Barriers to physical distancing among healthcare workers on an academic hospital unit during the coronavirus disease 2019 (COVID-19) pandemic.

BACKGROUND: Physical distancing among healthcare workers (HCWs) is an essential strategy in preventing HCW-to-HCWs transmission of severe acute respiratory coronavirus virus 2 (SARS-CoV-2). OBJECTIVE: To understand barriers to physical distancing among HCWs on an inpatient unit and identify strategies for improvement. DESIGN: Qualitative study including observations and semistructured interviews conducted over 3 months. SETTING: A non-COVID-19 adult general medical unit in an academic tertiary-care hospital. PARTICIPANTS: HCWs based on the unit. METHODS: We performed a qualitative study in which we (1) observed HCW activities and proximity to each other on the unit during weekday shifts July-October 2020 and (2) conducted semi-structured interviews of HCWs to understand their experiences with and perspectives of physical distancing in the hospital. Qualitative data were coded based on a human-factors engineering model. RESULTS: We completed 25 hours of observations and 20 HCW interviews. High-risk interactions often occurred during handoffs of care at shift changes and patient rounds, when HCWs gathered regularly in close proximity for at least 15 minutes. Identified barriers included spacing and availability of computers, the need to communicate confidential patient information, and the desire to maintain relationships at work. CONCLUSIONS: Physical distancing can be improved in hospitals by restructuring computer workstations, work rooms, and break rooms; applying visible cognitive aids; adapting shift times; and supporting rounds and meetings with virtual conferencing. Additional strategies to promote staff adherence to physical distancing include rewarding positive behaviors, having peer leaders model physical distancing, and encouraging additional safe avenues for social connection at a safe distance.

Improving physical distancing among healthcare workers in a pediatric intensive care unit.

BACKGROUND: Healthcare workers (HCWs) not adhering to physical distancing recommendations is a risk factor for acquisition of severe acute respiratory coronavirus virus 2 (SARS-CoV-2). The study objective was to assess the impact of interventions to improve HCW physical distancing on actual distance between HCWs in a real-life setting. METHODS: HCWs voluntarily wore proximity beacons to measure the number and intensity of physical distancing interactions between each other in a pediatric intensive care unit. We compared interactions before and after implementing a bundle of interventions including changes to the layout of workstations, cognitive aids, and individual feedback from wearable proximity beacons. RESULTS: Overall, we recorded 10,788 interactions within 6 feet (∼2 m) and lasting >5 seconds. The number of HCWs wearing beacons fluctuated daily and increased over the study period. On average, 13 beacons were worn daily (32% of possible staff; range, 2-32 per day). We recorded 3,218 interactions before the interventions and 7,570 interactions after the interventions began. Using regression analysis accounting for the maximum number of potential interactions if all staff had worn beacons on a given day, there was a 1% decline in the number of interactions per possible interactions in the postintervention period (incident rate ratio, 0.99; 95% confidence interval, 0.98-1.00; P = .02) with fewer interactions occurring at nursing stations, in workrooms and during morning rounds. CONCLUSIONS: Using quantitative data from wearable proximity beacons, we found an overall small decline in interactions within 6 feet between HCWs in a busy intensive care unit after a multifaceted bundle of interventions was implemented to improve physical distancing.

The case for wearable proximity devices to inform physical distancing among healthcare workers.

OBJECTIVE: Despite the importance of physical distancing in reducing SARS-CoV-2 transmission, this practice is challenging in healthcare. We piloted use of wearable proximity beacons among healthcare workers (HCWs) in an inpatient unit to highlight considerations for future use of trackable technologies in healthcare settings. MATERIALS AND METHODS: We performed a feasibility pilot study in a non-COVID adult medical unit from September 28 to October 28, 2020. HCWs wore wearable proximity beacons, and interactions defined as <6 feet for ≥5 s were recorded. Validation was performed using direct observations. RESULTS: A total of 6172 close proximity interactions were recorded, and with the removal of 2033 false-positive interactions, 4139 remained. The highest proportion of interactions occurred between 7:00 Am-9:00 Am. Direct observations of HCWs substantiated these findings. DISCUSSION: This pilot study showed that wearable beacons can be used to monitor and quantify HCW interactions in inpatient settings. CONCLUSION: Technology can be used to track HCW physical distancing.

