The rockefeller university

The food colorant Red 40 is an environmental risk factor for colitis development in mice with increased expression of interleukin (IL)-23. This immune response is mediated by CD4(+) T cells, but mechanistic insights into how these CD4(+) T cells trigger and perpetuate colitis have remained elusive. Here, using single-cell transcriptomic analysis, we found that several CD4(+) T-cell subsets are present in the intestines of colitic mice, including an interferon (IFN)-gamma-producing subset. In vivo challenge of primed mice with Red 40 promoted rapid activation of CD4(+) T cells and caused marked intestinal epithelial cell (IEC) apoptosis that was attenuated by depletion of CD4(+) cells and blockade of IFN-gamma. Ex vivo experiments showed that intestinal CD4(+) T cells from colitic mice directly promoted apoptosis of IECs and intestinal enteroids. CD4(+) T cell-mediated cytotoxicity was contact-dependent and required FasL, which promoted caspase-dependent cell death in target IECs. Genetic ablation of IFN-gamma constrained IL-23- and Red 40-induced colitis development, and blockade of IFN-gamma inhibited epithelial cell death in vivo. These results advance the understanding of the mechanisms regulating colitis development caused by IL-23 and food colorants and identify IFN-gamma(+) cytotoxic CD4(+) T cells as a new potential therapeutic target for colitis.

Biomolecular condensation constitutes an emerging mechanism for transcriptional regulation. Recent studies suggest that the co-condensation between transcription factors (TFs) and DNA can generate mechanical forces driving genome rearrangements. However, the reported forces generated by protein-DNA co-condensation are typically below one piconewton (pN), questioning its physiological significance. Moreover, the force-generating capacity of these condensates in the chromatin context remains unknown. Here, we show that Sox2, a nucleosome-binding pioneer TF, forms co-condensates with DNA and generates forces up to 7 pN, exerting considerable mechanical tension on DNA strands. We find that the disordered domains of Sox2 are required for maximum force generation but not for condensate formation. Furthermore, we show that nucleosomes dramatically attenuate the mechanical stress exerted by Sox2 by sequestering it from coalescing on bare DNA. Our findings reveal that TF-mediated DNA condensation can exert significant mechanical stress on the genome which can nonetheless be attenuated by the chromatin architecture. Here the authors used single-molecule imaging and manipulation to study the mechanical effects of transcription factor Sox2 co-condensation with DNA and chromatin. They found that Sox2 condensates exert a high level of mechanical stress on DNA, but this stress is dramatically attenuated by nucleosomes assembled on the DNA.

High-level expression of the transcription factor T-bet characterizes a phenotypically distinct murine B cell population known as "age-associated B cells" (ABCs). T-bet-deficient mice have reduced ABCs and impaired humoral immunity. We describe a patient with inherited T-bet deficiency and largely normal humoral immunity including intact somatic hypermutation, affinity maturation and memory B cell formation in vivo, and B cell differentiation into Ig-producing plasmablasts in vitro. Nevertheless, the patient exhibited skewed class switching to IgG1, IgG4, and IgE, along with reduced IgG2, both in vivo and in vitro. Moreover, T-bet was required for the in vivo and in vitro development of a distinct subset of human B cells characterized by reduced expression of CD21 and the concomitantly high expression of CD19, CD20, CD11c, FCRL5, and T-bet, a phenotype that shares many features with murine ABCs. Mechanistically, human T-bet governed CD21(lo)CD11C(hi) B cell differentiation by controlling the chromatin accessibility of lineage-defining genes in these cells: FAS, IL21R, SEC61B, DUSP4, DAPP1, SOX5, CD79B, and CXCR4. Thus, human T-bet is largely redundant for long-lived protective humoral immunity but is essential for the development of a distinct subset of human CD11c(hi)CD21(lo) B cells.

gamma delta T cells represent a substantial fraction of intestinal lymphocytes at homeostasis, but they also constitute a major lymphocyte population infiltrating colorectal cancers (CRCs); however, their temporal contribution to CRC development or progression remains unclear. Using human CRC samples and murine CRC models, we found that most gamma delta T cells in premalignant or nontumor colons exhibit cytotoxic markers, whereas tumor-infiltrating gamma delta T cells express a protumorigenic profile. These contrasting T cell profiles were associated with distinct T cell receptor (TCR)-V gamma delta gene usage in both humans and mice. Longitudinal intersectional genetics and antibody-dependent strategies targeting murine gamma delta T cells enriched in the epithelium at steady state led to heightened tumor development, whereas targeting gamma delta subsets that accumulate during CRC resulted in reduced tumor growth. Our results uncover temporal pro- and antitumor roles for gamma delta T cell subsets.

