COVID-19 infection and transmission includes complex sequence diversity.

SARS-CoV-2 whole genome sequencing has played an important role in documenting the emergence of polymorphisms in the viral genome and its continuing evolution during the COVID-19 pandemic. Here we present data from over 360 patients to characterize the complex sequence diversity of individual infections identified during multiple variant surges (e.g., Alpha and Delta). Across our survey, we observed significantly increasing SARS-CoV-2 sequence diversity during the pandemic and frequent occurrence of multiple biallelic sequence polymorphisms in all infections. This sequence polymorphism shows that SARS-CoV-2 infections are heterogeneous mixtures. Convention for reporting microbial pathogens guides investigators to report a majority consensus sequence. In our study, we found that this approach would under-report sequence variation in all samples tested. As we find that this sequence heterogeneity is efficiently transmitted from donors to recipients, our findings illustrate that infection complexity must be monitored and reported more completely to understand SARS-CoV-2 infection and transmission dynamics. Many of the nucleotide changes that would not be reported in a majority consensus sequence have now been observed as lineage defining SNPs in Omicron BA.1 and/or BA.2 variants. This suggests that minority alleles in earlier SARS-CoV-2 infections may play an important role in the continuing evolution of new variants of concern.

Genomic Biomarkers Can Provide a Deeper Understanding of Recurrent Pressure Injuries.

OBJECTIVE: To identify genetic biomarkers predisposing individuals with spinal cord injury (SCI) to recurrent pressure injuries (PIs). METHODS: Repeated measures of the transcriptome profile of veterans with SCI at three Veterans Spinal Cord Injuries and Disorders Centers. Exclusion criteria included having significant active systemic disease at time of enrollment. Researchers obtained comprehensive profiles of clinical and health factors and demographic information relevant to PI history at enrollment and at each follow-up visit by reviewing patients' medical charts. Whole blood samples were collected at 6- to 12-month intervals for 2 to 4 years. In addition to DNA profiling with whole genome sequencing of the patients, RNA sequencing was performed to assess pathways associated with PI risk. RESULTS: Whole genome sequencing analysis identified 260 genes that showed increased prevalence of single-nucleotide variations in exonic regions with high (>20) combined annotation-dependent depletion scores between persons with high versus low intramuscular adipose tissue levels when cross-referenced with persons who had recurrent PIs. Gene set enrichment analysis using Hallmark and KEGG (Kyoto Encyclopedia of Genes and Genomes) gene sets of these candidate genes revealed enrichment in genes encoding proteins involved in fatty acid metabolism (P < .01). Further, RNA sequencing revealed upregulated activity in biological senescence pathways and downregulated activity in antimicrobial protection pathways. CONCLUSIONS: Genomic biomarkers may complement electronic health records to support management of complex interactive health issues such as risk of recurrent PIs in people with SCI. These findings may also be leveraged for homogeneous phenotypic grouping of higher-risk individuals.

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a Patient Transport Van.

We report 2 episodes of potential SARS-CoV-2 transmission from infected van drivers to passengers despite masking and physical distancing. Whole-genome sequencing confirmed relatedness of driver and passenger SARS-CoV-2. With the heater operating, fluorescent microspheres were transported by airflow >3 meters from the front to the back of the van.

Prevalence and genetic variants of G6PD deficiency among two Malagasy populations living in Plasmodium vivax-endemic areas.

BACKGROUND: The prevalence and variants of G6PD deficiency in the Plasmodium vivax-endemic zones of Madagascar remain unknown. The admixed African-Austronesian origins of the Malagasy population make it probable that a heterogeneous mix of genetic variants with a spectrum of clinical severity will be circulating. This would have implications for the widespread use of P. vivax radical cure therapy. Two study populations in the P. vivax-endemic western foothills region of Madagascar were selected for G6PD screening. Both the qualitative fluorescent spot test and G6PD genotyping were used to screen all participants. RESULTS: A total of 365 unrelated male volunteers from the Tsiroanomandidy, Mandoto, and Miandrivazo districts of Madagascar were screened and 12.9% were found to be phenotypically G6PD deficient. Full gene sequencing of 95 samples identified 16 single nucleotide polymorphisms, which were integrated into a genotyping assay. Genotyping (n = 291) found one individual diagnosed with the severe G6PD Mediterranean C563T mutation, while the remaining G6PD deficient samples had mutations of African origin, G6PD A- and G6PD A. CONCLUSIONS: Deployment of P. vivax radical cure in Madagascar must be considerate of the risks presented by the observed prevalence of G6PDd prevalence. The potential morbidity associated with cumulative episodes of P. vivax clinical relapses requires a strategy for increasing access to safe radical cure. The observed dominance of African G6PDd haplotypes is surprising given the known mixed African-Austronesian origins of the Malagasy population; more widespread surveying of G6PDd epidemiology across the island would be required to characterize the distribution of G6PD haplotypes across Madagascar.

