Genes Involved in miRNA Biogenesis Are Not Downregulated in SARS-CoV-2 Infection.

miRNAs, small non-coding RNAs that regulate gene expression, are involved in various pathological processes, including viral infections. Virus infections may interfere with the miRNA pathway through the inhibition of genes involved in miRNA biogenesis. A reduction in the number and the levels of miRNAs expressed in nasopharyngeal swabs of patients with severe COVID-19 was lately observed by us, pointing towards the potential of miRNAs as possible diagnostic or prognostic biomarkers for predicting outcomes among patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The objective of the present study was to investigate whether SARS-CoV-2 infection influences the expression levels of messenger RNAs (mRNAs) of key genes involved in miRNA biogenesis. mRNA levels of AGO2, DICER1, DGCR8, DROSHA, and Exportin-5 (XPO5) were measured by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) in nasopharyngeal swab specimens from patients with COVID-19 and controls, as well as in cells infected with SARS-CoV-2 in vitro. Our data showed that the mRNA expression levels of AGO2, DICER1, DGCR8, DROSHA, and XPO5 were not significantly different in patients with severe COVID-19 when compared to patients with non-severe COVID-19 and controls. Similarly, the mRNA expression of these genes was not affected by SARS-CoV-2 infection in NHBE and Calu-3 cells. However, in Vero E6 cells, AGO2, DICER1, DGCR8, and XPO5 mRNA levels were slightly upregulated 24 h after infection with SARS-CoV-2. In conclusion, we did not find evidence for downregulation of mRNA levels of miRNA biogenesis genes during SARS-CoV-2 infection, neither ex vivo nor in vitro.

RETROSPECTIVE ANALYSIS OF THE COVID-19 PANDEMIC IN AN URBAN SAFETY-NET HOSPITAL IN ATLANTA, GA

SESSION TITLE: COVID-19 Infections: Issues During and After Hospitalization SESSION TYPE: Original Investigations PRESENTED ON: 10/17/2022 01:30 pm - 02:30 pm PURPOSE: We present a retrospective study at one of the largest public, safety-net hospitals in the United States to highlight the importance of codifying the impact of COVID-19 disparities in marginalized populations. We used the following metrics to draw conclusions: patient demographics, vaccination status, comorbid conditions, length of stay (LOS), readmission rates, and clinical outcome. METHODS: For this retrospective study, we used Slicer Dicer software (Epic Verona, WI), an Epic self-service reporting tool, to query clinical data and identified a cohort of 9,040 patients ≥ 18 years old diagnosed with COVID-10 at Grady Memorial Hospital in Atlanta from 1/1/21 to 12/31/21. Statistical significance was defined as p<0.05. RESULTS: Of the 9,040 patients, 54.7% were female (4,942) and 45.3% were male (4,096). The cohort median age was 51 (range 18 – 100) and 80.5% were African American (7,278/9,040). Double-dose vaccination rate was only 24.5% (2,215/9,040). 38.3% of patients diagnosed with COVID-19 were admitted (3,467/9,040) and among these patients 3.0% were re-admitted (107/3,467). The most prevalent comorbidities were essential hypertension (45.2%), diabetes (21.7%), and asthma (13.2%). Patients with these comorbidities were more likely to be discharged as opposed to being admitted. Patients with the following comorbidities were more likely to be admitted: Pulmonary hypertension (70% admission rate), COPD (64.9%), heart failure (61.0%), cancer (60.8%), atrial fibrillation (57.1%). Median LOS from admission was 4 days and there was no statistical difference among different comorbidities. We found higher mortality in COVID-19 patients with cancer (12.9%), atrial fibrillation (12.6%), heart failure (11.1%), pulmonary hypertension (10.1%) and COPD (9.1%) compared to patients with diabetes (7.5%), hypertension (6.7%), HIV (4.8%), DVT/PE (4.6%), or asthma (2.7%). When examining overall mortality based on self- reported race, we found that African American patients had a statistically significant higher mortality compared to Caucasian patients (p-value= 0.00454). CONCLUSIONS: Current retrospective study, which included COVID-19 patients with different comorbidities showed that COVID-19 patients with pulmonary hypertension have worse clinical outcomes compared to other comorbid conditions. CLINICAL IMPLICATIONS: Our findings suggest the importance of investigating COVID-19 disparities in marginalized populations to better understand the impact in these communities. All individuals should be encouraged to get vaccinated against COVID-19, especially those found to be at high risk of severe illness such as pulmonary hypertension. In this retrospective study, we found higher hospital admission rate and worse outcomes in patients with cancer, atrial fibrillation, heart failure, and pulmonary hypertension, as well as higher mortality among the African American patient population. DISCLOSURES: No relevant relationships by nicolas bakinde No relevant relationships by Suvrat Chandra No relevant relationships by Michelle Lee no disclosure on file for Mario Ponce;