SLC26A9 SNP rs7512462 is not associated with lung disease severity or lung function response to ivacaftor in cystic fibrosis patients with G551D-CFTR.

BACKGROUND: The CFTR modulator ivacaftor has been variably effective in treating individuals with cystic fibrosis (CF) who harbor CFTR gating variants such as G551D, as well as other classes of CFTR variants when used with other modulators. Because CFTR genotype does not fully explain this variability, defining genetic modifiers of response to modulator therapy is of particular interest to the field of individualized CF drug therapy. Previous studies have proposed that a variant in SLC26A9 (rs7512462) is associated with lung disease severity and with response to treatment with ivacaftor in individuals with CF who carry G551D or gating variants. METHODS: Given the implications for CF treatment, we re-examined the reported associations in three cohorts; patients enrolled in the Twin and Siblings study at Johns Hopkins University, the CF modifier study at the University of North Carolina at Chapel Hill, and the prospective G551D Observational (GOAL) study. The GOAL study was specifically designed to measure lung function response to ivacaftor. RESULTS: We find no association between SLC26A9 (rs7512462) genotype and lung disease severity (n = 272) or change in lung function at one-, three-, and six-month intervals following ivacaftor treatment(n = 141) in individuals with CF who carry at least one G551D variant. CONCLUSIONS: Our inability to replicate this association indicates that rs7512462 genotype should not be used in treatment decisions.

Increased expression of anion transporter SLC26A9 delays diabetes onset in cystic fibrosis.

Diabetes is a common complication of cystic fibrosis (CF) that affects approximately 20% of adolescents and 40%-50% of adults with CF. The age at onset of CF-related diabetes (CFRD) (marked by clinical diagnosis and treatment initiation) is an important measure of the disease process. DNA variants associated with age at onset of CFRD reside in and near SLC26A9. Deep sequencing of the SLC26A9 gene in 762 individuals with CF revealed that 2 common DNA haplotypes formed by the risk variants account for the association with diabetes. Single-cell RNA sequencing (scRNA-Seq) indicated that SLC26A9 is predominantly expressed in pancreatic ductal cells and frequently coexpressed with CF transmembrane conductance regulator (CFTR) along with transcription factors that have binding sites 5' of SLC26A9. These findings were replicated upon reanalysis of scRNA-Seq data from 4 independent studies. DNA fragments derived from the 5' region of SLC26A9-bearing variants from the low-risk haplotype generated 12%-20% higher levels of expression in PANC-1 and CFPAC-1 cells compared with the high- risk haplotype. Taken together, our findings indicate that an increase in SLC26A9 expression in ductal cells of the pancreas delays the age at onset of diabetes, suggesting a CFTR-agnostic treatment for a major complication of CF.

Genetic Modifiers of Cystic Fibrosis-Related Diabetes Have Extensive Overlap With Type 2 Diabetes and Related Traits.

CONTEXT: Individuals with cystic fibrosis (CF) develop a distinct form of diabetes characterized by ß-cell dysfunction and islet amyloid accumulation similar to type 2 diabetes (T2D), but generally have normal insulin sensitivity. CF-related diabetes (CFRD) risk is determined by both CFTR, the gene responsible for CF, and other genetic variants. OBJECTIVE: To identify genetic modifiers of CFRD and determine the genetic overlap with other types of diabetes. DESIGN AND PATIENTS: A genome-wide association study was conducted for CFRD onset on 5740 individuals with CF. Weighted polygenic risk scores (PRSs) for type 1 diabetes (T1D), T2D, and diabetes endophenotypes were tested for association with CFRD. RESULTS: Genome-wide significance was obtained for variants at a novel locus (PTMA) and 2 known CFRD genetic modifiers (TCF7L2 and SLC26A9). PTMA and SLC26A9 variants were CF-specific; TCF7L2 variants also associated with T2D. CFRD was strongly associated with PRSs for T2D, insulin secretion, postchallenge glucose concentration, and fasting plasma glucose, and less strongly with T1D PRSs. CFRD was inconsistently associated with PRSs for insulin sensitivity and was not associated with a PRS for islet autoimmunity. A CFRD PRS comprising variants selected from these PRSs (with a false discovery rate < 0.1) and the genome-wide significant variants was associated with CFRD in a replication population. CONCLUSIONS: CFRD and T2D have more etiologic and mechanistic overlap than previously known, aligning along pathways involving ß-cell function rather than insulin sensitivity. Two CFRD risk loci are unrelated to T2D and may affect multiple aspects of CF. An 18-variant PRS stratifies risk of CFRD in an independent population.