Whole-genome sequencing reveals absence of recent gene flow and separate demographic histories for Anopheles punctulatus mosquitoes in Papua New Guinea.

Anopheles mosquitoes are the vectors of several human diseases including malaria. In many malaria endemic areas, several species of Anopheles coexist, sometimes in the form of related sibling species that are morphologically indistinguishable. Determining the size and organization of Anopheles populations, and possible ongoing gene flow among them is important for malaria control and, in particular, for monitoring the spread of insecticide resistance alleles. However, these parameters have been difficult to evaluate in most Anopheles species due to the paucity of genetic data available. Here, we assess the extent of contemporary gene flow and historical variations in population size by sequencing and de novo assembling the genomes of wild-caught mosquitoes from four species of the Anopheles punctulatus group of Papua New Guinea. Our analysis of more than 50 Mb of orthologous DNA sequences revealed no evidence of contemporary gene flow among these mosquitoes. In addition, investigation of the demography of two of the An. punctulatus species revealed distinct population histories. Overall, our analyses suggest that, despite their similarities in morphology, behaviour and ecology, contemporary sympatric populations of An. punctulatus are evolving independently.

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 Among Residents and Employees in a Veterans Affairs Community Living Center: A 42-Month Prospective Cohort Study.

Background: Understanding routes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission in long-term care facilities is essential for the development of effective control measures. Methods: Between March 1, 2020, and August 31, 2023, we identified coronavirus disease 2019 (COVID-19) cases among residents and employees in a Veterans Affairs community living center that conducted routine screening for asymptomatic COVID-19. Contact tracing was conducted to identify suspected transmission events, and whole genome sequencing was performed to determine the relatedness of SARS-CoV-2 samples. Results: During the 42-month study period, 269 cases of COVID-19 were diagnosed, including 199 employees and 70 residents. A total of 48 (24.1%) employees and 30 (42.9%) residents were asymptomatic. Sequencing analysis provided support for multiple events in which employees transmitted SARS-CoV-2 to co-workers and residents. There was 1 episode of likely transmission of SARS-CoV-2 from one resident to another resident, but no documented transmissions from residents to employees. Conclusions: Transmission of SARS-CoV-2 in the community living center predominantly involved transmission from employees to co-workers and residents. There is a need for improved measures to prevent transmission of SARS-CoV-2 by healthcare personnel.

Cathepsin B is a novel gender-dependent determinant of cholesterol absorption from the intestine.

We used a mouse C57BL/6J×CASA/Rk intercross to map a locus on chromosome 14 that displayed a gender-dependent effect on cholesterol absorption from the intestine. Studies in congenic animals revealed a complex locus with multiple operating genetic determinants resulting in alternating gender-dependent phenotypic effects. Fine-mapping narrowed the locus to a critical 6.3 Mb interval. Female subcongenics, but not males, of the critical interval displayed a decrease of 33% in cholesterol absorption. RNA-Seq analysis of female subcongenic jejunum revealed that cysteine protease cathepsin B (Ctsb) is a candidate to explain the interval effect. Consistent with the phenotype in critical interval subcongenics, female Ctsb knockout mice, but not males, displayed a decrease of 31% in cholesterol absorption. Although studies in Ctsb knockouts revealed a gender-dependent effect on cholesterol absorption, further fine-mapping dismissed a role for Ctsb in determining the effect of the critical 6.3 Mb interval on cholesterol absorption.

