GotGlycans: Role of N343 Glycosylation on the SARS-CoV-2 S RBD Structure and Co-Receptor Binding Across Variants of Concern (preprint)

Glycosylation of the SARS-CoV-2 spike (S) protein represents a key target for viral evolution because it affects both viral evasion and fitness. Successful variations in the glycan shield are difficult to achieve though, as protein glycosylation is also critical to folding and to structural stability. Within this framework, the identification of glycosylation sites that are structurally dispensable can provide insight into the evolutionary mechanisms of the shield and inform immune surveillance. In this work we show through over 45 s of cumulative sampling from conventional and enhanced molecular dynamics (MD) simulations, how the structure of the immunodominant S receptor binding domain (RBD) is regulated by N-glycosylation at N343 and how the structural role of this glycan changes from WHu-1, alpha (B.1.1.7), and beta (B.1.351), to the delta (B.1.617.2) and omicron (BA.1 and BA.2.86) variants. More specifically, we find that the amphipathic nature of the N-glycan is instrumental to preserve the structural integrity of the RBD hydrophobic core and that loss of glycosylation at N343 triggers a specific and consistent conformational change. We show how this change allosterically regulates the conformation of the receptor binding motif (RBM) in the WHu-1, alpha and beta RBDs, but not in the delta and omicron variants, due to mutations that reinforce the RBD architecture. In support of these findings, we show that the binding of the RBD to monosialylated ganglioside co-receptors is highly dependent on N343 glycosylation in the WHu-1, but not in the delta RBD, and that affinity changes significantly across VoCs. Ultimately, the molecular and functional insight we provide in this work reinforces our understanding of the role of glycosylation in protein structure and function and it also allows us to identify the structural constraints within which the glycosylation site at N343 can become a hotspot for mutations in the SARS-CoV-2 S glycan shield.

Infectious disease surveillance needs for the United States: lessons from COVID-19 (preprint)

The COVID-19 pandemic has highlighted the need to upgrade systems for infectious disease surveillance and forecasting and modeling of the spread of infection, both of which inform evidence-based public health guidance and policies. Here, we discuss requirements for an effective surveillance system to support decision making during a pandemic, drawing on the lessons of COVID-19 in the U.S., while looking to jurisdictions in the U.S. and beyond to learn lessons about the value of specific data types. In this report, we define the range of decisions for which surveillance data are required, the data elements needed to inform these decisions and to calibrate inputs and outputs of transmission-dynamic models, and the types of data needed to inform decisions by state, territorial, local, and tribal health authorities. We define actions needed to ensure that such data will be available and consider the contribution of such efforts to improving health equity.

Assessment of Sociodemographic Factors Associated with Time to Self-reported COVID-19 Infection Among a Large Multi-Center Prospective Cohort Population in the Southeastern United States (preprint)

Objective: We aimed to investigate sociodemographic factors associated with self-reported COVID-19 infection. Methods: The study population is a multicenter prospective cohort of adult volunteers recruited from healthcare systems located in the mid-Atlantic and southern United States. Between April 2020 and October 2021 participants completed daily online questionnaires about symptoms, exposures, and risk behaviors related to COVID-19, including self-reports of positive SARS CoV-2 detection tests and COVID-19 vaccination. Analysis of time from study enrollment to self-reported COVID-19 infection used a time-varying mixed effects Cox-proportional hazards framework. Results: Overall, 1,603 of 27,214 study participants (5.9%) reported a positive COVID-19 test during the study period. The adjusted hazard ratio demonstrated lower risk for women, those with a graduate level degree, and smokers. A higher risk was observed for healthcare workers, those aged 18-34, those in rural areas, those from households where a member attends school or interacts with the public, and those who visited a health provider in the last year. Conclusions: Increased risk of self-reported COVID-19 was associated with specific demographic characteristics, which may help to inform targeted interventions for future pandemics.