Detecting interaction networks in the human microbiome with conditional Granger causality.

Human microbiome research is rife with studies attempting to deduce microbial correlation networks from sequencing data. Standard correlation and/or network analyses may be misleading when taken as an indication of taxon interactions because "correlation is neither necessary nor sufficient to establish causation"; environmental filtering can lead to correlation between non-interacting taxa. Unfortunately, microbial ecologists have generally used correlation as a proxy for causality although there is a general consensus about what constitutes a causal relationship: causes both precede and predict effects. We apply one of the first causal models for detecting interactions in human microbiome samples. Specifically, we analyze a long duration, high resolution time series of the human microbiome to decipher the networks of correlation and causation of human-associated microbial genera. We show that correlation is not a good proxy for biological interaction; we observed a weak negative relationship between correlation and causality. Strong interspecific interactions are disproportionately positive, whereas almost all strong intraspecific interactions are negative. Interestingly, intraspecific interactions also appear to act at a short timescale causing vast majority of the effects within 1-3 days. We report how different taxa are involved in causal relationships with others, and show that strong interspecific interactions are rarely conserved across two body sites whereas strong intraspecific interactions are much more conserved, ranging from 33% between the gut and right-hand to 70% between the two hands. Therefore, in the absence of guiding assumptions about ecological interactions, Granger causality and related techniques may be particularly helpful for understanding the driving factors governing microbiome composition and structure.

A Stoichioproteomic Analysis of Samples from the Human Microbiome Project.

Ecological stoichiometry (ES) uses organism-specific elemental content to explain differences in species life histories, species interactions, community organization, environmental constraints and even ecosystem function. Although ES has been successfully applied to a range of different organisms, most emphasis on microbial ecological stoichiometry focuses on lake, ocean, and soil communities. With the recent advances in human microbiome research, however, large amounts of data are being generated that describe differences in community composition across body sites and individuals. We suggest that ES may provide a framework for beginning to understand the structure, organization, and function of human microbial communities, including why certain organisms exist at certain locations, and how they interact with both the other microbes in their environment and their human host. As a first step, we undertake a stoichioproteomic analysis of microbial communities from different body sites. Specifically, we compare and contrast the elemental composition of microbial protein samples using annotated sequencing data from 690 gut, vaginal, oral, nares, and skin samples currently available through the Human Microbiome Project. Our results suggest significant differences in both the median and variance of the carbon, oxygen, nitrogen, and sulfur contents of microbial protein samples from different locations. For example, whereas proteins from vaginal sites are high in carbon, proteins from skin and nasal sites are high in nitrogen and oxygen. Meanwhile, proteins from stool (the gut) are particularly high in sulfur content. We interpret these differences in terms of the local environments at different human body sites, including atmospheric exposure and food intake rates.

Systematic Computational Identification of Variants That Activate Exonic and Intronic Cryptic Splice Sites.

We developed a variant-annotation method that combines sequence-based machine-learning classification with a context-dependent algorithm for selecting splice variants. Our approach is distinctive in that it compares the splice potential of a sequence bearing a variant with the splice potential of the reference sequence. After training, classification accurately identified 168 of 180 (93.3%) canonical splice sites of five genes. The combined method, CryptSplice, identified and correctly predicted the effect of 18 of 21 (86%) known splice-altering variants in CFTR, a well-studied gene whose loss-of-function variants cause cystic fibrosis (CF). Among 1,423 unannotated CFTR disease-associated variants, the method identified 32 potential exonic cryptic splice variants, two of which were experimentally evaluated and confirmed. After complete CFTR sequencing, the method found three cryptic intronic splice variants (one known and two experimentally verified) that completed the molecular diagnosis of CF in 6 of 14 individuals. CryptSplice interrogation of sequence data from six individuals with X-linked dyskeratosis congenita caused by an unknown disease-causing variant in DKC1 identified two splice-altering variants that were experimentally verified. To assess the extent to which disease-associated variants might activate cryptic splicing, we selected 458 pathogenic variants and 348 variants of uncertain significance (VUSs) classified as high confidence from ClinVar. Splice-site activation was predicted for 129 (28%) of the pathogenic variants and 75 (22%) of the VUSs. Our findings suggest that cryptic splice-site activation is more common than previously thought and should be routinely considered for all variants within the transcribed regions of genes.

