SARS-CoV-2 variants evolve convergent strategies to remodel the host response.

SARS-CoV-2 variants of concern (VOCs) emerged during the COVID-19 pandemic. Here, we used unbiased systems approaches to study the host-selective forces driving VOC evolution. We discovered that VOCs evolved convergent strategies to remodel the host by modulating viral RNA and protein levels, altering viral and host protein phosphorylation, and rewiring virus-host protein-protein interactions. Integrative computational analyses revealed that although Alpha, Beta, Gamma, and Delta ultimately converged to suppress interferon-stimulated genes (ISGs), Omicron BA.1 did not. ISG suppression correlated with the expression of viral innate immune antagonist proteins, including Orf6, N, and Orf9b, which we mapped to specific mutations. Later Omicron subvariants BA.4 and BA.5 more potently suppressed innate immunity than early subvariant BA.1, which correlated with Orf6 levels, although muted in BA.4 by a mutation that disrupts the Orf6-nuclear pore interaction. Our findings suggest that SARS-CoV-2 convergent evolution overcame human adaptive and innate immune barriers, laying the groundwork to tackle future pandemics.

A Multiplexed System for Quantitative Comparisons of Chromatin Landscapes.

Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here, we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of p300, EZH2, or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions, and drug treatments.

The proximal proteome of 17 SARS-CoV-2 proteins links to disrupted antiviral signaling and host translation.

Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2.

Impact of San Francisco's toy ordinance on restaurants and children's food purchases, 2011-2012.

INTRODUCTION: In 2011, San Francisco passed the first citywide ordinance to improve the nutritional standards of children's meals sold at restaurants by preventing the giving away of free toys or other incentives with meals unless nutritional criteria were met. This study examined the impact of the Healthy Food Incentives Ordinance at ordinance-affected restaurants on restaurant response (eg, toy-distribution practices, change in children's menus), and the energy and nutrient content of all orders and children's-meal-only orders purchased for children aged 0 through 12 years. METHODS: Restaurant responses were examined from January 2010 through March 2012. Parent-caregiver/child dyads (n = 762) who were restaurant customers were surveyed at 2 points before and 1 seasonally matched point after ordinance enactment at Chain A and B restaurants (n = 30) in 2011 and 2012. RESULTS: Both restaurant chains responded to the ordinance by selling toys separately from children's meals, but neither changed their menus to meet ordinance-specified nutrition criteria. Among children for whom children's meals were purchased, significant decreases in kilocalories, sodium, and fat per order were likely due to changes in children's side dishes and beverages at Chain A. CONCLUSION: Although the changes at Chain A did not appear to be directly in response to the ordinance, the transition to a more healthful beverage and default side dish was consistent with the intent of the ordinance. Study results underscore the importance of policy wording, support the concept that more healthful defaults may be a powerful approach for improving dietary intake, and suggest that public policies may contribute to positive restaurant changes.

Fife, a Drosophila Piccolo-RIM homolog, promotes active zone organization and neurotransmitter release.

Neuronal communication depends on the precisely orchestrated release of neurotransmitter at specialized sites called active zones (AZs). A small number of scaffolding and cytoskeletal proteins comprising the cytomatrix of the active zone (CAZ) are thought to organize the architecture and functional properties of AZs. The majority of CAZ proteins are evolutionarily conserved, underscoring the fundamental similarities in neurotransmission at all synapses. However, core CAZ proteins Piccolo and Bassoon have long been believed exclusive to vertebrates, raising intriguing questions about the conservation of the molecular mechanisms that regulate presynaptic properties. Here, we present the identification of a piccolo-rim-related gene in invertebrates, together with molecular phylogenetic analyses that indicate the encoded proteins may represent Piccolo orthologs. In accordance, we find that the Drosophila homolog, Fife, is neuronal and localizes to presynaptic AZs. To investigate the in vivo function of Fife, we generated a deletion of the fife locus. We find that evoked neurotransmitter release is substantially decreased in fife mutants and loss of fife results in motor deficits. Through morphological analysis of fife synapses, we identify underlying AZ abnormalities including pervasive presynaptic membrane detachments and reduced synaptic vesicle clustering. Our data demonstrate the conservation of a Piccolo-related protein in invertebrates and identify critical roles for Fife in regulating AZ structure and function. These findings suggest the CAZ is more conserved than previously thought, and open the door to a more complete understanding of how CAZ proteins regulate presynaptic structure and function through genetic studies in simpler model systems.

