DNA hypomethylation is a feature of epidermal cells from aged and sun-exposed skin, but the mechanisms responsible for this methylation loss are not known. Dnmt3a is the dominant de novo DNA methyltransferase in the skin; while epidermal Dnmt3a deficiency creates a premalignant state in which keratinocytes are more easily transformed by topical mutagens, the conditions responsible for this increased susceptibility to transformation are not well understood. Using whole genome bisulfite sequencing, we identified a focal, canonical DNA hypomethylation phenotype in the epidermal cells of Dnmt3a-deficient mice. Single-cell transcriptomic analysis revealed an increased proportion of cells with a proliferative gene expression signature, while other populations in the skin were relatively unchanged. Although total DNMT3A deficiency has not been described in human disease states, rare patients with an overgrowth syndrome associated with behavioral abnormalities and an increased risk of cancer often have heterozygous, germline mutations in DNMT3A that reduce its function (Tatton-Brown Rahman syndrome [TBRS]). We evaluated the DNA methylation phenotype of the skin from a TBRS patient with a germline DNMT3A^R882H mutation, which encodes a dominant-negative protein that reduces its methyltransferase function by ∼80%. We detected a focal, canonical hypomethylation phenotype that revealed considerable overlap with hypomethylated regions found in Dnmt3a-deficient mouse skin. Together, these data suggest that DNMT3A loss creates a premalignant epigenetic state associated with a hyperproliferative phenotype in the skin and further suggest that DNMT3A acts as a tumor suppressor in the skin.

Osteoarthritis (OA) is a painful and debilitating condition of synovial joints without any disease-modifying therapies [A. M. Valdes, T. D. Spector, Nat. Rev. Rheumatol. 7, 23–32 (2011)]. We previously identified mechanosensitive PIEZO channels, PIEZO1 and PIEZO2, both expressed in articular cartilage, to function in chondrocyte mechanotransduction in response to injury [W. Lee et al., Proc. Natl. Acad. Sci. U.S.A. 111, E5114–E5122 (2014); W. Lee, F. Guilak, W. Liedtke, Curr. Top. Membr. 79, 263–273 (2017)]. We therefore asked whether interleukin-1–mediated inflammatory signaling, as occurs in OA, influences Piezo gene expression and channel function, thus indicative of maladaptive reprogramming that can be rationally targeted. Primary porcine chondrocyte culture and human osteoarthritic cartilage tissue were studied. We found that interleukin-1α (IL-1α) up-regulated Piezo1 in porcine chondrocytes. Piezo1 expression was significantly increased in human osteoarthritic cartilage. Increased Piezo1 expression in chondrocytes resulted in a feed-forward pathomechanism whereby increased function of Piezo1 induced excess intracellular Ca²⁺ at baseline and in response to mechanical deformation. Elevated resting state Ca²⁺ in turn rarefied the F-actin cytoskeleton and amplified mechanically induced deformation microtrauma. As intracellular substrates of this OA-related inflammatory pathomechanism, in porcine articular chondrocytes exposed to IL-1α, we discovered that enhanced Piezo1 expression depended on p38 MAP-kinase and transcription factors HNF4 and ATF2/CREBP1. CREBP1 directly bound to the proximal PIEZO1 gene promoter. Taken together, these signaling and genetic reprogramming events represent a detrimental Ca²⁺-driven feed-forward mechanism that can be rationally targeted to stem the progression of OA.

Schlafen-11 (SLFN11) inactivation in ∼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4^CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1–CUL4^CDT2 ubiquitin ligase.

