Effects of endoscopic reprocessing on disinfection and its influential factors during coronavirus disease 2019 pandemic.

Objective To explore the effects of endoscopic reprocessing on disinfection and its influential factors under the coronavirus disease 2019 (COVID-19) pandemic. Methods A total of 450 endoscopes cleaned and disinfected according to Technical Specifications for Cleaning and Disinfection of Endoscopes from November 2019 to January 2020, and 450 endoscopes cleaned and disinfected according to The recommended procedure for cleaning and disinfection of gastrointestinal endoscopes during COVID-19 epidemic by Chinese Society of Digestive Endoscopology from February to April 2020 in the Second Affiliated Hospital of Chongqing Medical University were enrolled in the control group and observation group respectively by random number method. Both the control group and the observation group contained 200 gastroscopes, 200 enteroscopes and 50 ultrasound endoscopes. ATP fluorescence detection method and pour plate technique were used to evaluate the disinfection effect of endoscopes. Single factor analysis and multiple logistic regression were used to analyze the risk factors for unqualified sterilization after endoscopic reprocessing. Results The disinfection pass rates of gastroscopes, enteroscopes and ultrasound endoscopes in the observation group were not significantly different compared with those of the control group (P>0. 05). The sterilization pass rates and ATP test pass rates of gastroscopes, enteroscopes and ultrasound endoscopes in the observation group were significantly higher than those in the control group (all P<0. 05). Multivariate logistic regression analysis showed that non-strict implementation of endoscopic reprocessing (OR = 7. 96, 95%CI: 4. 55-22. 84, P<0. 001), non-standard operation (OR = 2. 26, 95%CI: 1. 24-5. 63, P<0. 001), insufficient concentration of disinfectant (OR = 5. 43, 95% CI: 2. 52-9. 02, P < 0. 001), insufficient concentration ratio of multi-enzyme solution (OR = 4. 38, 95% CI: 1. 95-8. 61, P < 0. 001), non-timely cleaning (OR= 2. 86, 95%CI: 1. 33-6. 42, P<0. 001), incomplete cleaning (OR = 3. 75, 95%CI: 1. 61-7. 49, P<0. 001) and improper endoscopic preservation (OR= 2. 12, 95%CI: 1. 36-4. 12, P<0. 001) were independent risk factors for unqualified sterilization after endoscopic reprocessing. Conclusion In COVID-19 pandemic, endoscope reprocessing can significantly improve the disinfection effect of endoscopes, worthy of further clinical promotion. The failure to strictly implement the reprocessing procedure is an important factor that may lead to unqualified sterilization.Copyright © 2021 The authors.

Perceived Attitudes Toward Vaccines, Trust and Vaccination Behaviors

Objectives: The decision-making process for taking vaccination is influenced by a multitude of factors such as individual beliefs concerning vaccinations, trust in contextual forces, and sociodemographic. This study established a model to understand the relationship between people's beliefs in the safety, importance and effectiveness of vaccines, their trust in the medical advice from the government and doctors and their behaviors of having their children vaccinated from infectious diseases in low-and-middle-income countries (LMIC). Method(s): We structured a structural equation model with two latent variables, Motivation and Trust, and their relationships with the vaccination taking behavior. Motivation is constructed by people's beliefs in the safety, importance and effectiveness of vaccines and trust is constructed by people's trust in government, medical providers and scientists. This study used the 2018 Wellcome Global Monitor dataset and focused on people in 80 LMIC. The countries were divided into eight geographic regions: Eastern Africa, Central & Southern Africa, Norther Africa & Middle East, Western Africa, Central Asia, Southeast Asia, South Asia and Southern& Eastern Europe. Result(s): The latent variable Motivation is significantly positively associated with parental vaccination behaviors in all geographic areas except for South Asia and Western Africa. South Asia is the only area where the trust in government and medical system, providers had a significant association with vaccination behavior and such association is positive. Conclusion(s): In most LMIC, positive attitudes about vaccines are associated with an improved vaccine rate. Increasing people's belief in vaccines' importance, safety and effectiveness will be essential both for boosting vaccination rates and scaling up a vaccine for COVID-19. In South Asia, trust in the government and the public health system are important in deciding taking vaccines. In these countries, policymakers need to think of ways to improve people's trust in the public health system and further effectively communicate important health messages.Copyright © 2023

SARS-CoV-2 Invasion and Pathogenesis of COVID-19: A Perspective of Viral Receptors, Bradykinin, and Purinergic System

The present chapter focuses on the mechanisms of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection, pathogenesis, and the possible therapeutic strategies targeted to the viral receptors, purinergic and kallikrein-kinin systems. SARS-CoV-2 spike protein binds with high affinity to the human ACE2 receptor on host cells, but it can also interact with other receptors and enzymes. Following viral infection, a plethora of subsequent molecular and cellular alterations occurs in the host. These alterations, which include the cytokine and bradykinin storms, as well as exacerbated ATP signalling, have been implicated in the genesis and progression of the signs and symptoms observed in COVID-19 patients. These routes and systems provide important targets for developing specific and effective anti-COVID-19 drugs, as well as reveal a novel understanding of pathogenesis and tropism of SARS-CoV-2. © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2022.