Integrative analyses highlight functional regulatory variants associated with neuropsychiatric diseases.

Noncoding variants of presumed regulatory function contribute to the heritability of neuropsychiatric disease. A total of 2,221 noncoding variants connected to risk for ten neuropsychiatric disorders, including autism spectrum disorder, attention deficit hyperactivity disorder, bipolar disorder, borderline personality disorder, major depression, generalized anxiety disorder, panic disorder, post-traumatic stress disorder, obsessive-compulsive disorder and schizophrenia, were studied in developing human neural cells. Integrating epigenomic and transcriptomic data with massively parallel reporter assays identified differentially-active single-nucleotide variants (daSNVs) in specific neural cell types. Expression-gene mapping, network analyses and chromatin looping nominated candidate disease-relevant target genes modulated by these daSNVs. Follow-up integration of daSNV gene editing with clinical cohort analyses suggested that magnesium transport dysfunction may increase neuropsychiatric disease risk and indicated that common genetic pathomechanisms may mediate specific symptoms that are shared across multiple neuropsychiatric diseases.

Surgical correction of third eyelid cartilage eversion in an Anglo-Nubian goat: A case report.

This case of everted third eyelid cartilage in a goat demonstrates that everted cartilage occurs in animals other than domestic dogs and cats. Everted cartilage in the goat can be treated successfully with surgical excision of the abnormally bent cartilage.

Comparative Efficacy of Topical Ophthalmic Ganciclovir and Oral Famciclovir in Cats with Experimental Ocular Feline Herpesvirus-1 Epithelial Infection.

Purpose: To determine the comparative efficacy of ganciclovir ophthalmic gel and famciclovir oral tablets in cats with experimentally induced ocular feline herpesvirus-1 (FHV-1) epithelial infection. Methods: A randomized, placebo-controlled trial was performed using 16 nonvaccinated, specific pathogen-free cats with experimental FHV-1 infection induced by topical ocular inoculation. Cats received topical ganciclovir 0.15% ophthalmic gel (1 drop 3 times daily, n = 6 cats), oral famciclovir (90 mg/kg twice daily, n = 6), or topical artificial tear gel (1 drop 3 times daily, n = 4) for 14 days. Cats were monitored after inoculation for 30 days. Ophthalmic examinations were performed every 2 days and ocular disease scores calculated. In vivo confocal microscopy was performed, and corneal leukocyte infiltrates quantified. Ocular samples for FHV-1 quantitative polymerase chain reaction (qPCR) and virus isolation assays were collected every 3 days. Hemograms and serum biochemistry panels were performed at intervals. Results: Clinical ocular disease scores and corneal leukocyte infiltrates were significantly lower in the ganciclovir and famciclovir groups compared with placebo, but no significant differences were detected between the antiviral treatment groups. Ocular viral loads determined by qPCR were significantly lower in the ganciclovir group compared with the placebo group, but there were no significant differences between the other study groups. Hemograms and biochemistry panels were unremarkable. Conclusion: Topical application of ganciclovir gel 3 times daily was well-tolerated and displayed similar efficacy at reducing clinical ocular disease scores and corneal inflammation as twice daily oral famciclovir treatment in cats with experimental ocular FHV-1 infection.

A cis-regulatory lexicon of DNA motif combinations mediating cell-type-specific gene regulation.

