the Shannon alpha-diversity (diversity), the Bray–Curtis beta-diversity (community structure), the Firmicutes/Bacteroidetes (F/B) ratio and individual genera were determined.

Results

We studied the gut microbiota of 1413 individuals (57.5% female, median age 62.6 years). The alpha-diversity was significantly lower up to 4 years after prescriptions of macrolides and lincosamides. It was also lower in the first year after the use of beta-lactams. The community structure (beta-diversity) of the microbiota was significantly different up to 4 years for macrolides and lincosamides, the first year for beta-lactams and at least the first year for quinolones. For the F/B ratio, drugs with a high anaerobic activity shifted the ratio toward Firmicutes in the first year whereas other antimicrobial drugs shifted the ratio toward Bacteroidetes.

Conclusion

Use of antimicrobial drugs is associated with a shift in the composition of the gut microbiota.These effects differ in strength and duration, depending on the antimicrobial drug group used. These findings should be considered when prescribing antimicrobial drugs.

Antihypertensive effects of exercise involve reshaping of gut microbiota and improvement of gut-brain axis in spontaneously hypertensive rat

Exercise (Ex) has long been recognized to produce beneficial effects on hypertension (HTN). This coupled with evidence of gut dysbiosis and an impaired gut-brain axis led us to hypothesize that reshaping of gut microbiota and improvement in impaired gut-brain axis would, in part, be associated with beneficial influence of exercise. Male spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats were randomized into sedentary, trained, and detrained groups. Trained rats underwent moderate-intensity exercise for 12 weeks, whereas, detrained groups underwent 8 weeks of moderate-intensity exercise followed by 4 weeks of detraining. Fecal microbiota, gut pathology, intestinal inflammation, and permeability, brain microglia and neuroinflammation were analyzed. We observed that exercise training resulted in a persistent decrease in systolic blood pressure in the SHR. This was associated with increase in microbial α diversity, altered β diversity, and enrichment of beneficial bacterial genera. Furthermore, decrease in the number of activated microglia, neuroinflammation in the hypothalamic paraventricular nucleus, improved gut pathology, inflammation, and permeability were also observed in the SHR following exercise. Interestingly, short-term detraining did not abolish these exercise-mediated improvements. Finally, fecal microbiota transplantation from exercised SHR into sedentary SHR resulted in attenuated SBP and an improved gut-brain axis. These observations support our concept that an impaired gut-brain axis is linked to HTN and exercise ameliorates this impairment to induce antihypertensive effects.

Ulcerative Colitis-associated E. coli pathobionts potentiate colitis in susceptible hosts

Ulcerative colitis (UC) is a chronic inflammatory condition linked to intestinal microbial dysbiosis, including the expansion of E. coli strains related to extra-intestinal pathogenic E. coli. These “pathobionts” exhibit pathogenic properties, but their potential to promote UC is unclear due to the lack of relevant animal models. Here, we established a mouse model using a representative UC pathobiont strain (p19A), and mice lacking single immunoglobulin and toll-interleukin 1 receptor domain (SIGIRR), a deficiency increasing susceptibility to gut infections. Strain p19A was found to adhere to the cecal mucosa of Sigirr -/- mice, causing modest inflammation. Moreover, it dramatically worsened dextran sodium sulfate-induced colitis. This potentiation was attenuated using a p19A strain lacking α-hemolysin genes, or when we targeted pathobiont adherence using a p19A strain lacking the adhesin FimH, or following treatment with FimH antagonists. Thus, UC pathobionts adhere to the intestinal mucosa, and worsen the course of colitis in susceptible hosts.

Exposure to soil environments during earlier life stages is distinguishable in the gut microbiome of adult mice

Environmental exposure during earlier life stages can govern the assembly and development of gut microbiota, yet it is insufficiently understood. In this study, ex-germ-free mice were cohoused with distinct soil-microbiota (from desert, steppe, and forest) beddings within 60 days after birth and subsequently transferred to new soil beddings from 60 to 90th day. Using metagenomic shotgun sequencing, firstly, we found soil microbes from natural environments (birthplace) greatly influenced the gut community assembly in the housing experiment. About 27% microbial species and 12% functional components that associated with birthplaces at Day 60 were still discriminatory of birthplaces after transferring mice to new environments. Moreover, prior soil-exposure types are associated with the magnitude of temporal microbiome change due to environmental shifts. The appropriate soil-exposure (e.g., steppe) might help mice gut microbiome adapt to changing environments or host development. Our study demonstrated the continuous soil-exposure history earlier is associated with the gut microbiome individuality and development later.