Deep resequencing of CFTR in 762 F508del homozygotes reveals clusters of non-coding variants associated with cystic fibrosis disease traits.

Extensive phenotypic variability is commonly observed in individuals with Mendelian disorders, even among those with identical genotypes in the disease-causing gene. To determine whether variants within and surrounding CFTR contribute to phenotypic variability in cystic fibrosis (CF), we performed deep sequencing of CFTR in 762 patients homozygous for the common CF-causing variant, F508del. In phase 1, ~200 kb encompassing CFTR and extending 10 kb 5' and 5 kb 3' of the gene was sequenced in 486 F508del homozygotes selected from the extremes of sweat chloride concentration. In phase 2, a 510 kb region, which included the entire topologically associated domain of CFTR, was sequenced in 276 F508del homozygotes drawn from extremes of lung function. An additional 163 individuals who carried F508del and a different CF-causing variant were sequenced to inform haplotype construction. Region-based burden testing of both common and rare variants revealed seven regions of significance (α=0.01), five of which overlapped known regulatory elements or chromatin interactions. Notably, the -80 kb locus known to interact with the CFTR promoter was associated with variation in both CF traits. Haplotype analysis revealed a single rare recombination event (1.9% frequency) in intron 15 of CFTR bearing the F508del variant. Otherwise, the majority of F508del chromosomes were markedly similar, consistent with a single origin of the F508del allele. Together, these high-resolution variant analyses of the CFTR locus suggest a role for non-coding regulatory motifs in trait variation among individuals carrying the common CF allele.

Loss of carbonic anhydrase XII function in individuals with elevated sweat chloride concentration and pulmonary airway disease.

Elevated sweat chloride levels, failure to thrive (FTT), and lung disease are characteristic features of cystic fibrosis (CF, OMIM #219700). Here we describe variants in CA12 encoding carbonic anhydrase XII in two pedigrees exhibiting CF-like phenotypes. Exome sequencing of a white American adult diagnosed with CF due to elevated sweat chloride, recurrent hyponatremia, infantile FTT and lung disease identified deleterious variants in each CA12 gene: c.908-1 G>A in a splice acceptor and a novel frameshift insertion c.859_860insACCT. In an unrelated consanguineous Omani family, two children with elevated sweat chloride, infantile FTT, and recurrent hyponatremia were homozygous for a novel missense variant (p.His121Gln). Deleterious CFTR variants were absent in both pedigrees. CA XII protein was localized apically in human bronchiolar epithelia and basolaterally in the reabsorptive duct of human sweat glands. Respiratory epithelial cell RNA from the adult proband revealed only aberrant CA12 transcripts and in vitro analysis showed greatly reduced CA XII protein. Studies of ion transport across respiratory epithelial cells in vivo and in culture revealed intact CFTR-mediated chloride transport in the adult proband. CA XII protein bearing either p.His121Gln or a previously identified p.Glu143Lys missense variant localized to the basolateral membranes of polarized Madin-Darby canine kidney (MDCK) cells, but enzyme activity was severely diminished when assayed at physiologic concentrations of extracellular chloride. Our findings indicate that loss of CA XII function should be considered in individuals without CFTR mutations who exhibit CF-like features in the sweat gland and lung.

Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants.

BACKGROUND: Analysis of the functional consequences and treatment response of rare CFTR variants is challenging due to the limited availability of primary airways cells. METHODS: A Flp recombination target (FRT) site for stable expression of CFTR was incorporated into an immortalized CF bronchial epithelial cell line (CFBE41o-). CFTR cDNA was integrated into the FRT site. Expression was evaluated by western blotting and confocal microscopy and function measured by short circuit current. RNA sequencing was used to compare the transcriptional profile of the resulting CF8Flp cell line to primary cells and tissues. RESULTS: Functional CFTR was expressed from integrated cDNA at the FRT site of the CF8Flp cell line at levels comparable to that seen in native airway cells. CF8Flp cells expressing WT-CFTR have a stable transcriptome comparable to that of primary cultured airway epithelial cells, including genes that play key roles in CFTR pathways. CONCLUSION: CF8Flp cells provide a viable substitute for primary CF airway cells for the analysis of CFTR variants in a native context.

Codon bias and the folding dynamics of the cystic fibrosis transmembrane conductance regulator.