Gene expression is controlled by transcription factors (TFs) that bind cognate DNA motif sequences in cis-regulatory elements (CREs). The combinations of DNA motifs acting within homeostasis and disease, however, are unclear. Gene expression, chromatin accessibility, TF footprinting, and H3K27ac-dependent DNA looping data were generated and a random-forest-based model was applied to identify 7,531 cell-type-specific cis-regulatory modules (CRMs) across 15 diploid human cell types. A co-enrichment framework within CRMs nominated 838 cell-type-specific, recurrent heterotypic DNA motif combinations (DMCs), which were functionally validated using massively parallel reporter assays. Cancer cells engaged DMCs linked to neoplasia-enabling processes operative in normal cells while also activating new DMCs only seen in the neoplastic state. This integrative approach identifies cell-type-specific cis-regulatory combinatorial DNA motifs in diverse normal and diseased human cells and represents a general framework for deciphering cis-regulatory sequence logic in gene regulation.

Mask use among health care workers and feelings of safety at work pre- and post- COVID-19 vaccine.

BACKGROUND: Correct mask use can prevent the spread of COVID-19 and hospitals require correct mask use. Despite this, there is variation in mask use among health care workers (HCW). Incorrect mask use may lead to increased infections and decreased feelings of safety. The purpose of this study was to determine variation in mask use among HCW as well as feelings of safety from exposure to COVID-19 when around colleagues before and after COVID-19 vaccine roll out. METHODS: This study used direct observation to assess mask use in patient-facing areas before and after COVID-19 vaccine. A staff survey was used to assess feelings of safety. RESULTS: Over 1,600 mask observations showed increased compliance from 94.6% to 97.5% (P = .001). Three hundred survey responses showed significantly increased feelings of safety (P < .001) after vaccine roll out, and 203 free-text responses with respondant reasoning were categorized into 6 themes. DISCUSSION: Understanding mask use behaviors and safety attitudes of HCW can help improve policies, workplace culture, and reduce HCW to HCW infections. CONCLUSIONS: Correct mask use was a highly adopted habit in patient-facing areas. The COVID-19 vaccine led to significantly increased feelings of safety among HCW, though the diverging narratives seen in the survey may be helpful to consider when crafting safety interventions.

An Opioid-free Anesthesia Protocol for Pediatric Strabismus Surgery: A Quality Improvement Project.

INTRODUCTION: This quality improvement (QI) project tracks a series of 2 Plan-Do-Study-Act (PDSA) cycles as we standardized and refined an ambulatory pediatric anesthesia strabismus protocol. We aimed to provide effective pain relief, reduce postoperative nausea and vomiting (PONV) rates, and be cost-efficient while minimizing perioperative opioids over 5 years. METHODS: We used statistical process control (SPC) charts to analyze real-world data captured from the medical record. We chose the following outcome and process measures to evaluate effectiveness: postoperative morphine rescue rate, maximum pain score in the postanesthesia care unit (PACU), and PONV rescue rate. We also used 2 balancing measures: postoperative length of stay (LOS) and total anesthesia time. We standardized our anesthesia protocol for our first PDSA cycle (April 2017) by removing intraoperative intravenous acetaminophen and utilizing fentanyl only. For the second PDSA cycle (January 2019), we replaced intraoperative fentanyl with dexmedetomidine. RESULTS: There was a total of 325 pediatric strabismus repair surgeries performed between April 2015 and July 2020. There was no special cause variation detected in the SPC charts for the family of measures chosen to measure effectiveness: postoperative morphine rescue rate, maximum pain score in the PACU, or the PONV rescue rate. The PONV rescue rate was 0 with the removal of opioids. Also, there was no special cause variation for the balancing measures: postoperative LOS or total anesthesia time. CONCLUSIONS: Throughout 2 PDSA cycles, this QI project enabled our team to standardize an opioid-free and cost-efficient anesthesia protocol for pediatric strabismus surgery over 5 years.