Maternal consumption of artificially sweetened beverages during pregnancy is associated with infant gut microbiota and metabolic modifications and increased infant body mass index

Artificial sweetener consumption by pregnant women has been associated with an increased risk of infant obesity, but the underlying mechanisms are unknown. We aimed to determine if maternal consumption of artificially sweetened beverages (ASB) during pregnancy is associated with modifications of infant gut bacterial community composition and function during the first year of life, and whether these alterations are linked with infant body mass index (BMI) at one year of age. We studied 100 infants from the prospective Canadian CHILD Cohort Study, selected based on maternal ASB consumption during pregnancy (50 non-consumers and 50 daily consumers). BMI was higher among ASB-exposed infants. Infant stool (16S rRNA gene sequencing) and urine (untargeted metabolomics) were acquired in early (3–4 months) and late (12 months) infancy. We identified four microbiome clusters, of which two recapitulated the maturation trajectory of the infant gut bacterial communities from immature (Cluster 1) to mature (Cluster 4) and two deviated from this trajectory (Clusters 2 and 3). Maternal ASB consumption did not differ between clusters, but was associated with community-level shifts in infant gut bacterial taxonomy structure and depletion of several Bacteroides sp. in Cluster 2. In the complete dataset, urine succinate and spermidine levels at 3 months were higher in ASB-exposed infants, and urine succinate was positively associated with BMI at one-year-old. Overall, gestational exposure to ASB was associated with gut microbiota structure in infants from Cluster 2, and gut microbiota structure was associated with infant BMI. Gestational exposure to ASB was positively associated with infant urine succinate and spermidine. Succinate was found to mediate 29% of the effect of ASB exposure on BMI at one-year-old, revealing a potential role of this metabolite in increased infant weight linked to gestational ASB consumption. As we face an unprecedented rise in childhood obesity, future studies should evaluate the causal relationships between maternal ASB consumption (a modifiable exposure), gut microbiota and metabolites, infant metabolism, and body composition.

From correlation to causality: the case of Subdoligranulum

Gut microbes are considered as major factors contributing to human health. Nowadays, the vast majority of the data available in the literature are mostly exhibiting negative or positive correlations between specific bacteria and metabolic parameters. From these observations, putative detrimental or beneficial effects are then inferred. Akkermansia muciniphila is one of the unique examples for which the correlations with health benefits have been causally validated in vivo in rodents and humans.

In this study, based on available metagenomic data in overweight/obese population and clinical variables that we obtained from two cohorts of individuals (n = 108) we identified several metagenomic species (MGS) strongly associated with A. muciniphila with one standing out: Subdoligranulum. By analyzing both qPCR and shotgun metagenomic data, we discovered that the abundance of Subdoligranulum was correlated positively with microbial richness and HDL-cholesterol levels and negatively correlated with fat mass, adipocyte diameter, insulin resistance, levels of leptin, insulin, CRP, and IL6 in humans.

Therefore, to further explore whether these strong correlations could be translated into causation, we investigated the effects of the unique cultivated strain of Subdoligranulum (Subdoligranulum variabile DSM 15176 ^T) in obese and diabetic mice as a proof-of-concept. Strikingly, there were no significant difference in any of the hallmarks of obesity and diabetes measured (e.g., body weight gain, fat mass gain, glucose tolerance, liver weight, plasma lipids) at the end of the 8 weeks of treatment. Therefore, the absence of effect following the supplementation with S. variabile indicates that increasing the intestinal abundance of this bacterium is not translated into beneficial effects in mice.

In conclusion, we demonstrated that despite the fact that numerous strong correlations exist between a given bacteria and health, proof-of-concept experiments are required to be further validated or not in vivo. Hence, an urgent need for causality studies is warranted to move from human observations to preclinical validations.