UP IN SMOKE: PATTERNS OF THC USE AND CANNABINOID HYPEREMESIS SYNDROME AT AN ACADEMIC MEDICAL CENTER

Background/Aim The prevalence of marijuana use has increased in the United States as many states have legalized its use. Cannabinoid hyperemesis syndrome (CHS) is an adverse effect that 17-30% of chronic users of marijuana will experience. The impact of the COVID- 19 pandemic on healthcare disruptions has been well established. The effect of the pandemic on vice-associated conditions has been described with increases in alcohol and substance related hospitalizations and mortality. Few studies have evaluated the effect of the COVID- 19 pandemic on CHS with regards to prevalence, admissions, readmissions, and healthcare burden. We sought to identify the impact of the COVID-19 pandemic on CHS using admissions and readmissions as metrics to evaluate healthcare burden. Methods Using Slicer-Dicer, an electronic medical record based self-service query tool, all cases of CHS requiring hospital admissions and those resulting in readmissions were recorded at the university's 3 hospital centers and stratified by gender, age, and location. Data was separated into pre-COVID (August 5, 2018 to April 5, 2020) and post-COVID (April 6, 2020 to October 5, 2021). Additionally, all positive cases of tetrahydrocannabinol (THC) tested were recorded and stratified by postal code. The primary outcome was identification of CHS cases requiring admission from the emergency department pre-COVID and post-COVID. The secondary outcome was identifying any differences in admission and readmission rates pre- COVID and post-COVID. Results A significant increase in total THC positive cases (p = <0.001) was seen with 2485 pre-COVID and 2936 post-COVID cases. 68 patients were diagnosed with CHS pre-COVID and 75 post-COVID. Cases requiring admission were 27.9% pre-COVID CHS and 30.7% post-COVID with a significant increase in admissions from one campus from 0% to 30.4% (p = 0.025). Pre-COVID CHS cases requiring readmission after an index admission was 31.6% and post-COVID was 26.1%. No significance was seen when stratifying the cohorts by gender and age. Discussion Our study shows a significant increase in CHS diagnoses and admissions with an associated significant increase in THC-positivity when comparing the pre-COVID and post-COVID cohorts. This is consistent with prior studies describing an increase in alcohol and substance use during the pandemic. Data from the Centers for Disease Control and Prevention also reveal a 30% increase in substance related deaths in 2020 when compared to 2019. Some suggest that factors related to the pandemic including social isolation stress, substance use in isolation, and decreased access to substance use treatment or programs are contributors. This study highlights the importance to identify this association to better understand and respond to pandemic-associated risk factors for substance use disorders to help alleviate its effect on healthcare burden.

Dysregulation of RNA interference components in COVID-19 patients.

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the novel coronavirus causing severe respiratory illness (COVID-19). This virus was initially identified in Wuhan city, a populated area of the Hubei province in China, and still remains one of the major global health challenges. RNA interference (RNAi) is a mechanism of post-transcriptional gene silencing that plays a crucial role in innate viral defense mechanisms by inhibiting the virus replication as well as expression of various viral proteins. Dicer, Drosha, Ago2, and DGCR8 are essential components of the RNAi system, which is supposed to be dysregulated in COVID-19 patients. This study aimed to assess the expression level of the mentioned mRNAs in COVID-19patients compared to healthy individuals. RESULTS: Our findings demonstrated that the expression of Dicer, Drosha, and Ago2 was statistically altered in COVID-19 patients compared to healthy subjects. Ultimately, the RNA interference mechanism as a crucial antiviral defense system was suggested to be dysregulated in COVID-19 patients.