Single-nuclei RNA sequencing characterizes cell types at the gene level. However, compared to single-cell approaches, many single-nuclei cDNAs are purely intronic, lack barcodes and hinder the study of isoforms. Here we present single-nuclei isoform RNA sequencing (SnISOr-Seq). Using microfluidics, PCR-based artifact removal, target enrichment and long-read sequencing, SnISOr-Seq increased barcoded, exon-spanning long reads 7.5-fold compared to naive long-read single-nuclei sequencing. We applied SnISOr-Seq to adult human frontal cortex and found that exons associated with autism exhibit coordinated and highly cell-type-specific inclusion. We found two distinct combination patterns: those distinguishing neural cell types, enriched in TSS-exon, exon-polyadenylation-site and non-adjacent exon pairs, and those with multiple configurations within one cell type, enriched in adjacent exon pairs. Finally, we observed that human-specific exons are almost as tightly coordinated as conserved exons, implying that coordination can be rapidly established during evolution. SnISOr-Seq enables cell-type-specific long-read isoform analysis in human brain and in any frozen or hard-to-dissociate sample.

This paper describes the plasma and memory antibody response in a cohort of SARS-CoV-2 Gamma-infected individuals in Brazil. Potent antibody neutralization was shown to be limited to Gamma and Beta, and epitope recognition skewed to Class 3 epitopes. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a global problem in part because of the emergence of variants of concern that evade neutralization by antibodies elicited by prior infection or vaccination. Here we report on human neutralizing antibody and memory responses to the Gamma variant in a cohort of hospitalized individuals. Plasma from infected individuals potently neutralized viruses pseudotyped with Gamma SARS-CoV-2 spike protein, but neutralizing activity against Wuhan-Hu-1-1, Beta, Delta, or Omicron was significantly lower. Monoclonal antibodies from memory B cells also neutralized Gamma and Beta pseudoviruses more effectively than Wuhan-Hu-1. 69% and 34% of Gamma-neutralizing antibodies failed to neutralize Delta or Wuhan-Hu-1. Although Class 1 and 2 antibodies dominate the response to Wuhan-Hu-1 or Beta, 54% of antibodies elicited by Gamma infection recognized Class 3 epitopes. The results have implications for variant-specific vaccines and infections, suggesting that exposure to variants generally provides more limited protection to other variants.

The Hippo signaling pathway acts as a brake on regeneration in many tissues. This cascade of kinases culminates in the phosphorylation of the transcriptional cofactors Yap and Taz, whose concentration in the nucleus consequently remains low. Various types of cellular signals can reduce phosphorylation, however, resulting in the accumulation of Yap and Taz in the nucleus and subsequently in mitosis. We earlier identified a small molecule, TRULI, that blocks the final kinases in the pathway, Lats1 and Lats2, and thus elicits proliferation of several cell types that are ordinarily postmitotic and aids regeneration in mammals In the present study, we present the results of chemical modification of the original compound and demonstrate that a derivative, TDI-011536, is an effective blocker of Lats kinases in vitro at nanomolar concentrations. The compound fosters extensive proliferation in retinal organoids derived from human induced pluripotent stem cells. Intraperitoneal administration of the substance to mice suppresses Yap phosphorylation for several hours and induces transcriptional activation of Yap target genes in the heart, liver, and skin. Moreover, the compound initiates the proliferation of cardiomyocytes in adult mice following cardiac cryolesions. After further chemical refinement, related compounds might prove useful in protective and regenerative therapies.

