The rockefeller university

In the type III CRISPR-Cas immune response of prokaryotes, infection triggers the production of cyclic oligoadenylates that bind and activate proteins that contain a CARF domain(1,2). Many type III loci are associated with proteins in which the CRISPR-associated Rossman fold (CARF) domain is fused to a restriction endonuclease-like domain(3,4). However, with the exception of the well-characterized Csm6 and Csx1 ribonucleases(5,6), whether and how these inducible effectors provide defence is not known. Here we investigated a type III CRISPR accessory protein, which we name cyclic-oligoadenylate-activated single-stranded ribonuclease and single-stranded deoxyribonuclease 1 (Card1). Card1 forms a symmetrical dimer that has a large central cavity between its CRISPR-associated Rossmann fold and restriction endonuclease domains that binds cyclic tetra-adenylate. The binding of ligand results in a conformational change comprising the rotation of individual monomers relative to each other to form a more compact dimeric scaffold, in which a manganese cation coordinates the catalytic residues and activates the cleavage of single-stranded-but not double-stranded-nucleic acids (both DNA and RNA). In vivo, activation of Card1 induces dormancy of the infected hosts to provide immunity against phage infection and plasmids. Our results highlight the diversity of strategies used in CRISPR systems to provide immunity. Structural analyses of the type III CRISPR accessory protein Card1, which induces dormancy in infected hosts to provide immunity against phage infection, reveal the mechanisms by which it cleaves single-stranded RNA and DNA.

Recent advances in single-particle cryogenic electron microscopy (cryo-EM) have enabled the structural determination of numerous protein assemblies at high resolution, yielding unprecedented insights into their function. However, despite its extraordinary capabilities, cryo-EM remains time-consuming and resource-intensive. It is therefore beneficial to have a means for rapidly assessing and optimizing the quality of samples prior to lengthy cryo-EM analyses. To do this, we have developed a native mass spectrometry (nMS) platform that provides rapid feedback on sample quality and highly streamlined biochemical screening. Because nMS enables accurate mass analysis of protein complexes, it is well suited to routine evaluation of the composition, integrity, and homogeneity of samples prior to their plunge-freezing on EM grids. We demonstrate the utility of our nMS-based platform for facilitating cryo-EM studies using structural characterizations of exemplar bacterial transcription complexes as well as the replication-transcription assembly from the SARS-CoV-2 virus that is responsible for the COVID-19 pandemic.

Notch signaling is critical for controlling a variety of cell fate decisions during metazoan development and homeostasis. This unique, highly conserved signaling pathway relies on cell-to-cell contact, which triggers the proteolytic release of the cytoplasmic domain of the membrane-anchored transcription factor Notch from the membrane. A disintegrin and metalloproteinase (ADAM) proteins are crucial for Notch activation by processing its S2 site. While ADAM10 cleaves Notch1 under physiological, ligand-dependent conditions, ADAM17 mainly cleaves Notch1 under ligand-independent conditions. However, the mechanism(s) that regulate the distinct contributions of these ADAMs in Notch processing remain unclear. Using cell-based assays in mouse embryonic fibroblasts (mEFs) lacking ADAM10 and/or ADAM17, we aimed to clarify what determines the relative contributions of ADAM10 and ADAM17 to ligand-dependent or ligand-independent Notch processing. We found that EDTA-stimulated ADAM17-dependent Notch1 processing is rapid and requires the ADAM17-regulators iRhom1 and iRhom2, whereas the Delta-like 4-induced ligand-dependent Notch1 processing is slower and requires ADAM10. The selectivity of ADAM17 for EDTA-induced Notch1 processing can most likely be explained by a preference for ADAM17 over ADAM10 for the Notch1 cleavage site and by the stronger inhibition of ADAM10 by EDTA. The physiological ADAM10-dependent processing of Notch1 cannot be compensated for by ADAM17 in Adam10-/- mEFs, or by other ADAMs shown here to be able to cleave the Notch1 cleavage site, such as ADAMs9, 12, and 19. Collectively, these results provide new insights into the mechanisms underlying the substrate selectivity of ADAM10 and ADAM17 towards Notch1.