Synonymous or silent mutations are often overlooked in genetic analyses for disease-causing mutations unless they are directly associated with potential splicing defects. More recent studies, however, indicate that some synonymous single polynucleotide polymorphisms (sSNPs) are associated with changes in protein expression, and in some cases, protein folding and function. The impact of codon usage and mRNA structural changes on protein translation rates and how they can affect protein structure and function is just beginning to be appreciated. Examples are given here that demonstrate how synonymous mutations alter the translational kinetics and protein folding and/or function. The mechanism for how this occurs is based on a model in which codon usage modulates the translational rate by introducing pauses caused by nonoptimal or rare codons or by introducing changes in the mRNA structure, and this in turn influences co-translational folding. Two examples of this include the multidrug resistance protein (p-glycoprotein) and the cystic fibrosis transmembrane conductance regulator gene (CFTR). CFTR is also used here as a model to illustrate how synonymous mutations can be examined using in silico predictive methods to identify which sSNPs have the potential to change protein structure. The methodology described here can be used to help identify "non-silent" synonymous mutations in other genes.

Experimental assessment of splicing variants using expression minigenes and comparison with in silico predictions.

Assessment of the functional consequences of variants near splice sites is a major challenge in the diagnostic laboratory. To address this issue, we created expression minigenes (EMGs) to determine the RNA and protein products generated by splice site variants (n = 10) implicated in cystic fibrosis (CF). Experimental results were compared with the splicing predictions of eight in silico tools. EMGs containing the full-length Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) coding sequence and flanking intron sequences generated wild-type transcript and fully processed protein in Human Embryonic Kidney (HEK293) and CF bronchial epithelial (CFBE41o-) cells. Quantification of variant induced aberrant mRNA isoforms was concordant using fragment analysis and pyrosequencing. The splicing patterns of c.1585-1G>A and c.2657+5G>A were comparable to those reported in primary cells from individuals bearing these variants. Bioinformatics predictions were consistent with experimental results for 9/10 variants (MES), 8/10 variants (NNSplice), and 7/10 variants (SSAT and Sroogle). Programs that estimate the consequences of mis-splicing predicted 11/16 (HSF and ASSEDA) and 10/16 (Fsplice and SplicePort) experimentally observed mRNA isoforms. EMGs provide a robust experimental approach for clinical interpretation of splice site variants and refinement of in silico tools.

Cigarette smoke induces epithelial to mesenchymal transition and increases the metastatic ability of breast cancer cells.

BACKGROUND: Recent epidemiological studies demonstrate that both active and involuntary exposure to tobacco smoke increase the risk of breast cancer. Little is known, however, about the molecular mechanisms by which continuous, long term exposure to tobacco smoke contributes to breast carcinogenesis because most previous studies have focused on short term treatment models. In this work we have set out to investigate the progressive transforming effects of tobacco smoke on non-tumorigenic mammary epithelial cells and breast cancer cells using in vitro and in vivo models of chronic cigarette smoke exposure. RESULTS: We show that both non-tumorigenic (MCF 10A, MCF-12A) and tumorigenic (MCF7) breast epithelial cells exposed to cigarette smoke acquire mesenchymal properties such as fibroblastoid morphology, increased anchorage-independent growth, and increased motility and invasiveness. Moreover, transplantation experiments in mice demonstrate that treatment with cigarette smoke extract renders MCF 10A cells more capable to survive and colonize the mammary ducts and MCF7 cells more prone to metastasize from a subcutaneous injection site, independent of cigarette smoke effects on the host and stromal environment. The extent of transformation and the resulting phenotype thus appear to be associated with the differentiation state of the cells at the time of exposure. Analysis by flow cytometry showed that treatment with CSE leads to the emergence of a CD44(hi)/CD24(low) population in MCF 10A cells and of CD44+ and CD49f + MCF7 cells, indicating that cigarette smoke causes the emergence of cell populations bearing markers of self-renewing stem-like cells. The phenotypical alterations induced by cigarette smoke are accompanied by numerous changes in gene expression that are associated with epithelial to mesenchymal transition and tumorigenesis. CONCLUSIONS: Our results indicate that exposure to cigarette smoke leads to a more aggressive and transformed phenotype in human mammary epithelial cells and that the differentiation state of the cell at the time of exposure may be an important determinant in the phenotype of the final transformed state.