The rockefeller university

Vimentin intermediate filaments (VIFs) form complex, tightly packed networks; due to this density, traditional imaging approaches cannot discern single-filament behavior. To address this, we developed and validated a sparse vimentin-SunTag labeling strategy, enabling single-particle tracking of individual VIFs and providing a sensitive, unbiased, and quantitative method for measuring global VIF motility. Using this approach, we define the steady-state VIF motility rate, showing a constant similar to 8% of VIFs undergo directed microtubule-based motion irrespective of subcellular location or local filament density. Significantly, our single-particle tracking approach revealed uncorrelated motion of individual VIFs within bundles, an observation seemingly at odds with conventional models of tightly cross-linked bundles. To address this, we acquired high-resolution focused ion beam scanning electron microscopy volumes of vitreously frozen cells and reconstructed three-dimensional VIF bundles, finding that they form only loosely organized, semi-coherent structures from which single VIFs frequently emerge to locally engage neighboring microtubules. Overall, this work demonstrates single VIF dynamics and organization in the cellular milieu for the first time.

Early antibody therapy can prevent severe SARS-CoV-2 infection (COVID-19). However, the effectiveness of COVID-19 convalescent plasma (CCP) therapy in treating severe COVID-19 remains inconclusive. To test a hypothesis that some CCP units are associated with a coagulopathy hazard in severe disease that offsets its benefits, we tracked 304 CCP units administered to 414 hospitalized COVID-19 patients to assess their association with the onset of unfavorable post-transfusion D-dimer trends. CCP recipients with increasing or persistently elevated D-dimer trajectories after transfusion experienced higher mortality than those whose D-dimer levels were persistently low or decreasing after transfusion. Within the CCP donor-recipient network, recipients with increasing or persistently high D-dimer trajectories were skewed toward association with a minority of CCP units. In in vitro assays, CCP from "higher-risk" units had higher cross-reactivity with the spike protein of human seasonal betacoronavirus OC43. "Higher-risk" CCP units also mediated greater Fc gamma receptor IIa signaling against cells expressing SARS-CoV-2 spike compared with "lower-risk" units. This study finds that post-transfusion activation of coagulation pathways during severe COVID-19 is associated with specific CCP antibody profiles and supports a potential mechanism of immune complex-activated coagulopathy.

Variants with large effect contribute to congenital heart disease (CHD). To date, recessive genotypes (RGs) have commonly been implicated through anecdotal ascertainment of consanguineous families and candidate gene-based analysis; the recessive contribution to the broad range of CHD phenotypes has been limited. We analyzed whole exome sequences of 5,424 CHD probands. Rare damaging RGs were estimated to contribute to at least 2.2% of CHD, with greater enrichment among laterality phenotypes (5.4%) versus other subsets (1.4%). Among 108 curated human recessive CHD genes, there were 66 RGs, with 54 in 11 genes with >1 RG, 12 genes with 1 RG, and 85 genes with zero. RGs were more prevalent among offspring of consanguineous union (4.7%, 32/675) than among nonconsanguineous probands (0.7%, 34/4749). Founder variants in GDF1 and PLD1 accounted for 74% of the contribution of RGs among 410 Ashkenazi Jewish probands. We identified genome-wide significant enrichment of RGs in C1orf127, encoding a likely secreted protein expressed in embryonic mouse notochord and associated with laterality defects. Single-cell transcriptomes from gastrulation-stage mouse embryos revealed enrichment of RGs in genes highly expressed in the cardiomyocyte lineage, including contractility-related genes MYH6, UNC45B, MYO18B, and MYBPC3 in probands with left-sided CHD, consistent with abnormal contractile function contributing to these malformations. Genes with significant RG burden account for 1.3% of probands, more than half the inferred total. These results reveal the recessive contribution to CHD, and indicate that many genes remain to be discovered, with each likely accounting for a very small fraction of the total.

The ongoing emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that reduce the effectiveness of antibody therapeutics necessitates development of next-generation antibody modalities that are resilient to viral evolution. Here, we characterized amino-terminal domain (NTD)- and receptor binding domain (RBD)-specific monoclonal antibodies previously isolated from coronavirus disease 2019 (COVID-19) convalescent donors for their activity against emergent SARS-CoV-2 VOCs. Among these, the NTD-specific antibody C1596 displayed the greatest breadth of binding to VOCs, with cryo-electron microscopy structural analysis revealing recognition of a distinct NTD epitope outside of the site i antigenic supersite. Given C1596's favorable binding profile, we designed a series of bispecific antibodies (bsAbs), termed CoV2-biRNs, that featured both NTD and RBD specificities. Two of the C1596-inclusive bsAbs, CoV2-biRN5 and CoV2-biRN7, retained potent in vitro neutralization activity against all Omicron variants tested, including XBB.1.5, BA.2.86, and JN.1, contrasting the diminished potency of parental antibodies delivered as monotherapies or as a cocktail. Furthermore, prophylactic delivery of CoV2-biRN5 reduced the viral load within the lungs of K18-hACE2 mice after challenge with SARS-CoV-2 XBB.1.5. In conclusion, NTD-RBD bsAbs offer promising potential for the design of resilient, next-generation antibody therapeutics against SARS-CoV-2 VOCs.