Background Bimekizumab is a monoclonal IgG1 antibody that selectively inhibits interleukin (IL)-17F in addition to IL-17A. This study investigated the efficacy and safety of bimekizumab in patients with moderate to severe plaque psoriasis, the effects of treatment withdrawal, and two maintenance dosing schedules over 56 weeks. Methods BE READY was a phase 3, multicentre, randomised, double-blind, placebo-controlled trial done at 77 sites (hospitals, clinics, private doctor's practices, and dedicated clinical research centres) in nine countries across Asia, Australia, Europe, and North America. Adult patients aged 18 years or older with moderate to severe plaque psoriasis were stratified by region and previous biologic exposure, and randomly assigned (4:1) to receive bimekizumab 320 mg every 4 weeks or placebo every 4 weeks by use of interactive response technology. Coprimary endpoints were the proportion of patients achieving 90% or greater improvement from baseline in the Psoriasis Area Severity Index (PASI90) and the proportion of patients achieving a score of 0 (clear) or 1 (almost clear) on the five-point Investigator's Global Assessment (IGA) scale at week 16 (non-responder imputation). Bimekizumab-treated patients achieving PASI90 at week 16 were re-allocated (1:1:1) to receive bimekizumab 320 mg every 4 weeks, every 8 weeks, or placebo for weeks 16-56. Efficacy analyses were done in the intention-to-treat population; the safety analysis set comprised all patients who received at least one dose of study treatment. This trial is registered with ClinicalTrials.gov (NCT03410992), and is now completed. Findings Between Feb 5, 2018, and Jan 7, 2020, 435 patients were randomly assigned to receive either bimekizumab 320 mg every 4 weeks (n=349) or placebo every 4 weeks (n=86). Coprimary endpoints were met: at week 16, 317 (91%) of 349 patients receiving bimekizumab 320 mg every 4 weeks achieved PASI90, compared with one (1%) of 86 patients receiving placebo (risk difference 89.8 [95% CI 86.1-93.4]; p<0.0001); and 323 (93%) of 349 patients receiving bimekizumab 320 mg every 4 weeks achieved an IGA score of 0 or 1 versus one (1%) of 86 patients receiving placebo (risk difference 91.5 [95% CI 88.0-94.9]; p<0.0001). Responses were maintained through to week 56 with bimekizumab 320 mg every 8 weeks and every 4 weeks. Treatment-emergent adverse events in the initial treatment period (up to week 16) were reported in 213 (61%) of 349 patients receiving bimekizumab 320 mg every 4 weeks and 35 (41%) of 86 patients receiving placebo every 4 weeks. From week 16 to week 56, treatment-emergent adverse events were reported in 78 (74%) of 106 patients receiving bimekizumab 320 mg every 4 weeks, 77 (77%) of 100 patients receiving bimekizumab 320 mg every 8 weeks, and 72 (69%) of 105 patients receiving placebo. Interpretation Bimekizumab showed high levels of response, which were durable over 56 weeks, with both maintenance dosing schedules (every 4 weeks and every 8 weeks). Moreover, bimekizumab was well tolerated, with no unexpected safety findings. Data presented here further support the therapeutic value of bimekizumab and inhibition of IL-17F in addition to IL-17A for patients with moderate to severe plaque psoriasis.

Mendelian Susceptibility to Mycobacterial diseases (MSMD) are a group of innate immune defects with more than 17 genes and 32 clinical phenotypes identified. Defects in the IFN-gamma mediated immunity lead to an increased susceptibility to intracellular pathogens like mycobacteria including attenuated Mycobacterium bovis-Bacillus Calmette-Guerin (BCG) vaccine strains and non-tuberculous environmental mycobacteria (NTM), Salmonella, fungi, parasites like Leishmania and some viruses, in otherwise healthy individuals. Mutations in the IL12R beta 1 gene are the commonest genetic defects identified. This retrospective study reports the clinical, immunological, and molecular characteristics of a cohort of 55 MSMD patients from 10 centers across India. Mycobacterial infection was confirmed by GeneXpert, Histopathology, and acid fast bacilli staining. Immunological workup included lymphocyte subset analysis, Nitro blue tetrazolium (NBT) test, immunoglobulin levels, and flow-cytometric evaluation of the IFN-gamma mediated immunity. Genetic analysis was done by next generation sequencing (NGS). Disseminated BCG-osis was the commonest presenting manifestation (82%) with a median age of presentation of 6 months due to the practice of BCG vaccination at birth. This was followed by infection with Salmonella and non-typhi Salmonella (13%), Cytomegalovirus (CMO (11%), Candida (7%), NTM (4%), and Histoplasma (2%). Thirty-six percent of patients in cohort were infected by more than one organism. This study is the largest cohort of MSMD patients reported from India to the best of our knowledge and we highlight the importance of work up for IL-12/IL-23/ISG15/IFN-gamma circuit in all patients with BCG-osis and suspected MSMD irrespective of age.

Neural network computations are usually assumed to emerge from patterns of fast electrical activity. Challenging this view, we show that a male fly's decision to persist in mating hinges on a biochemical computation that enables processing over minutes to hours. Each neuron in a recurrent network contains slightly different internal molecular estimates of mating progress. Protein kinase A (PKA) activity contrasts this internal measurement with input from the other neurons to represent accumulated evidence that the goal of the network has been achieved. When consensus is reached, PKA pushes the network toward a large-scale and synchronized burst of calcium influx that we call an eruption. Eruptions transform continuous deliberation within the network into an all-or-nothing output, after which the male will no longer sacrifice his life to continue mating. Here, biochemical activity, invisible to most large-scale recording techniques, is the key computational currency directing behavior and motivational state.