Variants of uncertain significance in multi-gene cancer panels: a comparison between 2017 and 2020 in a diverse urban population

Background: Multi-gene cancer panels, available since 2013, have been used in routine clinical practice for the past six years. A significant concern in the uptake of panel testing by both clinicians and patients is the possibility of receiving a variant of uncertain significance (VUS). There are no published standard VUS rates, however, labs have anecdotally reported a rate of 1–1.3% per gene. The VUS rate in cancer panels has fallen drastically since the introduction of testing. This rate may continue to fall with the implementation of data sharing platforms, like ClinVar. With the exception of founder mutations, pathogenic variant rates are similar across different ethnic groups, however, VUS rates are significantly higher in non-white ethnic groups. In 2017, our center assessed the VUS rate for our mostly urban, ethnically diverse patient population. At that time, out of 478 patients who underwent multi-gene cancer testing, 162 (33.8%) were found to have at least one VUS;the overall VUS to gene ratio was 1.64%. With this study, we aim to observe our current VUS rate in order to assess whether there is a decrease in VUS in our center similar to that observed nationally. Methods: As part of our routine clinical practice, the results of all patients referred for cancer genetic counseling for a personal or family history of cancer are maintained in a secure, HIPPA-compliant database. The entries of all patients who completed a genetics consultation in 2020 were reviewed. Patients were excluded from analysis if they did not pursue genetic testing or if they pursued single gene or single site testing. Patients who completed any multi-gene cancer panel testing were included. The number of genes included in the panels varied due to reported history and patient preference. Results were analyzed for presence or absence of pathogenic variants and VUS. Results: In 2020, 768 patients completed a cancer genetics consultation. The majority of our patients (624/768 or 81%) reside in the Bronx. 20 patients (3%) reside in other New York City (NYC) boroughs and the remaining 124 patients reside outside of NYC. Of the 768 patients, 450 (59%) had a personal history of cancer and 318 (41%) had a family history of cancer only. A total of 307 were excluded from analysis: 160 patients declined genetic testing. An additional 147 patients were excluded, (26 with abnormal results from testing performed by outside providers, 10 who pursued single site testing, 8 who canceled testing due to billing concerns, 48 who missed their blood draw appointments, and 17 who did not return their saliva kits). Moreover, 1 patient passed away before completing testing and 36 had results pending. Out of 461 patients who completed cancer panel testing, 51 (11%) were found to have a pathogenic variant and 171 (37%) were found to have at least one VUS;a total of 213 VUS were noted, as some patients were found to have more than one VUS. Of note,13 of these individuals were also found to have a pathogenic variant. A total of 17,613 genes were tested through all the cancer panels ordered, which yields a variant rate of 1.21%. The most common identified VUSs were in the following genes: ATM (18/171, 11%, one patient had 2 ATM VUSs);BRCA2 (12/171, 7%);APC (14/171, 8%, one patient had 2 APC VUSs);DICER1 (11/171 or 6%, one patient had 2 DICER1 VUSs);BARD1 (11/171, 6%);CHEK2, MSH2, and RAD51D (each with 10/171, 6%). Discussion: As compared to 2017, the number of patients receiving a VUS on their multi-gene cancer panel testing increased to 37%, as compared to 34%. The variant to gene ratio decreased, however, to 1.2% as compared to 1.6%. One likely explanation for this apparent contradiction is the fact that larger, more comprehensive cancer gene panels are being offered now as compared to the outset of the testing, as many patients and providers favored smaller, more targeted panels and many labs have increased the number of genes on the panels they offer. Therefore, while the overall variant rate per gene has decreased, more of our patients are faced with receiving a VUS result and coping with the anxiety and other negative psychological impacts. Limitations to these data include the COVID-19 pandemic. In 2020, with the suspension of elective procedures and scheduling limitations, the ability of our center to complete cancer consultations was suspended and/or significantly reduced for many months. Therefore, patients seen for cancer genetic counseling may have been more likely to have a personal history of cancer or a stronger family history of cancer, however, it is unclear if this would affect the noted VUS rate. Conclusions: This study highlights the importance of pre and posttest counseling as well as ongoing vigilance with VUS rates, especially in an ethnically diverse population. It also makes the sharing of data between clinical laboratories more imperative in order to attempt to further reduce these rates.

Computational search of hybrid human/SARS-CoV-2 dsRNA reveals unique viral sequences that diverge from those of other coronavirus strains.

