The rockefeller university

Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.

The world is totally dependent on medications. As science progresses, new, better, and cheaper drugs are needed more than ever. The pharmaceutical industry has been predominantly dependent on high-throughput screening (HTS) for the past three decades. Considering that the discovery rate has been relatively constant, can one hope for a much-needed sudden trend uptick? DNA-encoded libraries (DELs) and similar technologies, that have several orders of magnitude more screening power than HTS, and that we propose to group together under the umbrella term of high-power screening (HPS), are very well positioned to do exactly that. HPS also offers novel screening options such as parallel screening, ex vivo and in vivo screening, as well as a new path to druggable alternatives such as proteolysis targeting chimeras (PROTACs). Altogether, HPS unlocks novel powerful drug discovery avenues.

IMPORTANCE Ichthyoses are clinically and genetically heterogeneous disorders characterized by scaly skin. Despite decades of investigation identifying pathogenic variants in more than 50 genes, clear genotype-phenotype associations have been difficult to establish. OBJECTIVE To expand the genotypic and phenotypic spectra of ichthyosis and delineate genotype-phenotype associations. DESIGN, SETTING, AND PARTICIPANTS This cohort study recruited an international group of individuals with ichthyosis and describes characteristic and distinguishing features of common genotypes, including genotype-phenotype associations, during a 10-year period from June 2011 to July 2021. Participants of all ages, races, and ethnicities were included and were enrolled worldwide from referral centers and patient advocacy groups. A questionnaire to assess clinical manifestations was completed by those with a genetic diagnosis. MAIN OUTCOMES AND MEASURES Genetic analysis of saliva or blood DNA, a phenotyping questionnaire, and standardized clinical photographs. Descriptive statistics, such as frequency counts, were used to describe the cases in the cohort. Fisher exact tests identified significant genotype-phenotype associations. RESULTS Results were reported for 1000 unrelated individuals enrolled from around the world (mean [SD] age, 50.0 [34.0] years; 524 [52.4%] were female, 427 [42.7%] were male, and 49 [4.9%] were not classified); 75% were from the US, 12% from Latin America, 4% from Canada, 3% from Europe, 3% from Asia, 2% from Africa, 1% from the Middle East, and 1% from Australia and New Zealand. A total of 266 novel disease-associated variants in 32 genes were identified among 869 kindreds. Of these, 241 (91%) pathogenic variants were found through multiplex amplicon sequencing and 25 (9%) through exome sequencing. Among the 869 participants with a genetic diagnosis, 304 participants (35%) completed the phenotyping questionnaire. Analysis of clinical manifestations in these 304 individuals revealed that pruritus, hypohydrosis, skin pain, eye problems, skin odor, and skin infections were the most prevalent self-reported features. Genotype-phenotype association analysis revealed that the presence of a collodion membrane at birth (odds ratio [OR], 6.7; 95% CI, 3.0-16.7; P<.001), skin odor (OR, 2.8; 95% CI, 1.1-6.8; P=.02), hearing problems (OR, 2.9; 95% CI, 1.6-5.5; P<.001), eye problems (OR, 3.0; 95% CI, 1.5-6.0; P<.001), and alopecia (OR, 4.6; 95% CI, 2.4-9.0; P<.001) were significantly associated with TGM1 variants compared with other ichthyosis genotypes studied. Skin pain (OR, 6.8; 95% CI, 1.6-61.2; P=.002), odor (OR, 5.7; 95% CI, 2.0-19.7; P<.001), and infections (OR, 3.1; 95% CI, 1.4-7.7; P=.03) were significantly associated with KRT10 pathogenic variants compared with disease-associated variants in other genes that cause ichthyosis. Pathogenic variants were identified in 869 (86.9%) participants. Most of the remaining individuals had unique phenotypes, enabling further genetic discovery. CONCLUSIONS AND RELEVANCE This cohort study expands the genotypic and phenotypic spectrum of ichthyosis, establishing associations between clinical manifestations and genotypes. Collectively, the findings may help improve clinical assessment, assist with developing customized management plans, and improve clinical course prognostication.

Chronic mucocutaneous candidiasis is a genetically heterogeneous group of disorders, which are characterised by chronic or recurrent non-invasive skin, nail and mucous membrane infections caused by Candida. The increased susceptibility to Candida infections is due to a Th17-cell mediated immune detect with different gene mutations in the background. The isolated form of the disorder, referred to as chronic mucocutaneous candidiasis, presents primarily or only with mucocutaneous candidiasis. In contrast, the syndromic form of the disorder is characterised, besides the non-invasive Candida infections, by autoimmune disorders, which most commonly affect the endocrine system. Genetic tests are important in confirming the diagnosis, which in affected families would provide the opportunity for prenatal genetic testing. The authors present the main types of chronic mucocutaneous candidiasis, exploring the clinical aspects, diagnostic methods, and available therapies. Furthermore, the authors conclude the molecular genetic background and the currently known pathomechanism of the disorder.

The adenosine triphosphate (ATP) analog ATP gamma S often greatly slows or prevents enzymatic ATP hydrolysis. The eukaryotic CMG (Cdc45, Mcm2 to 7, GINS) replicative helicase is presumed unable to hydrolyze ATP gamma S and thus unable to perform DNA unwinding, as documented for certain other helicases. Consequently, ATP gamma S is often used to "preload" CMG onto forked DNA substrates without unwinding before adding ATP to initiate helicase activity. We find here that CMG does hydrolyze ATP gamma S and couples it to DNA unwinding. Indeed, the rate of unwinding of a 20- and 30-mer duplex fork of different sequences by CMG is only reduced 1- to 1.5-fold using ATP gamma S compared with ATP. These findings imply that a conformational change is the rate-limiting step during CMG unwinding, not hydrolysis. Instead of using ATP gamma S for loading CMG onto DNA, we demonstrate here that nonhydrolyzable adenylyl-imidodiphosphate (AMP-PNP) can be used to preload CMG onto a forked DNA substrate without unwinding.

