Symptomatic, clinical and biomarker associations for mortality in hospitalized COVID-19 patients enriched for African Americans.

BACKGROUND AND AIMS: Initial reports on US COVID-19 showed different outcomes in different races. In this study we use a diverse large cohort of hospitalized COVID-19 patients to determine predictors of mortality. METHODS: We analyzed data from hospitalized COVID-19 patients (n = 5852) between March 2020- August 2020 from 8 hospitals across the US. Demographics, comorbidities, symptoms and laboratory data were collected. RESULTS: The cohort contained 3,662 (61.7%) African Americans (AA), 286 (5%) American Latinx (LAT), 1,407 (23.9%), European Americans (EA), and 93 (1.5%) American Asians (AS). Survivors and non-survivors mean ages in years were 58 and 68 for AA, 58 and 77 for EA, 44 and 61 for LAT, and 51 and 63 for AS. Mortality rates for AA, LAT, EA and AS were 14.8, 7.3, 16.3 and 2.2%. Mortality increased among patients with the following characteristics: age, male gender, New York region, cardiac disease, COPD, diabetes mellitus, hypertension, history of cancer, immunosuppression, elevated lymphocytes, CRP, ferritin, D-Dimer, creatinine, troponin, and procalcitonin. Use of mechanical ventilation (p = 0.001), shortness of breath (SOB) (p < 0.01), fatigue (p = 0.04), diarrhea (p = 0.02), and increased AST (p < 0.01), significantly correlated with death in multivariate analysis. Male sex and EA and AA race/ethnicity had higher frequency of death. Diarrhea was among the most common GI symptom amongst AAs (6.8%). When adjusting for comorbidities, significant variables among the demographics of study population were age (over 45 years old), male sex, EA, and patients hospitalized in New York. When adjusting for disease severity, significant variables were age over 65 years old, male sex, EA as well as having SOB, elevated CRP and D-dimer. Glucocorticoid usage was associated with an increased risk of COVID-19 death in our cohort. CONCLUSION: Among this large cohort of hospitalized COVID-19 patients enriched for African Americans, our study findings may reflect the extent of systemic organ involvement by SARS-CoV-2 and subsequent progression to multi-system organ failure. High mortality in AA in comparison with LAT is likely related to high frequency of comorbidities and older age among AA. Glucocorticoids should be used carefully considering the poor outcomes associated with it. Special focus in treating patients with elevated liver enzymes and other inflammatory biomarkers such as CRP, troponin, ferritin, procalcitonin, and D-dimer are required to prevent poor outcomes.

Human Germline Genome Editing.

With CRISPR/Cas9 and other genome-editing technologies, successful somatic and germline genome editing are becoming feasible. To respond, an American Society of Human Genetics (ASHG) workgroup developed this position statement, which was approved by the ASHG Board in March 2017. The workgroup included representatives from the UK Association of Genetic Nurses and Counsellors, Canadian Association of Genetic Counsellors, International Genetic Epidemiology Society, and US National Society of Genetic Counselors. These groups, as well as the American Society for Reproductive Medicine, Asia Pacific Society of Human Genetics, British Society for Genetic Medicine, Human Genetics Society of Australasia, Professional Society of Genetic Counselors in Asia, and Southern African Society for Human Genetics, endorsed the final statement. The statement includes the following positions. (1) At this time, given the nature and number of unanswered scientific, ethical, and policy questions, it is inappropriate to perform germline gene editing that culminates in human pregnancy. (2) Currently, there is no reason to prohibit in vitro germline genome editing on human embryos and gametes, with appropriate oversight and consent from donors, to facilitate research on the possible future clinical applications of gene editing. There should be no prohibition on making public funds available to support this research. (3) Future clinical application of human germline genome editing should not proceed unless, at a minimum, there is (a) a compelling medical rationale, (b) an evidence base that supports its clinical use, (c) an ethical justification, and (d) a transparent public process to solicit and incorporate stakeholder input.

COVID-19 among African Americans and Hispanics: Does gastrointestinal symptoms impact the outcome?

