The rockefeller university

Interindividual clinical variability in the course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is vast. We report that at least 101 of 987 patients with life-threatening coronavirus disease 2019 (COVID-19) pneumonia had neutralizing immunoglobulin G (IgG) autoantibodies (auto-Abs) against interferon-omega (IFN-omega) (13 patients), against the 13 types of IFN-alpha (36), or against both (52) at the onset of critical disease; a few also had auto-Abs against the other three type I IFNs. The auto-Abs neutralize the ability of the corresponding type I IFNs to block SARS-CoV-2 infection in vitro. These auto-Abs were not found in 663 individuals with asymptomatic or mild SARS-CoV-2 infection and were present in only 4 of 1227 healthy individuals. Patients with auto-Abs were aged 25 to 87 years and 95 of the 101 were men. A B cell autoimmune phenocopy of inborn errors of type I IFN immunity accounts for lifethreatening COVID-19 pneumonia in at least 2.6% of women and 12.5% of men.

Congenital anomalies of the kidney and urinary tract (CAKUT) constitute one of the most frequent birth defects and represent the most common cause of chronic kidney disease in the first three decades of life. Despite the discovery of dozens of monogenic causes of CAKUT, most pathogenic pathways remain elusive. We performed whole-exome sequencing (WES) in 551 individuals with CAKUT and identified a heterozygous de novo stop-gain variant in ZMYM2 in two different families with CAKUT. Through collaboration, we identified in total 14 different heterozygous loss-of-function mutations in ZMYM2 in 15 unrelated families. Most mutations occurred de novo, indicating possible interference with reproductive function. Human disease features are replicated in X. tropicalis larvae with morpholino knockdowns, in which expression of truncated ZMYM2 proteins, based on individual mutations, failed to rescue renal and craniofacial defects. Moreover, heterozygous Zmym2-deficient mice recapitulated features of CAKUT with high penetrance. The ZMYM2 protein is a component of a transcriptional corepressor complex recently linked to the silencing of developmentally regulated endogenous retrovirus elements. Using protein-protein interaction assays, we show that ZMYM2 interacts with additional epigenetic silencing complexes, as well as confirming that it binds to FOXP1, a transcription factor that has also been linked to CAKUT. In summary, our findings establish that loss-of-function mutations of ZMYM2, and potentially that of other proteins in its interactome, as causes of human CAKUT, offering new routes for studying the pathogenesis of the disorder.

In order to evaluate whether seagulls living on the Lisbon coastline, Portugal, might be colonized and consequently represent potential spreaders of multidrug-resistant bacteria, a total of 88 gull fecal samples were screened for detection of extended-spectrum beta-lactamase (ESBL)- or carbapenemase-producing Enterobacteriaceae for methicillin-resistant Staphylococcus aureus (MRSA) and for vancomycin-resistant Enterococci (VRE). A large proportion of samples yielded carbapenemase- or ESBL-producing Enterobacteriaceae (16% and 55%, respectively), while only two MRSA and two VRE were detected. Mating-out assays followed by PCR and whole-plasmid sequencing allowed to identify carbapenemase and ESBL encoding genes. Among 24 carbapenemase-producing isolates, there were mainly Klebsiella pneumoniae (50%) and Escherichia coli (33%). OXA-181 was the most common carbapenemase identified (54%), followed by OXA-48 (25%) and KPC-2 (17%). Ten different ESBLs were found among 62 ESBL-producing isolates, mainly being CTX-M-type enzymes (87%). Co-occurrence in single samples of multiple ESBL- and carbapenemase producers belonging to different bacterial species was observed in some cases. Seagulls constitute an important source for spreading multidrug-resistant bacteria in the environment and their gut microbiota a formidable microenvironment for transfer of resistance genes within bacterial species.