Mitochondrial genome sequences reveal deep divergences among Anopheles punctulatus sibling species in Papua New Guinea.

BACKGROUND: Members of the Anopheles punctulatus group (AP group) are the primary vectors of human malaria in Papua New Guinea. The AP group includes 13 sibling species, most of them morphologically indistinguishable. Understanding why only certain species are able to transmit malaria requires a better comprehension of their evolutionary history. In particular, understanding relationships and divergence times among Anopheles species may enable assessing how malaria-related traits (e.g. blood feeding behaviours, vector competence) have evolved. METHODS: DNA sequences of 14 mitochondrial (mt) genomes from five AP sibling species and two species of the Anopheles dirus complex of Southeast Asia were sequenced. DNA sequences from all concatenated protein coding genes (10,770 bp) were then analysed using a Bayesian approach to reconstruct phylogenetic relationships and date the divergence of the AP sibling species. RESULTS: Phylogenetic reconstruction using the concatenated DNA sequence of all mitochondrial protein coding genes indicates that the ancestors of the AP group arrived in Papua New Guinea 25 to 54 million years ago and rapidly diverged to form the current sibling species. CONCLUSION: Through evaluation of newly described mt genome sequences, this study has revealed a divergence among members of the AP group in Papua New Guinea that would significantly predate the arrival of humans in this region, 50 thousand years ago. The divergence observed among the mtDNA sequences studied here may have resulted from reproductive isolation during historical changes in sea-level through glacial minima and maxima. This leads to a hypothesis that the AP sibling species have evolved independently for potentially thousands of generations. This suggests that the evolution of many phenotypes, such as insecticide resistance will arise independently in each of the AP sibling species studied here.

Pharmacogenomic variation in the Malagasy population: implications for the antimalarial drug primaquine metabolism.

Aim: Antimalarial primaquine (PQ) eliminates liver hypnozoites of Plasmodium vivax. CYP2D6 gene variation contributes to PQ therapeutic failure. Additional gene variation may contribute to PQ efficacy. Information on pharmacogenomic variation in Madagascar, with vivax malaria and a unique population admixture, is scanty. Methods: The authors performed genome-wide genotyping of 55 Malagasy samples and analyzed data with a focus on a set of 28 pharmacogenes most relevant to PQ. Results: Mainly, the study identified 110 coding or splicing variants, including those that, based on previous studies in other populations, may be implicated in PQ response and copy number variation, specifically in chromosomal regions that contain pharmacogenes. Conclusion: With this pilot information, larger genome-wide association analyses with PQ metabolism and response are substantially more feasible.

Vascular surgery patients with elevated neutrophil-to-lymphocyte ratios have downregulated neutrophil complement RNA expression.

Elevated neutrophil-to-lymphocyte ratio (NLR) in patients who undergo elective vascular surgery (EVS) have increased mortality independent of perioperative surgical outcome. To understand why high NLR is associated with higher mortality, we investigated neutrophil and lymphocyte transcriptome expression in patients undergoing EVS. Blood samples were collected from patients undergoing EVS and healthy donors for NLR calculation. RNA samples were isolated from patients' neutrophils and lymphocytes and divided into NLR_Low (<3) and NLR_High (≥3) groups (n = 6 each). Paired samples with the highest RNA integrity number (mean = 9.8 ± 0.4) were sequenced and analyzed for differential expression. Normalized data were inputted for downstream analysis using iPathwayGuide (AdvaitaBio) and gene set enrichment analysis using GenePattern and MSigDB (Broad Institute). There was no clinical difference between the patient groups with regard to clinical diagnosis, age, sex, history of hypertension, lipid abnormalities, diabetes mellitus, smoking, or statin use. The mean NLR was 4.37 ± 0.27 SEM in the NLR_High and 1.88 ± 0.16 for the NLR_Low groups. Significantly differentially expressed gene sets identified in the RNA sequence data were enriched highly (P = 1E-24) in the humoral immunity and complement systems. Neutrophils from NLR_High patients downregulated complement genes (C1QA, C1QB, C1QC, C1S, C2, CR2, C3AR1, C3, C8G, and C9 and complement regulatory genes CD59, SERPING1, C4BPA, CFH, and CFI). Downregulation of gene expressions of humoral immunity and complement within the neutrophils are associated with elevated NLR. It remains to be determined whether and how these changes contribute to increased late mortality previously observed in patients undergoing EVS.