Sensitive and accurate RT-qPCR tests are the primary diagnostic tools to identify SARS-CoV-2-infected patients. While many SARS-CoV-2 RT-qPCR tests are available, there are significant differences in test sensitivity, workflow (e.g. hands-on-time), gene targets and other functionalities that users must consider. Several publicly available protocols shared by reference labs and public health authorities provide useful tools for SARS-CoV-2 diagnosis, but many have shortcomings related to sensitivity and laborious workflows. Here, we describe a series of SARS-CoV-2 RT-qPCR tests that are originally based on the protocol targeting regions of the RNA-dependent RNA polymerase (RdRp) and envelope (E) coding genes developed by the Charite Berlin. We redesigned the primers/probes, utilized locked nucleic acid nucleotides, incorporated dual probe technology and conducted extensive optimizations of reaction conditions to enhance the sensitivity and specificity of these tests. By incorporating an RNase P internal control and developing multiplexed assays for distinguishing SARS-CoV-2 and influenza A and B, we streamlined the workflow to provide quicker results and reduced consumable costs. Some of these tests use modified enzymes enabling the formulation of a room temperature-stable master mix and lyophilized positive control, thus increasing the functionality of the test and eliminating cold chain shipping and storage. Moreover, a rapid, RNA extraction-free version enables high sensitivity detection of SARS-CoV-2 in about an hour using minimally invasive, self-collected gargle samples. These RT-qPCR assays can easily be implemented in any diagnostic laboratory and can provide a powerful tool to detect SARS-CoV-2 and the most common seasonal influenzas during the vaccination phase of the pandemic.

Objective: Lowering serum phosphorus in people on hemodialysis may improve their survival. However, prior studies have shown that restricting dietary protein intake, a major source of phosphorus, is associated with higher mortality. We hypothesized that a novel metric that incorporates both these values commensurately can improve survival prediction. Methods: We used serum phosphorous and normalized protein catabolic rate (nPCR), a surrogate of dietary protein intake, to form a new metric R that was used to examine the associations with mortality in 63,016 people on hemodialysis (HD) of one year after treatment initiation. Survival models were adjusted for case-mix, malnutrition-inflammation cachexia syndrome (MICS), and residual kidney function (RKF). Results: Individuals treated with hemodialysis were divided into five groups in accordance with R value. Group 1 included sick individuals with high phosphorous and low nPCR. Group 5 included individuals with low phosphorous and high nPCR. After 1-year follow-up, survival difference between the groups reflected R value, where an increase in R was associated with improved survival. The association of R with mortality was strengthened by adjustment in demographic variables and attenuated after adjustment to MICS. Mortality associations in accordance with R were not influenced by residual kidney function (RKF). Conclusion: The novel protein to phosphorus ratio score R predicts mortality in people on dialysis, probably reflecting both nutrition and inflammation state independent of RKF. The metric enables better phosphorus monitoring, although adequate dietary protein intake is ensured and may improve the prediction of outcomes in the clinical setting. (C) 2021 by the National Kidney Foundation, Inc. All rights reserved.

Pattern recognition receptors (PRRs) protect against microbial invasion by de-tecting specific molecular patterns found in pathogens and initiating an immune response. Although microbial-derived PRR ligands have been extensively charac-terized, the contribution and relevance of endogenous ligands to PRR activation remains overlooked. Here, we characterize the landscape of endogenous ligands that engage RIG-I-like receptors (RLRs) upon infection by different RNA viruses. In each infection, several RNAs transcribed by RNA polymerase III (Pol3) specif-ically engaged RLRs, particularly the family of Y RNAs. Sensing of Y RNAs was dependent on their mimicking of viral secondary structure and their 5'-triphos-phate extremity. Further, we found that HIV-1 triggered a VPR-dependent down -regulation of RNA triphosphatase DUSP11 in vitro and in vivo, inducing a tran-scriptome-wide change of cellular RNA 5'-triphosphorylation that licenses Y RNA immunogenicity. Overall, our work uncovers the contribution of endoge-nous RNAs to antiviral immunity and demonstrates the importance of this pathway in HIV-1 infection.