Tuberous sclerosis complex (TSC) is caused by mutations in either TSC1 or TSC2 genes and affects multiple organs, including kidney, lung, and brain. In the kidney, TSC presents with the enlargement of benign tumors (angiomyolipomata) and cysts, which eventually leads to kidney failure. The factors promoting cyst formation and tumor growth in TSC are incompletely understood. Here, we report that mice with principal cell-specific inactivation of Tsc1 develop numerous cortical cysts, which are overwhelmingly composed of hyperproliferating A-intercalated (A-IC) cells. RNA sequencing and confirmatory expression studies demonstrated robust expression of Forkhead Transcription Factor 1 (Foxi1) and its downstream targets, apical H⁺-ATPase and cytoplasmic carbonic anhydrase 2 (CAII), in cyst epithelia in Tsc1 knockout (KO) mice but not in Pkd1 mutant mice. In addition, the electrogenic 2Cl⁻/H⁺ exchanger (CLC-5) is significantly up-regulated and shows remarkable colocalization with H⁺-ATPase on the apical membrane of cyst epithelia in Tsc1 KO mice. Deletion of Foxi1, which is vital to intercalated cells viability and H⁺-ATPase expression, completely abrogated the cyst burden in Tsc1 KO mice, as indicated by MRI images and histological analysis in kidneys of Foxi1/Tsc1 double-knockout (dKO) mice. Deletion of CAII, which is critical to H⁺-ATPase activation, caused significant reduction in cyst burden and increased life expectancy in CAII/Tsc1 dKO mice vs. Tsc1 KO mice. We propose that intercalated cells and their acid/base/electrolyte transport machinery (H⁺-ATPase/CAII/CLC-5) are critical to cystogenesis, and their inhibition or inactivation is associated with significant protection against cyst generation and/or enlargement in TSC.

We evaluated the potential for a monoclonal antibody antagonist of the glucagon receptor (Ab-4) to maintain glucose homeostasis in type 1 diabetic rodents. We noted durable and sustained improvements in glycemia which persist long after treatment withdrawal. Ab-4 promoted β-cell survival and enhanced the recovery of insulin⁺ islet mass with concomitant increases in circulating insulin and C peptide. In PANIC-ATTAC mice, an inducible model of β-cell apoptosis which allows for robust assessment of β-cell regeneration following caspase-8–induced diabetes, Ab-4 drove a 6.7-fold increase in β-cell mass. Lineage tracing suggests that this restoration of functional insulin-producing cells was at least partially driven by α-cell-to-β-cell conversion. Following hyperglycemic onset in nonobese diabetic (NOD) mice, Ab-4 treatment promoted improvements in C-peptide levels and insulin⁺ islet mass was dramatically increased. Lastly, diabetic mice receiving human islet xenografts showed stable improvements in glycemic control and increased human insulin secretion.

Ever since the discovery of X-rays, tremendous efforts have been made to develop new imaging techniques for unlocking the hidden secrets of our world and enriching our understanding of it. X-ray differential phase contrast imaging, which measures the gradient of a sample’s phase shift, can reveal more detail in a weakly absorbing sample than conventional absorption contrast. However, normally only the gradient’s component in two mutually orthogonal directions is measurable. In this article, omnidirectional differential phase images, which record the gradient of phase shifts in all directions of the imaging plane, are efficiently generated by scanning an easily obtainable, randomly structured modulator along a spiral path. The retrieved amplitude and main orientation images for differential phase yield more information than the existing imaging methods. Importantly, the omnidirectional dark-field images can be simultaneously extracted to study strongly ordered scattering structures. The proposed method can open up new possibilities for studying a wide range of complicated samples composed of both heavy, strongly scattering atoms and light, weakly scattering atoms.

The characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral kinetics in hospitalized patients and its association with mortality is unknown. We analyzed death and nasopharyngeal viral kinetics in 655 hospitalized patients from the prospective French COVID cohort. The model predicted a median peak viral load that coincided with symptom onset. Patients with age ≥65 y had a smaller loss rate of infected cells, leading to a delayed median time to viral clearance occurring 16 d after symptom onset as compared to 13 d in younger patients (P < 10⁻⁴). In multivariate analysis, the risk factors associated with mortality were age ≥65 y, male gender, and presence of chronic pulmonary disease (hazard ratio [HR] > 2.0). Using a joint model, viral dynamics after hospital admission was an independent predictor of mortality (HR = 1.31, P < 10⁻³). Finally, we used our model to simulate the effects of effective pharmacological interventions on time to viral clearance and mortality. A treatment able to reduce viral production by 90% upon hospital admission would shorten the time to viral clearance by 2.0 and 2.9 d in patients of age <65 y and ≥65 y, respectively. Assuming that the association between viral dynamics and mortality would remain similar to that observed in our population, this could translate into a reduction of mortality from 19 to 14% in patients of age ≥65 y with risk factors. Our results show that viral dynamics is associated with mortality in hospitalized patients. Strategies aiming to reduce viral load could have an effect on mortality rate in this population.