Characteristics of gadolinium-based contrast media cancellation at the point of care: a 15-month assessment of FDA-inspired medication guides on gadolinium retention.

PURPOSE: To assess the effect of United States Food and Drug Administration (FDA)-inspired patient handouts, regarding gadolinium retention, on the frequency of patient-initiated cancellations of gadolinium-based contrast media (GBCM) at the point of care. METHODS: This is a single center retrospective cohort review of MRI examinations protocolled to receive GBCM but performed without GBCM from October 2017 to January 2019. The study period spans an FDA-inspired safety intervention (December 2017 to May 2018) to provide patients with a handout on gadolinium retention. Examinations were coded according to rationale for GBCM cancellation. Process control charts were created to statistically evaluate trends in GBCM cancellations over time. RESULTS: From October 2017 to January 2019, GBCM was canceled at the point of care for 0.22% (131/58,837; 95% CI 0.19-0.26) of examinations in which GBCM was indicated. This did not significantly vary during the study period. No cancellations (0/131) were due to stated concern for gadolinium retention. However, 38% (50/131; 95% CI 30-47) were canceled due to kidney impairment and 21% (28/131; 95% CI 13-30) were canceled for an unknown reason. CONCLUSION: FDA-inspired handouts informing patients about gadolinium retention did not significantly change the frequency of GBCM cancellation. GBCM cancellations at the point of care remain uncommon.

easyCLIP analysis of RNA-protein interactions incorporating absolute quantification.

Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP target RNAs. Here, we develop an ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing method, easyCLIP. easyCLIP provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize RNA libraries during sequencing library preparation. Measurement of >200 independent cross-link experiments across >35 proteins identifies an RNA cross-link rate threshold that distinguishes RBPs from non-RBPs and defines target RNAs as those with a complex frequency unlikely for a random protein. We apply easyCLIP to the 33 most recurrent cancer mutations across 28 RBPs, finding increased RNA binding per RBP molecule for KHDRBS2 R168C, A1CF E34K and PCBP1 L100P/Q cancer mutations. Quantitating RBP-RNA interactions can thus nominate proteins as RBPs and define the impact of specific disease-associated RBP mutations on RNA association.

The proximal proteome of 17 SARS-CoV-2 proteins links to disrupted antiviral signaling and host translation.

Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2. AUTHOR SUMMARY: SARS-CoV-2 is the latest pathogenic coronavirus to emerge as a public health threat. We create a database of proximal host proteins to 17 SARS-CoV-2 viral proteins. We validate that NSP1 is proximal to the EIF3 translation initiation complex and is a potent inhibitor of translation. We also identify ORF6 antagonism of RNA-mediate innate immune signaling. We produce a database of potential host targets of the viral protease NSP5, and create a fluorescence-based assay to screen cleavage of peptide sequences. We believe that this data will be useful for identifying roles for many of the uncharacterized SARS-CoV-2 proteins and provide insights into the pathogenicity of new or emerging coronaviruses.

The dynamic, combinatorial cis-regulatory lexicon of epidermal differentiation.

Transcription factors bind DNA sequence motif vocabularies in cis-regulatory elements (CREs) to modulate chromatin state and gene expression during cell state transitions. A quantitative understanding of how motif lexicons influence dynamic regulatory activity has been elusive due to the combinatorial nature of the cis-regulatory code. To address this, we undertook multiomic data profiling of chromatin and expression dynamics across epidermal differentiation to identify 40,103 dynamic CREs associated with 3,609 dynamically expressed genes, then applied an interpretable deep-learning framework to model the cis-regulatory logic of chromatin accessibility. This analysis framework identified cooperative DNA sequence rules in dynamic CREs regulating synchronous gene modules with diverse roles in skin differentiation. Massively parallel reporter assay analysis validated temporal dynamics and cooperative cis-regulatory logic. Variants linked to human polygenic skin disease were enriched in these time-dependent combinatorial motif rules. This integrative approach shows the combinatorial cis-regulatory lexicon of epidermal differentiation and represents a general framework for deciphering the organizational principles of the cis-regulatory code of dynamic gene regulation.