Mucosal and systemic immune dynamics associated with COVID-19 outcomes: a longitudinal prospective clinical study (preprint)

Rationale: COVID-19 severity varies widely; children and African Americans have low and high risk, respectively. Mechanistic data from these groups and the mucosa is lacking. Objectives: To quantify mucosal and systemic viral and immune dynamics in a diverse cohort to identify mechanisms underpinning COVID-19 severity and outcome predictors. Methods: In this prospective study of unvaccinated children and adults COVID-19 outcome was based on an ordinal clinical severity scale. We quantified viral RNA, antigens, antibodies, and cytokines by PCR, ELISA, and Luminex from 579 longitudinally collected blood and nasal specimens from 78 subjects including 45 women and used modeling to determine functional relationships between these data. Measurements and Main Results: COVID-19 induced unique immune responses in African Americans (n=26) and children (n=20). Mild outcome was associated with more effective coordinated responses whereas moderate and severe outcomes had rapid seroconversion, significantly higher antigen, mucosal sCD40L, MCP-3, MCP-1, MIP-1, and MIP-1{beta}, and systemic IgA, IgM, IL-6, IL-8, IL-10, IL-15, IL-1RA, and IP-10, and uncoordinated early immune responses that went unresolved. Mucosal IL-8, IL-1{beta}, and IFN-{gamma} with systemic IL-1RA and IgA predicted COVID-19 outcomes. Conclusions: We present novel mucosal data, biomarkers, and therapeutic targets from a diverse cohort. Based on our findings, children and African Americans with COVID-19 have significantly lower IL-6 and IL-17 levels which may reduce responsiveness to drugs targeting IL-6 and IL-17. Unregulated immune responses persisted indicating moderate to severe COVID-19 cases may require prolonged treatments. Reliance on slower acting adaptive responses may cause immune crisis for some adults who encounter a novel virus.

Determining compliance with the COVID-19 daily symptom monitoring tool used to identify suspected COVID-19 workers of the military health support formation employees of the South African national defence force

Introduction: the COVID-19 pandemic had prompted governments in many countries to enact laws and policies to combat the spread of COVID-19 at work. The DEL required every worker to be screened when they arrived at work. Screening methods included self-reporting symptoms using a symptom monitoring tool. This study aimed to determine compliance with the symptom monitoring tool by assessing the knowledge, attitude, and practice of the MHSF employees. Methods: a cross-sectional questionnaire was administered to the employees. Information related to demographic, COVID-19 exposure, knowledge of COVID-19 and the symptom monitoring tool, attitude towards the symptom monitoring tool and practices towards COVID-19 and the symptom monitoring tool was collected. Results: a total of 90 participants participated in the study. The majority (N=45;50%) of respondents were aged between 30 and 39 years old, with more female (N=50) than male (N=40) participants. The majority (N=51;56.7%) only had grade 12 as the highest level of education. There were 25% (N=10) of males and 20% (N=10) of females who contracted COVID-19. The relationship between the COVID-19 positive cases and the symptom monitoring tool identifying symptoms had a strong negative correlation (-0.932). Respondent's knowledge of COVID-19 and the symptom monitoring tool was moderate (72.4%), with the attitude to the symptom monitoring tool being moderate (63.3%) as well. However, the practices of the COVID-19 guidelines and the symptom monitoring tool were good (93.3%). Conclusion: the employees of the MHSF complied with the completion of the daily symptom monitoring tool. There was decent knowledge of COVID-19 and the symptom monitoring tool, with a moderate attitude and good practices towards COVID-19 and completing the tool. The tool was able to identify suspected COVID-19 cases, which possibly reduced the spread of the virus in the workplace. © Stellan Abraham John et al.

Discovery of Small Molecules Targeting the Frameshifting Element RNA in SARS-CoV-2 Viral Genome.

Targeting structured RNA elements in the SARS-CoV-2 viral genome with small molecules is an attractive strategy for pharmacological control over viral replication. In this work, we report the discovery of small molecules that target the frameshifting element (FSE) in the SARS-CoV-2 RNA genome using high-throughput small-molecule microarray (SMM) screening. A new class of aminoquinazoline ligands for the SARS-CoV-2 FSE are synthesized and characterized using multiple orthogonal biophysical assays and structure-activity relationship (SAR) studies. This work reveals compounds with mid-micromolar binding affinity (KD = 60 ± 6 µM) to the FSE RNA and supports a binding mode distinct from previously reported FSE binders MTDB and merafloxacin. In addition, compounds are active in in vitro dual-luciferase and in-cell dual-fluorescent-reporter frameshifting assays, highlighting the promise of targeting structured elements of RNAs with druglike compounds to alter expression of viral proteins.