Germinal centers (GCs) are microanatomical sites of B cell clonal expansion and antibody affinity maturation. Therein, B cells undergo the Darwinian process of somatic diversification and affinity-driven selection of immunoglobulins that produces the high-affinity antibodies essential for effective humoral immunity. Here, we review recent developments in the field of GC biology, primarily as it pertains to GCs induced by infection or immunization. First, we summarize the phenotype and function of the different cell types that compose the GC, focusing on GC B cells. Then, we review the cellular and molecular bases of affinity-dependent selection within the GC and the export of memory and plasma cells. Finally, we present an overview of the emerging field of GC clonal dynamics, focusing on how GC and post-GC selection shapes the diversity of antibodies secreted into serum.

The drivers of critical coronavirus disease 2019 (COVID-19) remain unknown. Given major confounding factors such as age and comorbidities, true mediators of this condition have remained elusive. We used a multi-omics analysis combined with artificial intelligence in a young patient cohort where major comorbidities were excluded at the onset. The cohort included 47 "critical" (in the intensive care unit under mechanical ventilation) and 25 "non-critical" (in a non-critical care ward) patients with COVID-19 and 22 healthy individuals. The analyses included whole-genome sequencing, whole-blood RNA sequencing, plasma and blood mononuclear cell proteomics, cytokine profiling, and high-throughput immunophenotyping. An ensemble of machine learning, deep learning, quantum annealing, and structural causal modeling were used. Patients with critical COVID-19 were characterized by exacerbated inflammation, perturbed lymphoid and myeloid compartments, increased coagulation, and viral cell biology. Among differentially expressed genes, we observed up-regulation of the metalloprotease ADAM9. This gene signature was validated in a second independent cohort of 81 critical and 73 recovered patients with COVID-19 and was further confirmed at the transcriptional and protein level and by proteolytic activity. Ex vivo ADAM9 inhibition decreased severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake and replication in human lung epithelial cells. In conclusion, within a young, otherwise healthy, cohort of individuals with COVID-19, we provide the landscape of biological perturbations in vivo where a unique gene signature differentiated critical from non-critical patients. We further identified ADAM9 as a driver of disease severity and a candidate therapeutic target.

Mouse kidney parvovirus (MKPV), a newly identified parvovirus of the genus Chaphamaparvovirus, causes inclusion body nephropathy in severely immunocompromised mice and is prevalent in research mouse colonies. As nonenveloped viruses, mammalian parvoviruses are stable and generally resist thermal inactivation; however, as a novel and highly divergent parvovirus, the thermal stability of MKPV is undefined. This study aimed to evaluate the ability of cage sanitization in a mechanical washer to eliminate MKPV. Cages contaminated by MKPV-infected mice were assigned to 1 of 3 treatment groups: 1) control (bedding change only); 2) sanitization in a tunnel washer (88 degrees C final rinse for 20 s); or 3) sanitization in a tunnel washer followed by autoclave sterilization (121 degrees C for 20 min). The presence of MKPV on the cage's interior surface was assessed by PCR of cage swab extracts collected before and after cage treatment. After treatment and swabbing, each cage housed 4 MKPV-negative CD1 mice. Each group of naive CD1 mice was assigned to one of the treatment groups and was housed in a cage from this group for two, 1 wk periods. At 12, 17, and 20 wk after the first exposure, renal tissue was collected from 1 test mouse per cage and assessed for MKPV by PCR. MKPV was detected by PCR on the surface of 63% of the pretreatment cages. All cages sanitized in a tunnel washer with or without sterilization were PCR negative after treatment. Seven of 10 mice housed in untreated cages contained a mouse positive for MKPV by 20 wk after exposure. None of the mice housed in cages sanitized in a tunnel washer with or without sterilization tested positive for MKPV at any time point. This study indicates that MKPV contaminated caging can result in MKPV infection of mice, and the use of a tunnel washer at the temperature and duration evaluated was sufficient to remove MKPV nucleic acid and prevent MKPV transmission.

HIV-1 infection produces a long-lived reservoir of latently infected CD4(+) T cells that represents the major barrier to HIV-1 cure. The reservoir contains both intact and defective proviruses, but only the proviruses that are intact can reinitiate infection upon cessation of antiretroviral therapy (ART). Here we combine four-color quantitative PCR and next-generation sequencing (Q4PCR) to distinguish intact and defective proviruses and measure reservoir content longitudinally in 12 infected individuals. Q4PCR differs from other PCR-based methods in that the amplified proviruses are sequence verified as intact or defective. Samples were collected systematically over the course of up to 10 y beginning shortly after the initiation of ART. The size of the defective reservoir was relatively stable with minimal decay during the 10-y observation period. In contrast, the intact proviral reservoir decayed with an estimated half-life of 4.9 y. Nevertheless, both intact and defective proviral reservoirs are dynamic. As a result, the fraction of intact proviruses found in expanded clones of CD4(+) T cells increases over time with a concomitant decrease in overall reservoir complexity. Thus, reservoir decay measurements by Q4PCR are quantitatively similar to viral outgrowth assay (VOA) and intact proviral DNA PCR assay (IPDA) with the addition of sequence information that distinguishes intact and defective proviruses and informs reservoir dynamics. The data are consistent with the notion that intact and defective proviruses are under distinct selective pressure, and that the intact proviral reservoir is progressively enriched in expanded clones of CD4(+) T cells resulting in diminishing complexity over time.