A core NRF2 gene set defined through comprehensive transcriptomic analysis predicts selective drug resistance and poor multi-cancer prognosis.

The NRF2-KEAP1 pathway plays an important role in the cellular response to oxidative stress but may also contribute to metabolic changes and drug resistance in cancer. We investigated the activation of NRF2 in human cancers and fibroblast cells through KEAP1 inhibition and cancer associated KEAP1/NRF2 mutations. We define a core set of 14 upregulated NRF2 target genes from seven RNA-Sequencing databases that we generated and analyzed, which we validated this gene set through analyses of published databases and gene sets. An NRF2 activity score based on expression of these core target genes correlates with resistance to drugs such as PX-12 and necrosulfonamide but not to paclitaxel or bardoxolone methyl. We validated these findings and also found NRF2 activation led to radioresistance in cancer cell lines. Finally, our NRF2 score is prognostic for cancer survival and validated in additional independent cohorts for novel cancers types not associated with NRF2-KEAP1 mutations. These analyses define a core NRF2 gene set that is robust, versatile, and useful as a NRF2 biomarker and for predicting drug resistance and cancer prognosis.

Investigation of a cluster of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in a hospital administration building.

OBJECTIVE: To investigate a cluster of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in employees working on 1 floor of a hospital administration building. METHODS: Contact tracing was performed to identify potential exposures and all employees were tested for SARS-CoV-2. Whole-genome sequencing was performed to determine the relatedness of SARS-CoV-2 samples from infected personnel and from control cases in the healthcare system with coronavirus disease 2019 (COVID-19) during the same period. Carbon dioxide levels were measured during a workday to assess adequacy of ventilation; readings >800 parts per million (ppm) were considered an indication of suboptimal ventilation. To assess the potential for airborne transmission, DNA-barcoded aerosols were released, and real-time polymerase chain reaction was used to quantify particles recovered from air samples in multiple locations. RESULTS: Between December 22, 2020, and January 8, 2021, 17 coworkers tested positive for SARS-CoV-2, including 13 symptomatic and 4 asymptomatic individuals. Of the 5 cluster SARS-CoV-2 samples sequenced, 3 were genetically related, but these employees denied higher-risk contacts with one another. None of the sequences from the cluster were genetically related to the 17 control sequences of SARS-CoV-2. Carbon dioxide levels increased during a workday but never exceeded 800 ppm. DNA-barcoded aerosol particles were dispersed from the sites of release to locations throughout the floor; 20% of air samples had >1 log10 particles. CONCLUSIONS: In a hospital administration building outbreak, sequencing of SARS-CoV-2 confirmed transmission among coworkers. Transmission occurred despite the absence of higher-risk exposures and in a setting with adequate ventilation based on monitoring of carbon dioxide levels.

Epigenetic effects of high-fat diet on intestinal tumorigenesis in C57BL/6J-Apc Min/+ mice.

Aim: Obesity and obesogenic diets might partly accelerate cancer development through epigenetic mechanisms. To determine these early effects, we investigated the impact of three days of a high-fat diet on epigenomic and transcriptomic changes in Apc Min/+ murine intestinal epithelia. Method: ChIP-Seq and RNA-Seq were performed on small intestinal epithelia of WT and Apc Min/+ male mice fed high-fat diet (HFD) or low-fat diet (LFD) for three days to identify genomic regions associated with differential H3K27ac levels as a marker of variant enhancer loci (VELs) as well as differentially expressed genes (DEGs). Results: Regarding epigenetic and transcriptomic changes, diet type (LFD vs. HFD) showed a significant impact, and genotype (WT vs.Apc Min/+) showed a small impact. Compared to LFD, HFD resulted in 1306 gained VELs, 230 lost VELs, 133 upregulated genes, and 127 downregulated genes in WT mice, with 1056 gained VELs, 371 lost VELs, 222 upregulated genes, and 182 downregulated genes in Apc Min/+ mice. Compared to the WT genotype, the Apc Min/+ genotype resulted in zero changed VELs for either diet type group, 21 DEGs for LFD, and 48 DEGs for HFD. Most gained VELs, and upregulated genes were associated with lipid metabolic processes. Gained VELs were also associated with Wnt signaling. Downregulated genes were associated with antigen presentation and processing. Conclusion: Three days of HFD-induced epigenomic and transcriptomic changes involving metabolic and immunologic pathways that may promote tumor growth in the genetically predisposed murine intestine without affecting key cancer signaling pathways.