Adaptive immune responses to SARS-CoV-2 persist in the pharyngeal lymphoid tissue of children.

Most studies of adaptive immunity to SARS-CoV-2 infection focus on peripheral blood, which may not fully reflect immune responses at the site of infection. Using samples from 110 children undergoing tonsillectomy and adenoidectomy during the COVID-19 pandemic, we identified 24 samples with evidence of previous SARS-CoV-2 infection, including neutralizing antibodies in serum and SARS-CoV-2-specific germinal center and memory B cells in the tonsils and adenoids. Single-cell B cell receptor (BCR) sequencing indicated virus-specific BCRs were class-switched and somatically hypermutated, with overlapping clones in the two tissues. Expanded T cell clonotypes were found in tonsils, adenoids and blood post-COVID-19, some with CDR3 sequences identical to previously reported SARS-CoV-2-reactive T cell receptors (TCRs). Pharyngeal tissues from COVID-19-convalescent children showed persistent expansion of germinal center and antiviral lymphocyte populations associated with interferon (IFN)-γ-type responses, particularly in the adenoids, and viral RNA in both tissues. Our results provide evidence for persistent tissue-specific immunity to SARS-CoV-2 in the upper respiratory tract of children after infection.

RNA Regulatory Networks 2.0.

The central role of RNA molecules in cell biology has been an expanding subject of study since the proposal of the "RNA world" hypothesis 60 years ago [...].

Adverse Outcomes Associated With Atrial Arrhythmias After Veno-Venous Extracorporeal Membrane Oxygenation.

Veno-venous extracorporeal membrane oxygenation (VV ECMO) is used as a treatment modality in those who fail to respond to conventional care. Hypoxia and medications used in the intensive care unit may increase risk for atrial arrhythmias (AA). This study aims to evaluate the impact of AA on post-VV ECMO outcome. A retrospective review of patients who were placed on VV ECMO between October 2016 and October 2021. One hundred forty-five patients were divided into two groups, AA and no AA. Baseline characteristic and potential risk factors were assessed. Uni- and multivariate analysis using logistic regression models were constructed to evaluate the predictors of mortality between groups. Survival between groups was estimated by the Kaplan-Meier method using the log-rank test. Advanced age with history of coronary artery disease and hypertension were associated with increased risk to develop AA post-VV ECMO placement ( p value < 0.05). Length on ECMO, time intubated, hospital length of stay, and sepsis were significantly increased in patients in the AA group ( p value < 0.05). There was no difference in the overall mortality between the two groups. AAs were associated with worse hospital course and complications but no difference in overall mortality rate. Age and cardiovascular disease seem to be predisposing risk factors for this. Further studies are needed to investigate potential strategies to prevent AAs development in this population.

Back and neck problems as well as disadvantageous ergonomic behavior patterns in university students: Concomitants of the pandemic?

Background: As a part of the coronavirus disease 2019 (COVID-19) lockdown measures, universities converted courses to digital formats, leading to remote studying. It is unclear how these measures affect university students in terms of musculoskeletal problems (i.e., neck and back pain), e.g., by non-ergonomically equipped home offices or reduced physical activity. Material(s) and Method(s): Students from Osnabruck University weresurveyed via fully standardized online questionnaires from early March to mid-April 2022 (6 weeks) about neck and back problems, movement behavior in home offices, and personal information. Result(s): Of 447 students who clicked on the link, 378 students (80.4% female, mean age: 24.1 +/- 4.2 years) answered the questionnaire (response rate: 84.6%). 299 (79.1%) students suffered from neck pain and 294 (77.8%) from back pain during the pandemic. 206 (54.4%) students generally used ergonomic furniture, 83 (22.0%) used ergonomic sitting furniture, 57 (15.1%) used a standing desk, and 212 (56.1%) used aids for a more comfortable use of laptops in home office. 203 (53.7%) students took movement breaks in home office, 207 (54.8%) worked out regularly, and 65 (17.2%) moved regularly in the fresh air. Conclusion(s): The present study found a high rate of neck and back pain amongst university students. Measures are needed to prevent neck and back pain in this group, considering that increased physical activity and enhanced home office equipment might be beneficial.Copyright © 2022 Elsevier GmbH

Ensuring Quality Care in the COVID-19 Era: Applying the Donabedian Model to Tertiary Wound Care Center Practices.