Both gene repressor (Polycomb-dependent) and activator (Polycomb-independent) functions of the Polycomb protein enhancer of zeste homolog 2 (EZH2) are implicated in cancer progression. EZH2 protein can be phosphorylated at various residues, such as threonine 487 (T487), by CDK1 kinase, and such phosphorylation acts as a Polycomb repressive complex 2 (PRC2) suppression “code” to mediate the gene repressor-to-activator switch of EZH2 functions. Here we demonstrate that the histone reader protein ZMYND8 is overexpressed in human clear cell renal cell carcinoma (ccRCC). ZMYND8 binds to EZH2, and their interaction is largely enhanced by CDK1 phosphorylation of EZH2 at T487. ZMYND8 depletion not only enhances Polycomb-dependent function of EZH2 in hypoxia-exposed breast cancer cells or von Hippel–Lindau (VHL)-deficient ccRCC cells, but also suppresses the FOXM1 transcription program. We further show that ZMYND8 is required for EZH2–FOXM1 interaction and is important for FOXM1-dependent matrix metalloproteinase (MMP) gene expression and EZH2-mediated migration and invasion of VHL-deficient ccRCC cells. Our results identify a previously uncharacterized role of the chromatin reader ZMYND8 in recognizing the PRC2-inhibitory phosphorylation “code” essential for the Polycomb-dependent to -independent switch of EZH2 functions. They also reveal an oncogenic pathway driving cell migration and invasion in hypoxia-inducible factor–activated (hypoxia or VHL-deficient) cancer.

COVID-19 transmits by droplets generated from surfaces of airway mucus during processes of respiration within hosts infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. We studied respiratory droplet generation and exhalation in human and nonhuman primate subjects with and without COVID-19 infection to explore whether SARS-CoV-2 infection, and other changes in physiological state, translate into observable evolution of numbers and sizes of exhaled respiratory droplets in healthy and diseased subjects. In our observational cohort study of the exhaled breath particles of 194 healthy human subjects, and in our experimental infection study of eight nonhuman primates infected, by aerosol, with SARS-CoV-2, we found that exhaled aerosol particles vary between subjects by three orders of magnitude, with exhaled respiratory droplet number increasing with degree of COVID-19 infection and elevated BMI-years. We observed that 18% of human subjects (35) accounted for 80% of the exhaled bioaerosol of the group (194), reflecting a superspreader distribution of bioaerosol analogous to a classical 20:80 superspreader of infection distribution. These findings suggest that quantitative assessment and control of exhaled aerosol may be critical to slowing the airborne spread of COVID-19 in the absence of an effective and widely disseminated vaccine.

Multiplex assays, involving the simultaneous use of multiple circulating tumor DNA (ctDNA) markers, can improve the performance of liquid biopsies so that they are highly predictive of cancer recurrence. We have developed a single-tube methylation-specific quantitative PCR assay (mqMSP) that uses 10 different methylation markers and is capable of quantitative analysis of plasma samples with as little as 0.05% tumor DNA. In a cohort of 179 plasma samples from colorectal cancer (CRC) patients, adenoma patients, and healthy controls, the sensitivity and specificity of the mqMSP assay were 84.9% and 83.3%, respectively. In a head-to-head comparative study, the mqMSP assay also performed better for detecting early-stage (stage I and II) and premalignant polyps than a published SEPT9 assay. In an independent longitudinal cohort of 182 plasma samples (preoperative, postoperative, and follow-up) from 82 CRC patients, the mqMSP assay detected ctDNA in 73 (89.0%) of the preoperative plasma samples. Postoperative detection of ctDNA (within 2 wk of surgery) identified 11 of the 20 recurrence patients and was associated with poorer recurrence-free survival (hazard ratio, 4.20; P = 0.0005). With subsequent longitudinal monitoring, 14 patients (70%) had detectable ctDNA before recurrence, with a median lead time of 8.0 mo earlier than seen with radiologic imaging. The mqMSP assay is cost-effective and easily implementable for routine clinical monitoring of CRC recurrence, which can lead to better patient management after surgery.