Microbial enterotypes beyond genus level: Bacteroides species as a predictive biomarker for weight change upon controlled intervention with arabinoxylan oligosaccharides in overweight subjects

Recent studies indicate that microbial enterotypes may influence the beneficial effects of wholegrain enriched diets including bodyweight regulation. In a 4-week intervention trial, overweight subjects were randomized to consume either arabinoxylan-oligosaccharides (AXOS) (10.4 g/d) from wheat bran or polyunsaturated fatty acids (PUFA) (3.6 g/d). In the present study, we have stratified the subjects participating in the intervention (n = 29) according to the baseline Prevotella-to-Bacteroides (P/B) ratios through a post-hoc analysis and applied a linear mixed model analysis to identify the influence of this P/B ratio on the differences in weight changes in the intervention arms. Following AXOS consumption (n = 15), the high P/B group showed no bodyweight changes [−0.14 kg (95% CI: −0.67; 0.38, p = .59)], while the low P/B group gained 0.65 kg (95% CI: 0.16; 1.14, p = .009). Consequently, a difference of −0.79 kg was found between P/B groups (95% CI: −1.51; −0.08, p = .030). No differences were found between P/B groups following PUFA consumption (0.61 kg, 95% CI: −0.13; 1.35, p = .10). Among the Bacteroides species, B. cellulosilyticus relative abundance exhibited the highest positive rank correlation (Kendall’s tau = 0.51, FDR p = .070) with 4-week weight change on AXOS, and such association was further supported by using supervised classification methods (Random Forest). We outlined several carbohydrate-active enzyme (CAZy) genes involved in xylan-binding and degradation to be enriched in B. cellulosilyticus genomes, as well as multiple accessory genes, suggesting a supreme AXOS-derived glycan scavenging role of such species. This post-hoc analysis, ensuring species and strain demarcation at the human gut microbiota, permitted to uncover the predictive role of Bacteroides species over P/B enterotype in weight gain during a fiber-based intervention. The results of this pilot trial pave the way for future assessments on fiber fermentation outputs from Bacteroides species affecting lipid metabolism in the host and with direct impact on adiposity, thus helping to design personalized interventions.

Pomegranate peel extract ameliorates the severity of experimental autoimmune encephalomyelitis via modulation of gut microbiota

Multiple sclerosis (MS) is a CNS autoimmune disease characterized by demyelination and inflammatory infiltration with a high disability rate. Increasing evidence has demonstrated the importance of gut microbiota as an environmental risk factor in MS and its animal model experimental autoimmune encephalomyelitis (EAE). Diet is the main determinant of gut microbiota composition and function, which greatly affects the shaping of microbial structure. Pomegranate peel, a waste product in the production of juice, is rich in health-promoting compounds. However, its individual constituents, immunoregulatory activities, and action associated with bacterial diversity in the gut microbiota are largely unknown. Here, the main nutrient ingredients of pomegranate peel extract (PPE) were identified as phenols, flavonoids, amino acids, carbohydrates, fatty acids, lipids, nucleotides, organic acids, alcohols, and vitamins via metabolomics evaluation. We found, for the first time, oral PPE (100 mg/kg/day) not only effectively relieves EAE, inhibits CNS inflammatory factor infiltration and myelin loss, but also reshapes gut microbiota. Furthermore, recipient EAE mice with fecal transplantation from the PPE-treated donor delayed the disease development significantly. 16S rRNA gene sequencing revealed the increased gut microbiota richness in PPE-treated group. Among them, Lactobacillaceae enriched significantly, while Alcaligenaceae and Acidaminococcacea decreased remarkably. In conclusion, our data demonstrated that gut microbiota mediated the beneficial effects of oral PPE on EAE, and provided new ideas for developing the prebiotic value of pomegranate peel for the treatment of autoimmune diseases.

The prebiotic effects of omega-3 fatty acid supplementation: A six-week randomised intervention trial