The COVID-19 pandemic has disrupted healthcare, with its far-reaching effects seeping into chronic disease evaluation and treatment. Our tertiary wound care center was specially designed to deliver the highest quality care to wounded patients. Pre-pandemic, we were able to ensure rapid treatment via validated protocols delivered by a co-localized multidisciplinary team within the hospital setting. The pandemic has disrupted our model's framework, and we have worked to adapt our workflow without sacrificing quality of care. Using the modified Donabedian model of quality assessment, we present an analysis of pre- and intra-pandemic characteristics of our center. In this way, we hope other providers can use this framework for identifying evolving problems within their practice so that quality care can continue to be delivered to all patients.

Proximity-Induced Nucleic Acid Degrader (PINAD) Approach to Targeted RNA Degradation Using Small Molecules.

Nature has evolved intricate machinery to target and degrade RNA, and some of these molecular mechanisms can be adapted for therapeutic use. Small interfering RNAs and RNase H-inducing oligonucleotides have yielded therapeutic agents against diseases that cannot be tackled using protein-centered approaches. Because these therapeutic agents are nucleic acid-based, they have several inherent drawbacks which include poor cellular uptake and stability. Here we report a new approach to target and degrade RNA using small molecules, proximity-induced nucleic acid degrader (PINAD). We have utilized this strategy to design two families of RNA degraders which target two different RNA structures within the genome of SARS-CoV-2: G-quadruplexes and the betacoronaviral pseudoknot. We demonstrate that these novel molecules degrade their targets using in vitro, in cellulo, and in vivo SARS-CoV-2 infection models. Our strategy allows any RNA binding small molecule to be converted into a degrader, empowering RNA binders that are not potent enough to exert a phenotypic effect on their own. PINAD raises the possibility of targeting and destroying any disease-related RNA species, which can greatly expand the space of druggable targets and diseases.

Creating a Process for the Implementation of Tiered Huddles in a Veterans Affairs Medical Center.

INTRODUCTION: In 2019, the Veteran's Health Administration began its journey in pursuit of becoming an enterprise-wide High Reliability Organization (HRO). Improving the delivery of safe, high quality patient care is a central focus of HROs. Requisite to meeting this goal is the timely identification and resolution of problems. This is best achieved by empowering and engaging both clinical and non-clinical staff across the healthcare organization through the promotion of robust collaboration and communication between various disciplines. Improved care coordination and increased accountability are two important subsequent outcomes. One method for accomplishing this is through the implementation of tiered huddles. MATERIALS AND METHODS: An extensive review of the current literature from 2013 until June 2021 was conducted for evidence highlighting the experiences of other healthcare organizations during implementation of huddles. Following the review, a tiered huddle proposal was developed and presented to the executive leadership team of a healthcare system for approval. Pilot testing of the tiered huddle implementation plan began in October 2021 over a 12-week period with three services. On average, the pilot services had between three to four tiers from frontline staff to the executive level of leadership. RESULTS: Over the 12-week period, out of the possible 120 tiered huddles that could have been conducted, 68% (n = 81) were completed. Of the tiered huddles conducted, 99% (n = 80) started and ended on time. During the pilot test, seven issues were identified by frontline staff: coordination of pre-procedural coronavirus testing, equipment/computer issues, rooms out of service, staffing levels, and lack of responsiveness from other departments. Issues related to staffing, unresponsiveness from other departments, and equipment concerns required elevation to a higher-level tier with no issues remaining open. Delays in patient care, or prolongation of shift hours for staff because of tiered huddles, was low at 2.5% (n = 2). For the duration of the pilot test, a total of 75 minutes accounted for shifts being extended among five staff members. CONCLUSIONS: The success of this initiative demonstrates the importance of thoughtfully creating a robust process when planning for the implementation of tiered huddles. The findings from this initiative will be of immense value with the implementation of tiered huddles across our healthcare system. We believe that this approach can be used by other healthcare institutions along their journey to improving patient safety and quality.