Ant colonies must assess the internal states of their members and coordinate their responses to changes in state. One important example of this is the sensing of colony hunger and the regulation of foraging behavior. In many ant species, workers' own nutritional states at least partially determine how much they forage, and poorly nourished workers usually forage more, while well-nourished workers remain inside the nest. Workers in some species, such as the clonal raider ant Ooceraea biroi, mostly forage in response to larval signals. Here, we ask whether O. biroi larvae directly affect worker nutrition, and whether nutritional states in turn regulate workers' foraging and feeding behavior. We find that larval signals do not detectably influence workers' nutritional states or feeding behavior. Unlike in most other ant species, however, when colonies forage in response to larval signals, better-nourished O. biroi workers forage more. This suggests evolutionary modifications to the nature and strength of the relationship between nutritional state and foraging behavior in some ants. Nonetheless, worker nutritional states regulate feeding behavior as expected, with workers eating in proportion to their level of food deprivation. We discuss the implications of these results for the life history of O. biroi and the evolution of foraging regulation in social insects more generally. We suggest that the decoupling of regulatory mechanisms for feeding and foraging has parallels in the evolutionary elaboration of animal multicellularity. Significance statement Foraging in social insects is a cooperative behavior: workers forage for the colony, rather than just for themselves. In most species, workers primarily use their own hunger as proxies for the colony's needs. However, some species use other sources of information. Clonal raider ants, for example, forage in response to signals from their larvae. Here, we ask whether they also forage when deprived of nutrition. Surprisingly, we find instead that they forage more when better fed, and that in unmanipulated colonies, larval signals override worker nutrition, suggesting that the regulation of foraging has been rewired in this species. We also find that workers feed in proportion to their nutrient deprivation, suggesting that the regulation of feeding has been conserved. We propose that the uncoupling of feeding and foraging machinery has parallels in the evolutionary elaboration of animal multicellularity.

Despite the strong genetic basis of psychiatric disorders, the underlying molecular mechanisms are largely unmapped. RNA-binding proteins (RBPs) are responsible for most post-transcriptional regulation, from splicing to translation to localization. RBPs thus act as key gatekeepers of cellular homeostasis, especially in the brain. However, quantifying the pathogenic contribution of noncoding variants impacting RBP target sites is challenging. Here, we leverage a deep learning approach that can accurately predict the RBP target site dysregulation effects of mutations and discover that RBP dysregulation is a principal contributor to psychiatric disorder risk. RBP dysregulation explains a substantial amount of heritability not captured by large-scale molecular quantitative trait loci studies and has a stronger impact than common coding region variants. We share the genome-wide profiles of RBP dysregulation, which we use to identify DDHD2 as a candidate schizophrenia risk gene. This resource provides a new analytical framework to connect the full range of RNA regulation to complex disease.

Two professional societies recently published opinions on the clinical management of "mosaic" results from preimplantation genetic testing for aneuploidy (PGT-A) in human blastocyst-stage embryos in associations with in vitro fertilization (IVF). We here point out three principal shortcomings: (i) Though a most recent societal opinion states that it should not be understood as an endorsement of the use of PGT-A, any discussion of how PGT-A should be clinically interpreted for all practical purposes does offer such an endorsement. (ii) The same guideline derived much of its opinion from a preceding guidance in favor of utilization of PGT-A that did not follow even minimal professional requirements for establishment of practice guidelines. (iii) Published guidelines on so-called "mosaic" embryos from both societies contradict basic biological characteristics of human preimplantation-stage embryos. They, furthermore, are clinically unvalidated and interpret results of a test, increasingly seen as harmful to IVF outcomes for many infertile women. Qualified professional organizations, therefore, should finally offer transparent guidelines about the utilization of PGT-A in association with IVF in general.

Focal segmental glomerulosclerosis (FSGS) is the main pathology underlying steroid-resistant nephrotic syndrome (SRNS) and a leading cause of chronic kidney disease. Monogenic forms of pediatric SRNS are predominantly caused by recessive mutations, while the contribution of de novo variants (DNVs) to this trait is poorly understood. Using exome sequencing (ES) in a proband with FSGS/SRNS, developmental delay, and epilepsy, we discovered a nonsense DNV in TRIM8, which encodes the E3 ubiquitin ligase tripartite motif containing 8. To establish whether TRIM8 variants represent a cause of FSGS, we aggregated exome/genome-sequencing data for 2,501 pediatric FSGS/SRNS-affected individuals and 48,556 control subjects, detecting eight heterozygous TRIM8 truncating variants in affected subjects but none in control subjects (p = 3.28 3 10(-11)). In all six cases with available parental DNA, we demonstrated de novo inheritance (p = 2.21 3 10(-15)). Reverse phenotyping revealed neurodevelopmental disease in all eight families. We next analyzed ES from 9,067 individuals with epilepsy, yielding three additional families with truncating TRIM8 variants. Clinical review revealed FSGS in all. All TRIM8 variants cause protein truncation clustering within the last exon between residues 390 and 487 of the 551 amino acid protein, indicating a correlation between this syndrome and loss of the TRIM8 C-terminal region. Wild-type TRIM8 overexpressed in immortalized human podocytes and neuronal cells localized to nuclear bodies, while constructs harboring patient-specific variants mislocalized diffusely to the nucleoplasm. Co-localization studies demonstrated that Gemini and Cajal bodies frequently abut a TRIM8 nuclear body. Truncating TRIM8 DNVs cause a neuro-renal syndrome via aberrant TRIM8 localization, implicating nuclear bodies in FSGS and developmental brain disease.