A novel method for determining microflora composition using dynamic phylogenetic analysis of 16S ribosomal RNA deep sequencing data.

Deep sequencing of the 16S rRNA gene provides a comprehensive view of bacterial communities in a particular environment and has expanded our ability to study the impact of the microflora on human health and disease. Current analysis methods rely on comparisons of the sequences generated with an expanding but limited set of annotated 16S rRNA sequences or phylogenic clustering of sequences based on arbitrary similarity cutoffs. We describe a novel approach to characterize bacterial composition using deep sequencing of 16S rRNA gene. Our method defines operational taxonomic units based on phylogenetic tree reconstruction and dynamic clustering of sequences using solely sequencing data. These OTUs can be used to identify differences in bacteria abundance between environments. This approach can perform better than previous phylogenetic methods and will significantly improve our understanding of the microfloral role on human diseases by providing a comprehensive analysis of the microbial composition from various bacterial communities.

Repositioning Fenofibrate to Reactivate p53 and Reprogram the Tumor-Immune Microenvironment in HPV+ Head and Neck Squamous Cell Carcinoma.

Human papillomavirus-associated head and neck squamous cell carcinoma (HPV+ HNSCC) is recognized as a distinct disease with unique etiology and clinical features. Current standard of care therapeutic modalities are identical for HPV+ and HPV- HNSCC and thus, there remains an opportunity to develop innovative pharmacologic approaches to exploit the inherent vulnerabilities of HPV+ HNSCC. In this study, using an inducible HPVE6E7 knockdown system, we found that HPV+ HNSCC cells are addicted to HPVE6E7, such that loss of these viral oncogenes impaired tumorigenicity in vitro and in vivo. A number of druggable pathways, including PPAR and Wnt, were modulated in response to HPVE6E7 loss. Fenofibrate showed significant anti-proliferative effects in a panel of HPV+ cancer cell lines. Additionally, fenofibrate impaired tumor growth as monotherapy and potentiated the activity of cisplatin in a pre-clinical HPV+ animal model. Systemic fenofibrate treatment induced p53 protein accumulation, and surprisingly, re-programmed the tumor-immune microenvironment to drive immune cell infiltration. Since fenofibrate is FDA-approved with a favorable long-term safety record, repositioning of this drug, as a single agent or in combination with cisplatin or checkpoint blockade, for the HPV+ HNSCC setting should be prioritized.

Use of whole-genome sequencing to investigate a cluster of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in emergency department personnel.

Several recent reports have raised concern that infected coworkers may be an important source of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) acquisition by healthcare personnel. In a suspected outbreak among emergency department personnel, sequencing of SARS-CoV-2 confirmed transmission among coworkers. The suspected 6-person outbreak included 2 distinct transmission clusters and 1 unrelated infection.

KLF15 controls brown adipose tissue transcriptional flexibility and metabolism in response to various energetic demands.

Brown adipose tissue (BAT) is a specialized metabolic organ responsible for non-shivering thermogenesis. Recently, its activity has been shown to be critical in systemic metabolic health through its utilization and consumption of macronutrients. In the face of energetically demanding states, metabolic flexibility and systemic coordination of nutrient partitioning is requisite for health and survival. In this study, we elucidate BAT's differential transcriptional adaptations in response to multiple nutrient challenges and demonstrate its context-dependent prioritization of lipid, glucose, and amino acid metabolism. We show that the transcription factor Krüppel-like factor 15 (KLF15) plays a critical role in BAT metabolic flexibility. BAT-specific loss of KLF15 results in widespread changes in circulating metabolites and severely compromised thermogenesis in response to high energy demands, indicative of impaired nutrient utilization and metabolic flexibility. Together, our data demonstrate KLF15 in BAT plays an indispensable role in partitioning resources to maintain homeostasis and ensure survival.