The Influence of COVID-19 Disease on Pre-Analytical Blood Sample Haemolysis Rates in Three Acute Medical Units: An Interrupted Time Series Analysis

The COVID-19 pandemic impacted delivery of health services. The aim of our study was to determine the impact of COVID-19 disease on pre-analytical blood sample haemolysis by modelling the daily haemolysis rates variations pre and post COVID-19 infections. Ethics approval was obtained prior to study commencing. Interrupted Time Series data analysis was conducted on UK National Health Service Acute Admissions Unit 25-month (1 February 2019 to 28 February 2021) biochemistry (total and haemolysed) blood sample dataset. Interruption was set on 23 March 2021, the start of the first UK lockdown. Daily haemolysis rate (% samples haemolysed) data were fitted with a spline curve to determine influence of haemolysis rates on short or medium-term temporal trends. Linear regression was performed so as to determine long-term temporal trends pre- and post-intervention. There were 32,316 biochemistry blood sample results: 19,058 pre and 13,258 (342 days) from the post-intervention period. Overall median daily haemolysis rate was 7.3% (range: 0-30.6%), 7.7% pre-intervention versus 6.5% post-intervention (p<0.0001). The proportion of haemolysis cases negatively correlated with the number of samples processed (rho=0.09;p=0.01). The pre-intervention slope was -1.70 %.y-1, y intercept 9.04%;post-intervention slope was -1.88%.y-1, y intercept was 10.2%;with no difference in either the slope (p=0.87) or intercept (p=0.16). There was no association between short-term variation in haemolysis rates with changes in practice due to COVID-19 disease and the disease itself. The negative correlation between haemolysis rate and the number of samples processed highlights the importance of continued venepuncture practice to facilitate haemolysis rate reduction.

Safety of COVID-19 vaccines in multiple sclerosis: A systematic review and meta-analysis.

BACKGROUND: Data are sparse regarding the safety of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines in patients with multiple sclerosis (MS). OBJECTIVE: To estimate (1) the pooled proportion of MS patients experiencing relapse among vaccine recipients; (2) the rate of transient neurological worsening, adverse events, and serious adverse events; (3) the previous outcomes of interest for different SARS-CoV-2 vaccine types. METHODS: Systematic review and meta-analysis of pharmacovigilance registries and observational studies. RESULTS: Nineteen observational studies comprising 14,755 MS patients who received 23,088 doses of COVID-19 vaccines were included. Mean age was 43.3 years (95% confidence interval (CI): 40-46.6); relapsing-remitting, secondary-progressive, primary-progressive MS and clinically isolated syndrome were diagnosed in 82.6% (95% CI: 73.9-89.8), 12.6% (95% CI: 6.3-20.8), 6.7% (95% CI: 4.2-9.9), and 2.9% (95% CI: 1-5.9) of cases, respectively. The pooled proportion of MS patients experiencing relapse at a mean time interval of 20 days (95% CI: 12-28.2) from vaccination was 1.9% (95% CI: 1.3%-2.6%; I2 = 78%), with the relapse risk being independent of the type of administered SARS-CoV-2-vaccine (p for subgroup differences = 0.7 for messenger RNA (mRNA), inactivated virus, and adenovector-based vaccines). After vaccination, transient neurological worsening was observed in 4.8% (95% CI: 2.3%-8.1%) of patients. Adverse events and serious adverse events were reported in 52.8% (95% CI: 46.7%-58.8%) and 0.1% (95% CI: 0%-0.2%) of vaccinations, respectively. CONCLUSION: COVID-19 vaccination does not appear to increase the risk of relapse and serious adverse events in MS. Weighted against the risks of SARS-CoV-2-related complications and MS exacerbations, these safety data provide compelling pro-vaccination arguments for MS patients.

SARS-CoV-2 Spike N-Terminal Domain Engages 9-O-Acetylated α2-8-Linked Sialic Acids.