The role of the RNAi/Dicer/Ago system in degrading RNA viruses has been elusive in mammals in the past, which has prompted authors to think that interferon (IFN) synthesis is essential in this clade, relegating the RNAi defense strategy against viral infection as an accessory function. However, recent publications highlight the existence of abundant viral small interference and micro RNAs (VsiRNAs and VmiRNAs) in both cell-line and whole organism based experiments, indicating a contribution of these molecules in host responses and/or viral replication. We explore the theoretical possibility that RNAi triggered by SARS-CoV-2 might degrade some host transcripts in the opposite direction, although this hypothesis seems counterintuitive. The SARS-CoV-2 genome was therefore computationally searched for exact intrapairing within the viral RNA and exact hybrid pairing with the human transcriptome over a minimum of 20 bases in length. Minimal segments of 20-base lengths of SARS-CoV-2 RNA were found based on the theoretical matching with existing complementary strands in the human host transcriptome. Few human genes potentially annealing with SARS-CoV-2 RNA, including mitochondrial deubiquitinase USP30, the subunit of ubiquitin protein ligase complex FBXO21 and two long noncoding RNAs, were retrieved. The hypothesis that viral-originated RNAi might mediate degradation of host transcriptome messages was corroborated by published high throughput sequencing of RNA from infected tissues and cultured cells, clinical observation and phylogenetic comparative analysis, indicating a strong specificity of these SARS-CoV-2 hybrid pairing sequences for human genomes.

Targeting SARS CoV2 (Indian isolate) genome with miRNA: An in silico study.

The newly identified SARS CoV2 has become a global pandemic since December 2019. Various researchers are trying to design a vaccine candidate against the virus. On the other hand, another group is focussing on repurposing approved or clinically tested drugs for treatment. However, there is always a search for alternative therapies. Thus, we propose an alternative approach apart from chemotherapy that is the usage of miRNA as novel antisense therapy to cure SARS CoV2 infected patients. To address the objective, miRNAs have been designed by targeting the genome of SARS CoV2 (Indian isolate). First, the open reading frames in the viral genome have been identified, and the proteins encoded by those open reading frames have been predicted. Using computational biology, several miRNAs have been designed and their probability to bind to a viral gene has been predicted. In addition, miRNA target mining in the host cell has been done to rule out the possibility of non-specific binding of the miRNAs. The miRNAs having the highest chances to bind to the viral genome have been converted into pre-miRNAs, and their interaction with dicer endoribonuclease has been studied by molecular docking. Results revealed that the pre-miRNAs interact with the RNAse III 2 domain of dicer. Thus, it is predicted that the pre-miRNAs after delivery to the infected host cell will be processed by dicer to generate mature miRNAs that will target the SARS CoV2 viral genome. Therefore, miRNA therapy can be an alternative approach for the treatment of SARS CoV2 infection.

mTOR inhibition and p53 activation, microRNAs: The possible therapy against pandemic COVID-19.

mTOR is a serine-threonine kinase and participates in cell proliferation, cellular metabolism was found to be activated during Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infection and replication. During viral replication mTOR, downstream target genes such as ribosomal protein S6 kinase beta 1 (S6K1) and Eukaryotic translational initiation factor 4E-binding protein1 (4-E-BP1) are activated result in ribosome biosynthesis and efficient protein synthesis. In plasmacytoid dendritic cells (pDCs), mTOR plays a key role in the association of adapter protein myeloid differentiation primary response gene 88 (MyD88), Toll-like receptor 9 (TLR9) and interferon regulatory factor (IRF-7) leading to the transcriptional activation of type-I interferon (IFN) genes. Viruses also inactivate the interferon α (IFN-α) pathway by impairing the IRF-7 mediated activation of IFN-α gene transcription. Thus, mammalian target of rapamycin (mTOR) inhibitors can help in suppressing the early stages of viral infection and replication. Interestingly, the key tumor-suppressor p53 protein will undergo degradation by virus-encoded E3 ubiquitin ligase Ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) leading to an increased viral survival in host cells. Thus, the mTOR inhibitors and p53 activators or microRNAs that functions as p53 and can target 3'-UTR of mTOR and RPS6KB1 might effectively inhibit viral replication in the human respiratory tract and lung cells.