Prebiotics are compounds in food that benefit health via affecting the gut microbiome. Omega-3 fatty acids have been associated with differences in gut microbiome composition and are widely accepted to have health benefits, although recent large trials have been inconclusive. We carried out a 6-week dietary intervention comparing the effects of daily supplementation with 500 mg of omega-3 versus 20 g of a well-characterized prebiotic, inulin. Inulin supplementation resulted in large increases in Bifidobacterium and Lachnospiraceae. In contrast, omega-3 supplementation resulted in significant increases in Coprococcus spp. and Bacteroides spp, and significant decreases in the fatty-liver associated Collinsella spp. On the other hand, similar to the results with inulin supplementation which resulted in significant increases in butyrate, iso-valerate, and iso-butyrate (p < .004), omega-3 supplementation resulted in significant increases in iso-butyrate and isovalerate (p < .002) and nearly significant increases in butyrate (p < .053). Coprococcus, which was significantly increased post-supplementation with omega-3, was found to be positively associated with iso-butyric acid (Beta (SE) = 0.69 (0.02), P = 1.4 x 10⁻³) and negatively associated with triglyceride-rich lipoproteins such as VLDL (Beta (SE) = −0.381 (0.01), P = .001) and VLDL-TG (Beta (SE) = −0.372 (0.04), P = .001) after adjusting for confounders. Dietary omega-3 alters gut microbiome composition and some of its cardiovascular effects appear to be potentially mediated by its effect on gut microbial fermentation products indicating that it may be a prebiotic nutrient.

The gut bacterium Extibacter muris produces secondary bile acids and influences liver physiology in gnotobiotic mice

Extibacter muris is a newly described mouse gut bacterium which metabolizes cholic acid (CA) to deoxycholic acid (DCA) via 7α-dehydroxylation. Although bile acids influence metabolic and inflammatory responses, few in vivo models exist for studying their metabolism and impact on the host. Mice were colonized from birth with the simplified community Oligo-MM¹² with or without E. muris. As the metabolism of bile acids is known to affect lipid homeostasis, mice were fed either a low- or high-fat diet for eight weeks before sampling and analyses targeting the gut and liver. Multiple Oligo-MM¹² strains were capable of deconjugating primary bile acids in vitro. E. muris produced DCA from CA either as pure compound or in mouse bile. This production was inducible by CA in vitro. Ursodeoxycholic, chenodeoxycholic, and β-muricholic acid were not metabolized under the conditions tested. All gnotobiotic mice were stably colonized with E. muris, which showed higher relative abundances after HF diet feeding. The presence of E. muris had minor, diet-dependent effects on Oligo-MM¹² communities. The secondary bile acids DCA and surprisingly LCA and their taurine conjugates were detected exclusively in E. muris-colonized mice. E. muris colonization did not influence body weight, white adipose tissue mass, liver histopathology, hepatic aspartate aminotransferase, or blood levels of cholesterol, insulin, and paralytic peptide (PP). However, proteomics revealed shifts in hepatic pathways involved in amino acid, glucose, lipid, energy, and drug metabolism in E. muris-colonized mice. Liver fatty acid composition was substantially altered by dietary fat but not by E. muris.In summary, E. muris stably colonized the gut of mice harboring a simplified community and produced secondary bile acids, which affected proteomes in the liver. This new gnotobiotic mouse model can now be used to study the pathophysiological role of secondary bile acids in vivo.

Enteral broad-spectrum antibiotics antagonize the effect of fecal microbiota transplantation in preterm pigs

Preterm infants are at risk of multiple morbidities including necrotizing enterocolitis (NEC). Suspected NEC patients receive intravenous antibiotics (AB) to prevent sepsis, although enteral AB is arguably more effective at reducing NEC but is rarely used due to the risk of AB resistance. Fecal microbiota transplantation (FMT) has shown protective effects against NEC in animal experiments, but the interaction between AB and FMT has not been investigated in neonates. We hypothesized that administration of enteral AB followed by rectal FMT would effectively prevent NEC with negligible changes in AB resistance and systemic immunity. Using preterm piglets, we examined host and gut microbiota responses to AB, FMT, or a sequential combination thereof, with emphasis on NEC development. In a saline-controlled experiment, preterm piglets (n = 67) received oro-gastric neomycin (50 mg/kg/d) and amoxicillin-clavulanate (50/12.5 mg/kg/d) (hereafter AB) for four days after cesarean delivery, and were subsequently given rectal FMT from healthy suckling piglet donors. Whereas AB protected the stomach and small intestine, and FMT primarily protected the colon, the sequential combination treatment surprisingly provided no NEC protection. Furthermore, minor changes in the gut microbiota composition were observed in response to either treatment, although AB treatment decreased species diversity and increased AB resistance among coliform bacteria and Enterococci, which were both partly reversed by FMT. Besides, enteral AB treatment suppressed cellular and functional systemic immune development, which was not prevented by subsequent FMT. We discovered an antagonistic relationship between enteral AB and FMT in terms of NEC development. The outcome may depend on choice of AB compounds, FMT composition, doses, treatment duration, and administration routes, but these results challenge the applicability of enteral AB and FMT in preterm infants.