SARS-CoV-2 viruses engage ACE2 as a functional receptor with their spike protein. The S1 domain of the spike protein contains a C-terminal receptor binding domain (RBD) and an N-terminal domain (NTD). The NTD of other coronaviruses includes a glycan binding cleft. However, for the SARS-CoV-2 NTD, protein-glycan binding was only observed weakly for sialic acids with highly sensitive methods. Amino acid changes in the NTD of variants of concern (VoC) show antigenic pressure, which can be an indication of NTD-mediated receptor binding. Trimeric NTD proteins of SARS-CoV-2, alpha, beta, delta, and omicron did not reveal a receptor binding capability. Unexpectedly, the SARS-CoV-2 beta subvariant strain (501Y.V2-1) NTD binding to Vero E6 cells was sensitive to sialidase pretreatment. Glycan microarray analyses identified a putative 9-O-acetylated sialic acid as a ligand, which was confirmed by catch-and-release ESI-MS, STD-NMR analyses, and a graphene-based electrochemical sensor. The beta (501Y.V2-1) variant attained an enhanced glycan binding modality in the NTD with specificity toward 9-O-acetylated structures, suggesting a dual-receptor functionality of the SARS-CoV-2 S1 domain, which was quickly selected against. These results indicate that SARS-CoV-2 can probe additional evolutionary space, allowing binding to glycan receptors on the surface of target cells.

National trends in prescription drug expenditures and projections for 2023.

PURPOSE: To report historical patterns of pharmaceutical expenditures, to identify factors that may influence future spending, and to predict growth in drug spending in 2023 in the United States, with a focus on the nonfederal hospital and clinic sectors. METHODS: Historical patterns were assessed by examining data on drug purchases from manufacturers using the IQVIA National Sales Perspectives database. Factors that may influence drug spending in hospitals and clinics in 2023 were reviewed, including new drug approvals, patent expirations, and potential new policies or legislation. Focused analyses were conducted for biosimilars, cancer drugs, diabetes medications, generics, COVID-19 pandemic influence, and specialty drugs. For nonfederal hospitals, clinics, and overall (all sectors), estimates of growth of pharmaceutical expenditures in 2023 were based on a combination of quantitative analyses and expert opinion. RESULTS: In 2022, overall pharmaceutical expenditures in the US grew 9.4% compared to 2021, for a total of $633.5 billion. Utilization (a 5.9% increase), price (a 1.7% increase) and new drugs (a 1.8% increase) drove this increase. Adalimumab was the top-selling drug in 2022, followed by semaglutide and apixaban. Drug expenditures were $37.2 billion (a 5.9% decrease) and $116.9 billion (a 10.4% increase) in nonfederal hospitals and clinics, respectively. In clinics, new products and increased utilization growth drove growth, with a small impact from price changes. In nonfederal hospitals, a drop in utilization led to a decrease in expenditures, with price changes and new drugs contributing to growth in spending. Several new drugs that will influence spending have been or are expected to be approved in 2023. Specialty and cancer drugs will continue to drive expenditures along with the evolution of the COVID-19 pandemic. CONCLUSION: For 2023, we expect overall prescription drug spending to rise by 6.0% to 8.0%, whereas in clinics and hospitals we anticipate increases of 8.0% to 10.0% and 1.0% to 3.0%, respectively, compared to 2022. These national estimates of future pharmaceutical expenditure growth may not be representative of any particular health system because of the myriad of local factors that influence actual spending.

Parental compliance and reasons for COVID-19 Vaccination among American children.

COVID-19 vaccination rates among children have stalled, while new coronavirus strains continue to emerge. To improve child vaccination rates, policymakers must better understand parental preferences and reasons for COVID-19 vaccination among their children. Cross-sectional surveys were administered online to 30,174 US parents with at least one child of COVID-19 vaccine eligible age (5-17 years) between January 1 and May 9, 2022. Participants self-reported willingness to vaccinate their child and reasons for refusal, and answered additional questions about demographics, pandemic related behavior, and vaccination status. Willingness to vaccinate a child for COVID-19 was strongly associated with parental vaccination status (multivariate odds ratio 97.9, 95% confidence interval 86.9-111.0). The majority of fully vaccinated (86%) and unvaccinated (84%) parents reported concordant vaccination preferences for their eligible child. Age and education had differing relationships by vaccination status, with higher age and education positively associated with willingness among vaccinated parents. Among all parents unwilling to vaccinate their children, the two most frequently reported reasons were possible side effects (47%) and that vaccines are too new (44%). Unvaccinated parents were much more likely to list a lack of trust in government (41% to 21%, p < .001) and a lack of trust in scientists (34% to 19%, p < .001) as reasons for refusal. Cluster analysis identified three groups of unwilling parents based on their reasons for refusal to vaccinate, with distinct concerns that may be obscured when analyzed in aggregate. Factors associated with willingness to vaccinate children and reasons for refusal may inform targeted approaches to increase vaccination.