BACKGROUND: The coronavirus disease 2019 (COVID-19) disproportionately affected African Americans (AA) and Hispanics (HSP). AIM: To analyze the significant effectors of outcome in African American patient population and make special emphasis on gastrointestinal (GI) symptoms, laboratory values and comorbidities. METHODS: We retrospectively evaluated the medical records of 386 COVID-19 positive patients admitted at Howard University Hospital between March and May 2020. We assessed the symptoms, including the GI manifestations, comorbidities, and mortality, using logistic regression analysis. RESULTS: Of these 386 COVID-19 positive patients, 257 (63.7%) were AAs, 102 (25.3%) HSP, and 26 (6.45%) Whites. There were 257 (63.7%) AA, 102 (25.3%) HSP, 26 (6.45%) Whites. The mean age was 55.6 years (SD = 18.5). However, the mean age of HSP was the lowest (43.7 years vs 61.2 for Whites vs 60 for AAs). The mortality rate was highest among the AAs (20.6%) and lowest among HSP (6.9%). Patients with shortness of breath (SOB) (OR2 = 3.64, CI = 1.73-7.65) and elevated AST (OR2 = 8.01, CI = 3.79-16.9) elevated Procalcitonin (OR2 = 8.27, CI = 3.95-17.3), AST (OR2 = 8.01, CI = 3.79-16.9), ferritin (OR2 = 2.69, CI = 1.24-5.82), and Lymphopenia (OR2 = 2.77, CI = 1.41-5.45) had a high mortality rate. Cough and fever were common but unrelated to the outcome. Hypertension and diabetes mellitus were the most common comorbidities. Glucocorticoid treatment was associated with higher mortality (OR2 = 5.40, CI = 2.72-10.7). Diarrhea was prevalent (18.8%), and GI symptoms did not affect the outcome. CONCLUSION: African Americans in our study had the highest mortality as they consisted of an older population and comorbidities. Age is the most important factor along with SOB in determining the mortality rate. Overall, elevated liver enzymes, ferritin, procalcitonin and C-reactive protein were associated with poor prognosis. GI symptoms did not affect the outcome. Glucocorticoids should be used judiciously, considering the poor outcomes associated with it. Attention should also be paid to monitor liver function during COVID-19, especially in AA and HSP patients with higher disease severity.

Single-reaction genomic amplification accelerates sequencing and vaccine production for classical and Swine origin human influenza a viruses.

Pandemic influenza A viruses that emerge from animal reservoirs are inevitable. Therefore, rapid genomic analysis and creation of vaccines are vital. We developed a multisegment reverse transcription-PCR (M-RTPCR) approach that simultaneously amplifies eight genomic RNA segments, irrespective of virus subtype. M-RTPCR amplicons can be used for high-throughput sequencing and/or cloned into modified reverse-genetics plasmids via regions of sequence identity. We used these procedures to rescue a contemporary H3N2 virus and a swine origin H1N1 virus directly from human swab specimens. Together, M-RTPCR and the modified reverse-genetics plasmids that we designed streamline the creation of vaccine seed stocks (9 to 12 days).

Saccharomyces cerevisiae ubiquitin-like protein Rub1 conjugates to cullin proteins Rtt101 and Cul3 in vivo.

In Saccharomyces cerevisiae, the ubiquitin-like protein Rub1p (related to ubiquitin 1 protein) covalently attaches to the cullin protein Cdc53p (cell division cycle 53 protein), a subunit of a class of ubiquitin E3 ligases named SCF (Skp1-Cdc53-F-box protein) complex. We identified Rtt101p (regulator of Ty transposition 101 protein, where Ty stands for transposon of yeast), initially found during a screen for proteins to confer retrotransposition suppression, and Cul3p (cullin 3 protein), a protein encoded by the previously uncharacterized open reading frame YGR003w, as two new in vivo targets for Rub1p conjugation. These proteins show significant identity with Cdc53p and, therefore, are cullin proteins. Modification of Cul3p is eliminated by deletion of the Rub1p pathway through disruption of either RUB1 or its activating enzyme ENR2 / ULA1. The same disruptions in the Rub pathway decreased the percentage of total Rtt101p that is modified from approx. 60 to 30%. This suggests that Rtt101p has an additional RUB1 - and ENR2 -independent modification. All modified forms of Rtt101p and Cul3p were lost when a single lysine residue in a conserved region near the C-terminus was replaced by an arginine residue. These results suggest that this lysine residue is the site of Rub1p-dependent and -independent modifications in Rtt101p and of Rub1p-dependent modification in Cul3p. An rtt101 Delta strain was hypersensitive to thiabendazole, isopropyl ( N -3-chlorophenyl) carbamate and methyl methanesulphonate, but rub1 Delta strains were not. Whereas rtt101 Delta strains exhibited a 14-fold increase in Ty1 transposition, isogenic rub1 Delta strains did not show statistically significant increases. Rtt101K791Rp, which cannot be modified, complemented for Rtt101p function in a transposition assay. Altogether, these results suggest that neither the RUB1 -dependent nor the RUB1 -independent form of Rtt101p is required for Rtt101p function. The identification of additional Rub1p targets in S. cerevisiae suggests an expanded role for Rub in this organism.