Changes in dietary fiber intake in mice reveal associations between colonic mucin O-glycosylation and specific gut bacteria

The colonic mucus layer, comprised of highly O-glycosylated mucins, is vital to mediating host-gut microbiota interactions, yet the impact of dietary changes on colonic mucin O-glycosylation and its associations with the gut microbiota remains unexplored. Here, we used an array of omics techniques including glycomics to examine the effect of dietary fiber consumption on the gut microbiota, colonic mucin O-glycosylation and host physiology of high-fat diet-fed C57BL/6J mice. The high-fat diet group had significantly impaired glucose tolerance and altered liver proteome, gut microbiota composition, and short-chain fatty acid production compared to normal chow diet group. While dietary fiber inclusion did not reverse all high fat-induced modifications, it resulted in specific changes, including an increase in the relative abundance of bacterial families with known fiber digesters and a higher propionate concentration. Conversely, colonic mucin O-glycosylation remained similar between the normal chow and high-fat diet groups, while dietary fiber intervention resulted in major alterations in O-glycosylation. Correlation network analysis revealed previously undescribed associations between specific bacteria and mucin glycan structures. For example, the relative abundance of the bacterium Parabacteroides distasonis positively correlated with glycan structures containing one terminal fucose and correlated negatively with glycans containing two terminal fucose residues or with both an N-acetylneuraminic acid and a sulfate residue. This is the first comprehensive report of the impact of dietary fiber on the colonic mucin O-glycosylation and associations of these mucosal glycans with specific gut bacteria.

Metagenomics reveals impact of geography and acute diarrheal disease on the Central Indian human gut microbiome

Background

The Central Indian gut microbiome remains grossly understudied. Herein, we sought to investigate the burden of antimicrobial resistance and diarrheal diseases, particularly Clostridioides difficile, in rural-agricultural and urban populations in Central India, where there is widespread unregulated antibiotic use. We utilized shotgun metagenomics to comprehensively characterize the bacterial and viral fractions of the gut microbiome and their encoded functions in 105 participants.

Results

We observed distinct rural-urban differences in bacterial and viral populations, with geography exhibiting a greater influence than diarrheal status. Clostridioides difficile disease was more commonly observed in urban subjects, and their microbiomes were enriched in metabolic pathways relating to the metabolism of industrial compounds and genes encoding resistance to 3^rd generation cephalosporins and carbapenems. By linking phages present in the microbiome to their bacterial hosts through CRISPR spacers, phage variation could be directly related to shifts in bacterial populations, with the auxiliary metabolic potential of rural-associated phages enriched for carbon and amino acid energy metabolism.

Conclusions

We report distinct differences in antimicrobial resistance gene profiles, enrichment of metabolic pathways and phage composition between rural and urban populations, as well as a higher burden of Clostridioides difficile disease in the urban population. Our results reveal that geography is the key driver of variation in urban and rural Indian microbiomes, with acute diarrheal disease, including C. difficile disease exerting a lesser impact. Future studies will be required to understand the potential role of dietary, cultural, and genetic factors in contributing to microbiome differences between rural and urban populations.

Neonatal gut colonization by Bifidobacterium is associated with higher childhood cytokine responses

The gut microbiota is a major stimulus for the immune system, and late acquisition of bacteria and/or reduced complexity of the gut flora may delay adaptive immune maturation. However, it is unknown how the gut bacterial colonization pattern in human infants is related to T cell activation during early childhood. We followed 65 Swedish children in the FARMFLORA cohort, from birth up to 3 years of age. In fecal samples collected at several time points during the first year of life, the gut colonization pattern was investigated with the use of both 16S rRNA next generation sequencing (NGS) and culture-based techniques. This was related to production of IL-13, IL-5, IL-6, TNF, IL-1β and IFN-γ by PHA-stimulated fresh mononuclear cells and to proportions of CD4⁺ T cells that expressed CD45RO at 36 months of age. Both NGS and culture-based techniques showed that colonization by Bifidobacterium at 1 week of age associated with higher production of IL-5, IL-6, IL-13, TNF and IL-1β at 36 months of age. By contrast, gut colonization by Enterococcus, Staphylococcus aureus or Clostridium in early infancy related inversely to induced IL-13, IL-5 and TNF at 3 years of age. Infants with elder siblings produced more cytokines and had a larger fraction of CD45RO⁺ T cells compared to single children. However, controlling for these factors did not abolish the effect of colonization by Bifidobacterium on immune maturation. Thus, gut colonization in early infancy affects T cell maturation and Bifidobacterium may be especially prone to induce infantile immune maturation.