Pediatric Somatic Symptom and Related Disorders: Primary Care Provider Perspectives.

Somatization, or physical symptoms that are inconsistent with a physiological cause that may or may not involve an identified stressor, is common in outpatient pediatrics. When these symptoms persist, they can impair function and progress to a somatic symptom and related disorder (SSRD), resulting in increased health care use and increased demands on primary care providers (PCPs). We performed a needs assessment among PCPs to better understand how best to support providers caring for children with SSRDs. Pediatric PCPs (n = 77) were surveyed to better understand their training, experience, perceptions, and practices of SSRD care. Findings indicate that PCPs have limited training in SSRD care but express interest in learning more. Many barriers to effective care were reported. We hope to use these findings to develop training materials and support services for pediatric PCPs managing SSRDs.

A B Cell Regulome Links Notch to Downstream Oncogenic Pathways in Small B Cell Lymphomas.

Gain-of-function Notch mutations are recurrent in mature small B cell lymphomas such as mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), but the Notch target genes that contribute to B cell oncogenesis are largely unknown. We performed integrative analysis of Notch-regulated transcripts, genomic binding of Notch transcription complexes, and genome conformation data to identify direct Notch target genes in MCL cell lines. This B cell Notch regulome is largely controlled through Notch-bound distal enhancers and includes genes involved in B cell receptor and cytokine signaling and the oncogene MYC, which sustains proliferation of Notch-dependent MCL cell lines via a Notch-regulated lineage-restricted enhancer complex. Expression of direct Notch target genes is associated with Notch activity in an MCL xenograft model and in CLL lymph node biopsies. Our findings provide key insights into the role of Notch in MCL and other B cell malignancies and have important implications for therapeutic targeting of Notch-dependent oncogenic pathways.

Adaptive Chromatin Remodeling Drives Glioblastoma Stem Cell Plasticity and Drug Tolerance.

Glioblastoma, the most common and aggressive malignant brain tumor, is propagated by stem-like cancer cells refractory to existing therapies. Understanding the molecular mechanisms that control glioblastoma stem cell (GSC) proliferation and drug resistance may reveal opportunities for therapeutic interventions. Here we show that GSCs can reversibly transition to a slow-cycling, persistent state in response to targeted kinase inhibitors. In this state, GSCs upregulate primitive developmental programs and are dependent upon Notch signaling. This transition is accompanied by widespread redistribution of repressive histone methylation. Accordingly, persister GSCs upregulate, and are dependent on, the histone demethylases KDM6A/B. Slow-cycling cells with high Notch activity and histone demethylase expression are present in primary glioblastomas before treatment, potentially contributing to relapse. Our findings illustrate how cancer cells may hijack aspects of native developmental programs for deranged proliferation, adaptation, and tolerance. They also suggest strategies for eliminating refractory tumor cells by targeting epigenetic and developmental pathways.

Single-molecule decoding of combinatorially modified nucleosomes.

Different combinations of histone modifications have been proposed to signal distinct gene regulatory functions, but this area is poorly addressed by existing technologies. We applied high-throughput single-molecule imaging to decode combinatorial modifications on millions of individual nucleosomes from pluripotent stem cells and lineage-committed cells. We identified definitively bivalent nucleosomes with concomitant repressive and activating marks, as well as other combinatorial modification states whose prevalence varies with developmental potency. We showed that genetic and chemical perturbations of chromatin enzymes preferentially affect nucleosomes harboring specific modification states. Last, we combined this proteomic platform with single-molecule DNA sequencing technology to simultaneously determine the modification states and genomic positions of individual nucleosomes. This single-molecule technology has the potential to address fundamental questions in chromatin biology and epigenetic regulation.