Notch-Regulated Dendritic Cells Restrain Inflammation-Associated Colorectal Carcinogenesis.

Conventional dendritic cells (cDC) play a central role in T-cell antitumor responses. We studied the significance of Notch-regulated DC immune responses in a mouse model of colitis-associated colorectal cancer in which there is epithelial downregulation of Notch/Hes1 signaling. This defect phenocopies that caused by GMDS (GDP-mannose 4,6-dehydratase) mutation in human colorectal cancers. We found that, although wild-type immune cells restrained dysplasia progression and decreased the incidence of adenocarcinoma in chimeric mice, the immune system with Notch2 deleted in all blood lineages or in only DCs promoted inflammation-associated transformation. Notch2 signaling deficiency not only impaired cDC terminal differentiation, but also downregulated CCR7 expression, reduced DC migration, and suppressed antigen cross-presentation to CD8+ T cells. Transfer of Notch-primed DCs restrained inflammation-associated dysplasia progression. Consistent with the mouse data, we observed a correlation between infiltrating cDC1 and Notch2 signaling in human colorectal cancers and found that GMDS-mutant colorectal cancers showed decreased CCR7 expression and suppressed cDC1 signature gene expression. Suppressed cDC1 gene signature expression in human colorectal cancer was associated with a poor prognosis. In summary, our study supports an important role for Notch2 signaling in cDC1-mediated antitumor immunity and indicates that Notch2-controlled DCs restrain inflammation-associated colon cancer development in mice.

Muscle Krüppel-like factor 15 regulates lipid flux and systemic metabolic homeostasis.

Skeletal muscle is a major determinant of systemic metabolic homeostasis that plays a critical role in glucose metabolism and insulin sensitivity. By contrast, despite being a major user of fatty acids, and evidence that muscular disorders can lead to abnormal lipid deposition (e.g., nonalcoholic fatty liver disease in myopathies), our understanding of skeletal muscle regulation of systemic lipid homeostasis is not well understood. Here we show that skeletal muscle Krüppel-like factor 15 (KLF15) coordinates pathways central to systemic lipid homeostasis under basal conditions and in response to nutrient overload. Mice with skeletal muscle-specific KLF15 deletion demonstrated (a) reduced expression of key targets involved in lipid uptake, mitochondrial transport, and utilization, (b) elevated circulating lipids, (c) insulin resistance/glucose intolerance, and (d) increased lipid deposition in white adipose tissue and liver. Strikingly, a diet rich in short-chain fatty acids bypassed these defects in lipid flux and ameliorated aspects of metabolic dysregulation. Together, these findings establish skeletal muscle control of lipid flux as critical to systemic lipid homeostasis and metabolic health.

Oligodendroglial glycolytic stress triggers inflammasome activation and neuropathology in Alzheimer's disease.

Myelin degeneration and white matter loss resulting from oligodendrocyte (OL) death are early events in Alzheimer's disease (AD) that lead to cognitive deficits; however, the underlying mechanism remains unknown. Here, we find that mature OLs in both AD patients and an AD mouse model undergo NLR family pyrin domain containing 3 (NLRP3)-dependent Gasdermin D-associated inflammatory injury, concomitant with demyelination and axonal degeneration. The mature OL-specific knockdown of dynamin-related protein 1 (Drp1; a mitochondrial fission guanosine triphosphatase) abolishes NLRP3 inflammasome activation, corrects myelin loss, and improves cognitive ability in AD mice. Drp1 hyperactivation in mature OLs induces a glycolytic defect in AD models by inhibiting hexokinase 1 (HK1; a mitochondrial enzyme that initiates glycolysis), which triggers NLRP3-associated inflammation. These findings suggest that OL glycolytic deficiency plays a causal role in AD development. The Drp1-HK1-NLRP3 signaling axis may be a key mechanism and therapeutic target for white matter degeneration in AD.