RhoA GTPase phosphorylated at tyrosine 42 by src kinase binds to β-catenin and contributes transcriptional regulation of vimentin upon Wnt3A

2020-12-25T00:00

In the Wnt canonical pathway, Wnt3A has been known to stabilize β-catenin. In the non-canonical Wnt signaling pathway, Wnt is known to activate Rho GTPases. The correlation between canonical and non-canonical pathways by Wnt signaling, however, has not been well elucidated. Here, we identified that Wnt3A promoted superoxide generation, leading to Tyr42 phosphorylation of RhoA through activations of c-Src and Rho-dependent coiled coil kinase 2 (ROCK2) and phosphorylation of p47phox, a component of NADPH oxidase. Wnt3A also induced accumulation of β-catenin along with activations of RhoA and ROCK1. Concurrently, ROCK1 was able to phosphorylate GSK-3β at Ser9, which phosphorylated Src at Ser51 and Ser492 residues, leading to Src inactivation through dephosphorylation of Tyr416 during the late period of Wnt3A treatment. Meanwhile, p-Tyr42 RhoA bound to β-catenin via the N-terminal domain of β-catenin, thereby leading to the nuclear translocation of p-Tyr42 RhoA/β-catenin complex. Notably, p-Tyr42 RhoA as well as β-catenin was associated with the promoter of Vim, leading to increased expression of vimentin. In addition, stomach cancer patients harboring higher expressed p-Tyr42 Rho levels revealed the much poorer survival probability. Therefore, we propose that p-Tyr42 RhoA is crucial for transcriptional regulation of specific target genes in the nucleus by binding to their promoters and involved in tumorigenesis.

•

ROCK2 phosphorylates p47phox, a component of NADPH oxidase, leading to superoxide production upon Wnt3A.

•

Wnt3A upregulates not only β-catenin but also p-Tyr42 RhoA through Src, which is activated by superoxide.

•

p-Tyr42 RhoA/β-catenin complex translocates to nucleus and binds to the promoter of Vim, leading to vimentin expression.

•

ROCK1 phosphorylates GSK-3β at Ser9, resulting in Ser493 and Ser51 phosphorylation of Src and its inactivation for desensitizing Wnt3A.

•

Cancer patients harboring high p-Tyr42 RhoA level reveal the much poorer survival probability.

HIV TAT-mediated microglial senescence: Role of SIRT3-dependent mitochondrial oxidative stress

2020-12-23T00:00

The advent of combined antiretroviral treatment (cART) as a treatment for HIV-1 infection has not only resulted in a dramatic decrease in the peripheral viral load but has also led to increased life expectancy of the infected individuals. Paradoxically, increased lifespan is accompanied with higher prevalence of age-related comorbidities, including HIV-associated neurocognitive disorders (HAND). Present study was aimed at exploring the role of HIV TAT protein in mediating microglial mitochondrial oxidative stress, ultimately resulting in neuroinflammation and microglial senescence. Our findings demonstrated that exposure of mouse primary microglial cells (mPMs) to HIV TAT protein resulted in a senescence-like phenotype, that was characterized by elevated expression of both p16 and p21 proteins, increased numbers of senescence-associated-β-galactosidase positive cells, augmented cell-cycle arrest, increased release of proinflammatory cytokines and decreased telomerase activity. Additionally, exposure of mPMs to HIV TAT also resulted downregulation of SIRT3 with a concomitant increase in mitochondrial oxidative stress. Dual luciferase reporter assay identified miR-505 as a novel target of SIRT3, which was upregulated in mPMs exposed to HIV TAT. Furthermore, transient transfection of mPMs with either the SIRT3 plasmid or miRNA-505 inhibitor upregulated the expression of SIRT3 and mitochondrial antioxidant enzymes, with a concomitant decrease in microglial senescence. These in vitro findings were also validated in the prefrontal cortices and striatum of HIV transgenic rats as well as cART-treated HIV-infected individuals. In summary, this study underscores a yet undiscovered novel mechanism(s) underlying HIV TAT-mediated induction of senescence phenotype in microglia, involving the miR-505-SIRT3 axis-mediated induction of mitochondrial oxidative stress.