Inborn deficiencies of the alternative pathway (AP) of the complement system have been associated with life-threatening infections, mainly by encapsulated bacteria. Complete factor D (FD) deficiencies have been reported in only seven families in the literature. We report two new cases of biochemically and genetically confirmed complete FD deficiency, including the first in a Down syndrome patient. The index cases respectively suffered from severe H. influenza and N. meningitidis infections. Their FD activity was undetectable but was restored by adding recombinant human FD. FD levels were undetectable in the plasma of both patients using ELISA. Genetic analysis of the CFD gene identified a homozygous missense variant p.M40R in one patient, and compound heterozygous variants-a nonsense mutation p.Cys148* and a splice site variant c.212+2T>G-in the other. Patients with Down syndrome are more susceptible to infections, but this case highlights the importance of investigating the complement system, particularly the AP, even in those with Down syndrome or other secondary immune deficiencies. A familial study should follow if a congenital deficiency is found. The natural history of patients with inherited complete FD deficiency underscores the necessity of preventive measures against encapsulated bacteria for those receiving therapeutic MASP-3 or FD inhibitors.

Multiplexed imaging is a powerful approach in spatial biology, although it is complex, expensive and labor-intensive. Here, we present the IBEX Knowledge-Base, a central resource for reagents, protocols and more, to enhance knowledge sharing, optimization and innovation of spatial proteomics techniques.

Mycobacterium tuberculosis (Mtb), a leading cause of death from infection, completes its life cycle entirely in humans except for transmission through the air. To begin to understand how Mtb survives aerosolization, we mimicked liquid and atmospheric conditions experienced by Mtb before and after exhalation using a model aerosol fluid (MAF) based on the water- soluble, lipidic, and cellular constituents of necrotic tuberculosis lesions. MAF induced drug tolerance in Mtb, remodeled its transcriptome, and protected Mtb from dying in microdroplets desiccating in air. Yet survival was not passive: Mtb appeared to rely on hundreds of genes to survive conditions associated with transmission. Essential genes subserving proteostasis offered most protection. A large number of conventionally nonessential genes appeared to contribute as well, including genes encoding proteins that resemble antidesiccants. The candidate transmission survival genome of Mtb may offer opportunities to reduce transmission of tuberculosis.

The intestinal immune system must concomitantly tolerate food and commensals and protect against pathogens. Antigen-presenting cells (APCs) orchestrate these immune responses by presenting luminal antigens to CD4+ T cells and inducing their differentiation into regulatory (peripheral regulatory T cell) or inflammatory [T helper (Th) cell] subsets. We used a proximity labeling method (LIPSTIC) to identify APCs that presented dietary antigens under tolerizing and inflammatory conditions and to understand cellular mechanisms by which tolerance to food is induced and can be disrupted by infection. Helminth infections disrupted tolerance induction proportionally to the reduction in the ratio between tolerogenic APCs-including migratory dendritic cells (cDC1s) and Ror gamma t+ APCs-and inflammatory APCs, which were primarily cDC2s. These inflammatory cDC2s expanded by helminth infection did not present dietary antigens, thus avoiding diet-specific Th2 responses.

Males of many species have evolved behavioral traits to both attract females and repel rivals. Here, we explore mate selection in Drosophila from both the male and female perspective to shed light on how these key components of sexual selection-female choice and male-male competition-work in concert to guide reproductive strategies. We find that male flies fend off competing suitors by interleaving their courtship of a female with aggressive wing flicks, which both repel competitors and generate a "song"that obscures the female's auditory perception of other potential mates. Two higher-order circuit nodes-P1a and pC1x neurons-are coordinately recruited to allow males to flexibly interleave these agonistic actions with courtship displays, assuring they persistently pursue females until their rival falters. Together, our results suggest that female mating decisions are shaped by male-male interactions, underscoring how a male's ability to subvert his rivals is central to his reproductive success.

Changing a circuit dynamically, without actually changing the hardware itself, is called reconfiguration, and is of great importance due to its manifold technological applications. Circuit reconfiguration appears to be a feature of the cerebral cortex, so understanding the dynamical principles underlying self-reconfiguration may prove of import to elucidate brain function. We present a very simple example of dynamical reconfiguration: a family of networks whose signal pathways can be switched on the fly, only through use of their inputs, with no changes to their synaptic weights. These are single-layer convolutional recurrent network with local unitary synaptic weights and a smooth sigmoidal activation function. We generate traveling waves using the high spatiotemporal frequencies of the input, and we use the low spatiotemporal frequencies of the input to landscape the ongoing activity, channeling said traveling waves through an input-specified spatial pattern. This mechanism uses inherent properties of marginally stable, dynamically critical systems, which are a direct consequence of their unitary convolution kernels: every network in the family can do this. We show these networks solve the classical connectedness detection problem, by allowing signal propagation only along the regions to be evaluated for connectedness, and forbidding it elsewhere.