Pathological remodeling of the heart is a hallmark of chronic heart failure (HF) and these structural changes further perpetuate the disease. Cardiac fibroblasts are the critical cell type that is responsible for maintaining the structural integrity of the heart. Stress conditions, such as a myocardial infarction (MI), can activate quiescent fibroblasts into synthetic and contractile myofibroblasts. G protein-coupled receptor kinase 5 (GRK5) is an important mediator of cardiovascular homeostasis through dampening of GPCR signaling, and is expressed in the heart and up-regulated in human HF. Of note, GRK5 has been demonstrated to translocate to the nucleus in cardiomyocytes in a calcium-calmodulin (Ca²⁺-CAM)-dependent manner, promoting hypertrophic gene transcription through activation of nuclear factor of activated T cells (NFAT). Interestingly, NFAT is also involved in fibroblast activation. GRK5 is highly expressed and active in cardiac fibroblasts; however, its pathophysiological role in these crucial cardiac cells is unknown. We demonstrate using adult cardiac fibroblasts that genetic deletion of GRK5 inhibits angiotensin II (AngII)-mediated fibroblast activation. Fibroblast-specific deletion of GRK5 in mice led to decreased fibrosis and cardiac hypertrophy after chronic AngII infusion or after ischemic injury compared to nontransgenic littermate controls (NLCs). Mechanistically, we show that nuclear translocation of GRK5 is involved in fibroblast activation. These data demonstrate that GRK5 is a regulator of fibroblast activation in vitro and cardiac fibrosis in vivo. This adds to previously published data which demonstrate the potential beneficial effects of GRK5 inhibition in the context of cardiac disease.

The outbreak of COVID-19 caused by SARS-CoV-2 has resulted in more than 50 million confirmed cases and over 1 million deaths worldwide as of November 2020. Currently, there are no effective antivirals approved by the Food and Drug Administration to contain this pandemic except the antiviral agent remdesivir. In addition, the trimeric spike protein on the viral surface is highly glycosylated and almost 200,000 variants with mutations at more than 1,000 positions in its 1,273 amino acid sequence were reported, posing a major challenge in the development of antibodies and vaccines. It is therefore urgently needed to have alternative and timely treatments for the disease. In this study, we used a cell-based infection assay to screen more than 3,000 agents used in humans and animals, including 2,855 small molecules and 190 traditional herbal medicines, and identified 15 active small molecules in concentrations ranging from 0.1 nM to 50 μM. Two enzymatic assays, along with molecular modeling, were then developed to confirm those targeting the virus 3CL protease and the RNA-dependent RNA polymerase. Several water extracts of herbal medicines were active in the cell-based assay and could be further developed as plant-derived anti–SARS-CoV-2 agents. Some of the active compounds identified in the screen were further tested in vivo, and it was found that mefloquine, nelfinavir, and extracts of Ganoderma lucidum (RF3), Perilla frutescens, and Mentha haplocalyx were effective in a challenge study using hamsters as disease model.

5-Methylcytosine (5mC) is an important type of epigenetic modification. Bisulfite sequencing (BS-seq) has limitations, such as severe DNA degradation. Using single molecule real-time sequencing, we developed a methodology to directly examine 5mC. This approach holistically examined kinetic signals of a DNA polymerase (including interpulse duration and pulse width) and sequence context for every nucleotide within a measurement window, termed the holistic kinetic (HK) model. The measurement window of each analyzed double-stranded DNA molecule comprised 21 nucleotides with a cytosine in a CpG site in the center. We used amplified DNA (unmethylated) and M.SssI-treated DNA (methylated) (M.SssI being a CpG methyltransferase) to train a convolutional neural network. The area under the curve for differentiating methylation states using such samples was up to 0.97. The sensitivity and specificity for genome-wide 5mC detection at single-base resolution reached 90% and 94%, respectively. The HK model was then tested on human–mouse hybrid fragments in which each member of the hybrid had a different methylation status. The model was also tested on human genomic DNA molecules extracted from various biological samples, such as buffy coat, placental, and tumoral tissues. The overall methylation levels deduced by the HK model were well correlated with those by BS-seq (r = 0.99; P < 0.0001) and allowed the measurement of allele-specific methylation patterns in imprinted genes. Taken together, this methodology has provided a system for simultaneous genome-wide genetic and epigenetic analyses.