•

HIV TAT induces senescence-like phenotype in microglia.

•

HIV TAT decreases SIRT3 with concomitant increase of mitochondrial ROS.

•

Overexpression of SIRT3 attenuated HIV TAT-mediated microglial senescence.

•

miR-505 negatively regulate SIRT3 expression.

•

miR-505 inhibition prevents SIRT3-mediated mitochondria stress and glial senescence.

Nova Reader - Subject

Inhibition of the PP2A activity by the histone chaperone ANP32B is long-range allosterically regulated by respiratory cytochrome c

Activation of NRF2 by APE1/REF1 is redox-dependent in Barrett's related esophageal adenocarcinoma cells

Background

Results

Conclusion

AQP8 is a crucial H2O2 transporter in insulin-producing RINm5F cells

Decreased availability of nitric oxide and hydrogen sulfide is a hallmark of COVID-19

Background

Methods

Results

Conclusion

LRRc17 controls BMSC senescence via mitophagy and inhibits the therapeutic effect of BMSCs on ovariectomy-induced bone loss

Intestinal microbiota drives cholestasis-induced specific hepatic gene expression patterns

Development of gut microbiota and bifidobacterial communities of neonates in the first 6 weeks and their inheritance from mother

Sulforaphane exposure impairs contractility and mitochondrial function in three-dimensional engineered heart tissue

Sustained IKKβ phosphorylation and NF-κB activation by superoxide-induced peroxynitrite-mediated nitrotyrosine modification of B56γ3 and PP2A inactivation

Cutaneous antimicrobial peptides: New “actors” in pollution related inflammatory conditions

Skeletal muscle-specific Keap1 disruption modulates fatty acid utilization and enhances exercise capacity in female mice

Therapeutic wavelengths of ultraviolet B radiation activate apoptotic, circadian rhythm, redox signalling and key canonical pathways in psoriatic epidermis

Endogenous generation of nitro-fatty acid hybrids having dual nitrate ester (RONO2) and nitroalkene (RNO2) substituents

Lysosomal degradation ensures accurate chromosomal segregation to prevent chromosomal instability

The proppin Bcas3 and its interactor KinkyA localize to the early phagophore and regulate autophagy

Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5

TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium

Intestinal Cetobacterium and acetate modify glucose homeostasis via parasympathetic activation in zebrafish

Coxiella effector protein CvpF subverts RAB26-dependent autophagy to promote vacuole biogenesis and virulence

Prediction of the spread of Corona-virus carrying droplets in a bus - A computational based artificial intelligence approach

Macrophage migration inhibitory factor (MIF) enhances hypochlorous acid production in phagocytic neutrophils

Background

Methods

Results

Conclusion

Gut bacteriophage dynamics during fecal microbial transplantation in subjects with metabolic syndrome

The Impact of Migration on the Gut Metagenome of South Asian Canadians

NOX4 promotes ferroptosis of astrocytes by oxidative stress-induced lipid peroxidation via the impairment of mitochondrial metabolism in Alzheimer's diseases

Dexmedetomidine ameliorates endotoxin-induced acute lung injury in vivo and in vitro by preserving mitochondrial dynamic equilibrium through the HIF-1a/HO-1 signaling pathway

Quiescin sulfhydryl oxidase 1 promotes sorafenib-induced ferroptosis in hepatocellular carcinoma by driving EGFR endosomal trafficking and inhibiting NRF2 activation

Htd2 deficiency-associated suppression of α-lipoic acid production provokes mitochondrial dysfunction and insulin resistance in adipocytes

Cohousing-mediated microbiota transfer from milk bioactive components-dosed mice ameliorate colitis by remodeling colonic mucus barrier and lamina propria macrophages

Expansion and persistence of antibiotic-specific resistance genes following antibiotic treatment