The characterization of specific metaboliteprotein interactions is important in chemical biology and drug discovery. For example, nuclear receptors (NRs) are a family of ligand-activated transcription factors that regulate diverse physiological processes in animals and are key targets for therapeutic development. However, the identification and characterization of physiological ligands for many NRs remains challenging, because of limitations in domain-specific analysis of ligand binding in cells. To address these limitations, we developed a domain-specific covalent chemical reporter for peroxisome proliferator-activated receptors (PPARs) and demonstrated its utility to screen and characterize the potency of candidate NR ligands in live cells. These studies demonstrate targeted and domain-specific chemical reporters provide excellent tools to evaluate endogenous and exogenous (diet, microbiota, therapeutics) ligands of PPARs in mammalian cells, as well as additional protein targets for further investigation.

DNA repair is a highly dynamic process in which the actual damage recognition process occurs through an amazing dance between the DNA duplex containing the lesion and the DNA repair proteins. Single molecule investigations have revealed that DNA repair proteins solve the speed-stability paradox, of rapid search versus stable complex formation, by conformational changes induced in both the damaged DNA and the repair proteins. Using Rad4, XPA, PARP1, APE1, OGG1 and UV-DDB as examples, we have discovered how these repair proteins limit their travel on DNA, once a lesion is encountered through a process of anomalous diffusion. We have also observed how PARP1 and APE1, as well as UV-DDB and OGG1 or APE1, co-localize dynamically at sites near DNA damage. This review highlights how our group has greatly benefited from our productive collaborations with Sam Wilson's research group.

Adeno-associated virus (AAV) vectors are a leading platform for gene-based therapies for both monogenic and complex acquired disorders. The success of AAV gene transfer highlights the need to answer outstanding clinical questions of safety, durability, and the nature of the human immune response to AAV vectors. Here, we present longitudinal follow-up data of subjects who participated in the first trial of a systemically delivered AAV vector. Adult males (n = 7) with severe hemophilia B received an AAV2 vector at doses ranging from 8 x 10(10) to 2 x 10(12) vg/kg to target hepatocyte-specific expression of coagulation factor IX; a subset (n = 4) was followed for 12-15 years post-vector administration. No major safety concerns were observed. There was no evidence of sustained hepatic toxicity or development of hepatocellular carcinoma as assessed by liver transaminase values, serum alpha-fetoprotein, and liver ultrasound. Subjects demonstrated persistent, increased AAV neutralizing antibodies (NAbs) to the infused AAV serotype 2 (AAV2) as well as all other AAV serotypes tested (AAV5 and AAV8) for the duration of follow-up. These data represent the longest available longitudinal follow-up data of subjects who received intravascular AAV and support the preliminary safety of intravascular AAV administration at the doses tested in adults. Data demonstrate, for the first time, the persistence of high-titer, multi-serotype cross-reactive AAV NAbs for up to 15 years postAAV vector administration. Our observations are broadly applicable to the development of AAV-mediated gene therapy.

Adeno-associated virus (AAV) vector serotypes vary in their ability to transduce hepatocytes from different species. Chimeric mouse models harboring human hepatocytes have shown translational promise for liver-directed gene therapies. However, many variables that influence human hepatocyte transduction and transgene expression in such models remain poorly defined. Here, we aimed to test whether three experimental conditions influence AAV transgene expression in immunodeficient, fumaryl-acetoactetate-hydrolase-deficient (Fah(-/-)) chimeric mice repopulated with primary human hepatocytes. We examined the effects of the murine liver injury cycle, human donor variability, and vector doses on hepatocyte transduction with various AAV serotypes expressing a green fluorescent protein (GFP). We determined that the timing of AAV vector challenge in the liver injury cycle resulted in up to 7-fold differences in the percentage of GFP expressing human hepatocytes. The GFP+ hepatocyte frequency varied 7-fold between human donors without, however, changing the relative transduction efficiency between serotypes for an individual donor. There was also a clear relationship between AAV vector doses and human hepatocyte transduction and transgene expression. We conclude that several experimental variables substantially affect human hepatocyte transduction in the Fah(-/-) chimera model, attention to which may improve reproducibility between findings from different laboratories.