Reverse genetics plasmid for cloning unstable influenza A virus gene segments.

Reverse genetics approaches that enable the generation of recombinant influenza A viruses entirely from plasmids are invaluable for studies on virus replication, morphogenesis, pathogenesis, or transmission. Furthermore, influenza virus reverse genetics is now critical for the development of new vaccines for this human and animal pathogen. Periodically, influenza gene segments are unstable within plasmids in bacteria. The PB2 gene segment of a highly pathogenic avian H5 influenza virus A/Turkey/Ontario/7732/1966 (Ty/Ont) was unstable in commonly available cloning plasmids (e.g., pcDNA3.1/V5-His-TOPO) and in standard influenza virus reverse genetics plasmids (e.g., pHH21), which contain high copy origins of replication. Thus, a low-copy influenza reverse genetics plasmid (pGJ3C3) was developed to enable rapid cloning of unstable influenza A virus genes using ligation-independent recombination-based cloning. The unstable Ty/Ont PB2 gene segment was efficiently cloned using the pGJ3C3 plasmid and this clone was used to rescue a recombinant Ty/Ont virus. This low copy reverse genetics plasmid will be useful for cloning other unstable segments of influenza A viruses in order to rescue recombinant viruses, which will facilitate basic studies and vaccine seed stock production.

S-phase checkpoint pathways stimulate the mobility of the retrovirus-like transposon Ty1.

The mobility of the Ty1 retrotransposon in the yeast Saccharomyces cerevisiae is restricted by a large collection of proteins that preserve the integrity of the genome during replication. Several of these repressors of Ty1 transposition (Rtt)/genome caretakers are orthologs of mammalian retroviral restriction factors. In rtt/genome caretaker mutants, levels of Ty1 cDNA and mobility are increased; however, the mechanisms underlying Ty1 hypermobility in most rtt mutants are poorly characterized. Here, we show that either or both of two S-phase checkpoint pathways, the replication stress pathway and the DNA damage pathway, partially or strongly stimulate Ty1 mobility in 19 rtt/genome caretaker mutants. In contrast, neither checkpoint pathway is required for Ty1 hypermobility in two rtt mutants that are competent for genome maintenance. In rtt101delta mutants, hypermobility is stimulated through the DNA damage pathway components Rad9, Rad24, Mec1, Rad53, and Dun1 but not Chk1. We provide evidence that Ty1 cDNA is not the direct target of the DNA damage pathway in rtt101delta mutants; instead, levels of Ty1 integrase and reverse transcriptase proteins, as well as reverse transcriptase activity, are significantly elevated. We propose that DNA lesions created in the absence of Rtt/genome caretakers trigger S-phase checkpoint pathways to stimulate Ty1 reverse transcriptase activity.

Activation of a LTR-retrotransposon by telomere erosion.

Retrotransposons can facilitate repair of broken chromosomes, and therefore an important question is whether the host can activate retrotransposons in response to chromosomal lesions. Here we show that Ty1 elements, which are LTR-retrotransposons in Saccharomyces cerevisiae, are mobilized when DNA lesions are created by the loss of telomere function. Inactivation of telomerase in yeast results in progressive shortening of telomeric DNA, eventually triggering a DNA-damage checkpoint that arrests cells in G2/M. A fraction of cells, termed survivors, recover from arrest by forming alternative telomere structures. When telomerase is inactivated, Ty1 retrotransposition increases substantially in parallel with telomere erosion and then partially declines when survivors emerge. Retrotransposition is stimulated at the level of Ty1 cDNA synthesis, causing cDNA levels to increase 20-fold or more before survivors form. This response is elicited through a signaling pathway that includes Rad24, Rad17, and Rad9, three components of the DNA-damage checkpoint. Our findings indicate that Ty1 retrotransposons are activated as part of the cellular response to telomere dysfunction.