B cells have been engineered ex vivo to express an HIV-1 broadly neutralizing antibody (bNAb). B-cell reprograming may be scientifically and therapeutically useful, but current approaches limit B-cell repertoire diversity and disrupt the organization of the heavy-chain locus. A more diverse and physiologic B-cell repertoire targeting a key HIV-1 epitope could facilitate evaluation of vaccines designed to elicit bNAbs, help identity more potent and bioavailable bNAb variants, or directly enhance viral control in vivo. Here we address the challenges of generating such a repertoire by replacing the heavy-chain CDR3 (HCDR3) regions of primary human B cells. To do so, we identified and utilized an uncharacterized Cas12a ortholog that recognizes PAM motifs present in human and murine JH genes. We also optimized the design of 200 nucleotide homology-directed repair templates (HDRT) by minimizing the required 3'-5' resection of the HDRT-complementary strand. Using these techniques, we edited primary human B cells to express a hemagglutinin epitope tag and the HCDR3 regions of the bNAbs PG9 and CH01. Those edited with bNAb HCDR3 efficiently bound trimeric HIV-1 antigens, implying they could affinity mature in vivo in response to the same antigens. This approach generates diverse B-cell repertoires recognizing a key HIV-1 neutralizing epitope.

The mechanisms that determine the final topology of skeletal muscles remain largely unknown. We have been developing Drosophila body wall musculature as a model to identify and characterize the pathways that control muscle size, shape, and orientation during embryogenesis (Johnson et al., 2013; Williams et al., 2015; Yang et al., 2020a; Yang et al., 2020b). Our working model argues muscle morphogenesis is regulated by (1) extracellular guidance cues that direct muscle cells toward muscle attachment sites, and (2) contact dependent interactions between muscles and tendons. While we have identified several pathways that regulate muscle morphogenesis, our understanding is far from complete. Here we report the results of a recent EMS-based forward genetic screen that identified a myriad of loci not previously associated with muscle morphogenesis. We recovered new alleles of known muscle morphogenesis genes, including bsd, kon, ths, and tum, arguing our screening strategy was effective and efficient. We also identified and sequenced new alleles of salm, barr, and ptc that presumably disrupt independent pathways directing muscle morphogenesis. Equally as important, our screen shows that at least 11 morphogenetic loci remain to be identified. This screen has developed exciting new tools to study muscle morphogenesis, which may provide future insights into the mechanisms that determine skeletal muscle topology.

Laboratory experiments have revealed the meteorological sensitivity of the virus of the coronavirus disease 2019 (COVID-19). However, no consensus has been reached about how the meteorological conditions modulate the virus transmission as it is constrained more often by non-meteorological factors. Here, we find that the non-meteorological factors constrain statistically-least the growth rate of cumulative confirmed cases in a country when the cases arrive around 2500-3000. The least-constrained growth rate correlates with the near-surface ultraviolet flux and temperature significantly (correlation coefficients r=-0.55±0.08 and -0.45±0.08 at p 10-5, respectively). In response to increases of 1W/m2 ultraviolet and 1°C temperature, the growth rate decreases by 0.33±.11% and 0.18±.08% per day, respectively. The response to the ultraviolet flux exhibits a delay by about 7 days, providing an independent measure of the incubation period. Our quantifications imply a seasonality of COVID-19 and a high risk of a pandemic resurgence in the upcoming boreal winter, suggesting a need for seasonal adaption in public policies.

Purpose Pyroptosis has vital roles in tumorigenesis and cancer development; however, its relationship with cervical squamous cell cancer (CSCC) remains unexplored. In this study, we aimed to develop a CSCC prediction signature related to pyroptosis. Patients and Methods Consensus clustering analysis was conducted to detect pyroptosis-related subclusters for CSCC. Next, differentially expressed genes (DEGs) between subclusters were identified. Univariate, least absolute shrinkage and selection operator, and stepwise multivariate Cox regression analyses were applied to establish a prognostic model and a nomogram drawn. Additionally, functional enrichment analysis, tumor mutation burden, and immune characteristics associated with this signature were investigated. Results We constructed a seven-gene signature that functions as an independent predictor of prognosis in CSCC using data from The Cancer Genome Atlas. Patients with CSCC were divided into two groups based on median risk score, and patients in the low-risk group had significantly longer survival time than those in the high-risk group. Our findings were validated using Gene Expression Omnibus cohort data. We also established a nomogram, to expand the clinical applicability of our findings. The seven gene signature was associated with various molecular pathways, tumor mutation status, and immune microenvironment. Conclusion The pyroptosis-related risk signature consisting of seven genes developed here represents a potential robust biomarker for predicting prognosis and immunotherapy response in patients with CSCC.

Pathogenic variants in Tropomyosin 2 (TPM2), which encodes a skeletal muscle specific actin binding protein essential for sarcomere function, cause a spectrum of musculoskeletal disorders including Nemaline Myopathy, Cap Myopathy, congenital fiber type disproportion, and distal arthrogrypsosis (DA). TPM2-related disorders have not been modeled in vivo, so we expressed a series of dominant, pathogenic TPM2 variants in Drosophila embryos and found two variants, K49Del and E122K, disrupted muscle morphogenesis and muscle function. Transient overexpression of K49Del and E122K also disrupted muscle morphogenesis in zebrafish. We further developed a benchmarked overexpression assay in zebrafish to characterize TPM2 variants that we identified in DA patients, and found these variants caused musculoskeletal defects similar to those of the known pathogenic variants. In addition, the severity of musculoskeletal phenotypes in zebrafish expressing TPM2 variants correlated with the severity of clinical phenotypes observed in DA patients. Our study establishes transient overexpression in zebrafish as an efficient platform to characterize variants of uncertain significance in vivo, and argues our assays can predict the clinical severity of musculoskeletal associated variants.