Mathematical and experimental approaches are used to investigate the mechanical forces that shape the tumour architecture of two different common forms of skin cancer: basal cell carcinomas and invasive squamous cell carcinomas. Loss of normal tissue architecture is a hallmark of oncogenic transformation(1). In developing organisms, tissues architectures are sculpted by mechanical forces during morphogenesis(2). However, the origins and consequences of tissue architecture during tumorigenesis remain elusive. In skin, premalignant basal cell carcinomas form 'buds', while invasive squamous cell carcinomas initiate as 'folds'. Here, using computational modelling, genetic manipulations and biophysical measurements, we identify the biophysical underpinnings and biological consequences of these tumour architectures. Cell proliferation and actomyosin contractility dominate tissue architectures in monolayer, but not multilayer, epithelia. In stratified epidermis, meanwhile, softening and enhanced remodelling of the basement membrane promote tumour budding, while stiffening of the basement membrane promotes folding. Additional key forces stem from the stratification and differentiation of progenitor cells. Tumour-specific suprabasal stiffness gradients are generated as oncogenic lesions progress towards malignancy, which we computationally predict will alter extensile tensions on the tumour basement membrane. The pathophysiologic ramifications of this prediction are profound. Genetically decreasing the stiffness of basement membranes increases membrane tensions in silico and potentiates the progression of invasive squamous cell carcinomas in vivo. Our findings suggest that mechanical forces-exerted from above and below progenitors of multilayered epithelia-function to shape premalignant tumour architectures and influence tumour progression.

Simple Summary Lung neuroendocrine neoplasms (NENs) are a subset of lung cancer that is difficult to diagnose. MicroRNAs (miRNAs) are small RNA molecules that are valuable markers in many cancers. In this study, we generated miRNA profiles for 55 preserved lung NEN samples (14 typical carcinoid (TC), 15 atypical carcinoid (AC), 11 small cell lung carcinoma (SCLC), and 15 large cell neuroendocrine carcinoma (LCNEC)), and randomly assigned them to either discovery or validation sets. We used machine learning and data mining algorithms to identify important miRNA that can distinguish between the types. Using the miRNAs identified with these algorithms, we were able to distinguish between carcinoids (TC and AC) and neuroendocrine carcinomas (SCLC and LCNEC) in the discovery set with 93% accuracy; in the validation set, we were able to distinguish between these groups with 100% accuracy. Using the same machine learning and data mining techniques, we also identified miRNAs that can distinguish between TC and AC, and SCLC and LCNEC, however more samples are needed to validate these findings. Lung neuroendocrine neoplasms (NENs) can be challenging to classify due to subtle histologic differences between pathological types. MicroRNAs (miRNAs) are small RNA molecules that are valuable markers in many neoplastic diseases. To evaluate miRNAs as classificatory markers for lung NENs, we generated comprehensive miRNA expression profiles from 14 typical carcinoid (TC), 15 atypical carcinoid (AC), 11 small cell lung carcinoma (SCLC), and 15 large cell neuroendocrine carcinoma (LCNEC) samples, through barcoded small RNA sequencing. Following sequence annotation and data preprocessing, we randomly assigned these profiles to discovery and validation sets. Through high expression analyses, we found that miR-21 and -375 are abundant in all lung NENs, and that miR-21/miR-375 expression ratios are significantly lower in carcinoids (TC and AC) than in neuroendocrine carcinomas (NECs; SCLC and LCNEC). Subsequently, we ranked and selected miRNAs for use in miRNA-based classification, to discriminate carcinoids from NECs. Using miR-18a and -155 expression, our classifier discriminated these groups in discovery and validation sets, with 93% and 100% accuracy. We also identified miR-17, -103, and -127, and miR-301a, -106b, and -25, as candidate markers for discriminating TC from AC, and SCLC from LCNEC, respectively. However, these promising findings require external validation due to sample size.