Opa1 relies on cristae preservation and ATP synthase to curtail reactive oxygen species accumulation in mitochondria

The intestinal microbiota and metabolites in patients with anorexia nervosa

Bacteroides uniformis combined with fiber amplifies metabolic and immune benefits in obese mice

Methionine restriction alleviates age-associated cognitive decline via fibroblast growth factor 21

bFGF alleviates diabetes-associated endothelial impairment by downregulating inflammation via S-nitrosylation pathway

Far Upstream Binding Protein 1 (FUBP1) participates in translational regulation of Nrf2 protein under oxidative stress

The lncRNA Malat1 regulates microvascular function after myocardial infarction in mice via miR-26b-5p/Mfn1 axis-mediated mitochondrial dynamics

Rationale

Methods and results

Conclusions

Taurine rescues mitochondria-related metabolic impairments in the patient-derived induced pluripotent stem cells and epithelial-mesenchymal transition in the retinal pigment epithelium

MKL1 cooperates with p38MAPK to promote vascular senescence, inflammation, and abdominal aortic aneurysm

Divergent trajectories of cellular bioenergetics, intermediary metabolism and systemic redox status in survivors and non-survivors of critical illness

Background

Methods

Results

Conclusion

mtROS-mediated Akt/AMPK/mTOR pathway was involved in Copper-induced autophagy and it attenuates Copper-induced apoptosis in RAW264.7 mouse monocytes

Progression from Monoclonal gammopathy of undetermined significance of the immunoglobulin M class (IgM-MGUS) to Waldenstrom Macroglobulinemia is associated with an alteration in lipid metabolism

Endogenous SO2-dependent Smad3 redox modification controls vascular remodeling

Identification of proteins and cellular pathways targeted by 2-nitroimidazole hypoxic cytotoxins

Dietary vitamin K is remodeled by gut microbiota and influences community composition

Links between gut microbiome composition and fatty liver disease in a large population sample

Association between antibiotics and gut microbiome dysbiosis in children: systematic review and meta-analysis

Gut microbiota shape the inflammatory response in mice with an epithelial defect

Intestinal and systemic inflammation induced by symptomatic and asymptomatic enterotoxigenic E. coli infection and impact on intestinal colonization and ETEC specific immune responses in an experimental human challenge model

Sirt3-mediated mitophagy regulates AGEs-induced BMSCs senescence and senile osteoporosis

Binding of the human antioxidation protein α1-microglobulin (A1M) to heparin and heparan sulfate. Mapping of binding site, molecular and functional characterization, and co-localization in vivo and in vitro

Elevated levels of oxidized nucleosides in individuals with the JAK2V617F mutation from a general population study

Formation of protein cross-links by singlet oxygen-mediated disulfide oxidation

Reductively modified albumin attenuates DSS-Induced mouse colitis through rebalancing systemic redox state

Effects of colon-targeted vitamins on the composition and metabolic activity of the human gut microbiome– a pilot study

The gut virome in Irritable Bowel Syndrome differs from that of controls

Tempol ameliorates polycystic ovary syndrome through attenuating intestinal oxidative stress and modulating of gut microbiota composition-serum metabolites interaction

Ethnic variability associating gut and oral microbiome with obesity in children

Endothelial specific deletion of HMGB1 increases blood pressure and retards ischemia recovery through eNOS and ROS pathway in mice☆

Roux-en-Y gastric bypass surgery in Zucker rats induces bacterial and systemic metabolic changes independent of caloric restriction-induced weight loss

The rectal mucosal but not fecal microbiota detects subclinical ulcerative colitis

Involvement of the microbiota-gut-brain axis in chronic restraint stress: disturbances of the kynurenine metabolic pathway in both the gut and brain

AQP8 is a crucial H₂O₂ transporter in insulin-producing RINm5F cells

Endogenous generation of nitro-fatty acid hybrids having dual nitrate ester (RONO₂) and nitroalkene (RNO₂) substituents

Endogenous SO₂-dependent Smad3 redox modification controls vascular remodeling

Binding of the human antioxidation protein α₁-microglobulin (A1M) to heparin and heparan sulfate. Mapping of binding site, molecular and functional characterization, and co-localization in vivo and in vitro

Endothelial specific deletion of HMGB1 increases blood pressure and retards ischemia recovery through eNOS and ROS pathway in mice^☆