Recent progress in online detection methods of bioaerosols

The aerosol transmission of coronavirus disease in 2019, along with the spread of other respiratory diseases, caused significant loss of life and property;it impressed upon us the importance of real-time bioaerosol detection. The complexity, diversity, and large spatiotemporal variability of bioaerosols and their external/internal mixing with abiotic components pose challenges for effective online bioaerosol monitoring. Traditional methods focus on directly capturing bioaerosols before subsequent time-consuming laboratory analysis such as culture-based methods, preventing the high-resolution time-based characteristics necessary for an online approach. Through a comprehensive literature assessment, this review highlights and discusses the most commonly used real-time bioaerosol monitoring techniques and the associated commercially available monitors. Methods applied in online bioaerosol monitoring, including adenosine triphosphate bioluminescence, laser/light-induced fluorescence spectroscopy, Raman spectroscopy, and bioaerosol mass spectrometry are summarized. The working principles, characteristics, sensitivities, and efficiencies of these real-time detection methods are compared to understand their responses to known particle types and to contrast their differences. Approaches developed to analyze the substantial data sets obtained by these instruments and to overcome the limitations of current real-time bioaerosol monitoring technologies are also introduced. Finally, an outlook is proposed for future instrumentation indicating a need for highly revolutionized bioaerosol detection technologies.

Pediatric Cardiac Screening Data Warehouse: A National Public Health Resource

Background: Cardiac screening of youth for prevention of sudden cardiac death in the young (SCDY) has been debated due to the absence of large population-specific screening data with outcomes. Despite years of screening by US public screening groups (PSG), there is minimal coordination of effort and no standardized methods for real-world data collection. Objective(s): To understand the methods, quality, outcomes, and best practices of youth screening, the Cardiac Safety Research Consortium Pediatric Cardiology Working Group, in collaboration with FDA and PSGs, developed and enabled a scalable system to collect a uniform pediatric cardiac screening dataset including digital ECGs and post-screening electronic follow-up data. Method(s): Front end data collection (figure) was developed to include use of a universal unique ID system to align paper/digital collection of health and ECG data. PSGs use secure data transfer portals for digital ECG data upload for conversion to device-agnostic standardized FDA format to store in the national pediatric cardiac screening data warehouse. Follow-up data are obtained at designated post-screening intervals (one week, one and 3 months for pilot study) using initial text message contact followed by electronic consent (REDCap) and answering online health surveys. Result(s): Fourteen PSGs in ten states participated in the pilot study. PSG warehouse data include 33840 retrospective ECG datasets collected from 2010 to 2021 containing limited screened history/symptoms but demographics similar to US census as follows: Age 13-30y, Male/Female 57/43%, Asian 6%, Black 19%, Native American <1%, Pacific Islander <1%, White 68%, Other 4%;Hispanic/Non-Hispanic 27%/79%. Individual PSG site demographics reflected local populations. Prospective data collection since 2021 include >4000 uniform screening datasets (age, sex, race, ethnicity, ht, wt, screening H&P, COVID history, medications, digital ECG with results, screening outcome, and, if applicable, ECHO results). Follow up participation allowing initial cellular contact was high (avg 73%, range 51-91%/screening). Conclusion(s): Establishment of a national pediatric cardiac data warehouse enables large-scale aggregation of pediatric cardiac screening information to address deficits in the understanding and prevention of SCDY. This large real-world dataset will help establish normative data for pediatric ECGs which can facilitate development of new diagnostic tools such as machine learning and support pediatric drug and device development. [Formula presented]Copyright © 2023

Covid-19 Vaccination Impact on Atrial Arrhythmias - Real-World Evidence from the Certitude Registry

Background: The COVID-19 vaccines were developed unprecedentedly and have proven safe and efficacious in reducing transmissibility and severe infection. The impact of mRNA-based COVID-19 vaccines on atrial arrhythmias (AA) incidence is unknown. Objective(s): To analyze the incidence of AA after COVID-19 vaccination in patients with a cardiac implantable electronic device (CIED). Method(s): BIOTRONIK Home Monitoring data and Medicare claims data from CERTITUDE patients implanted with a CIED between 2010-20 were utilized to identify recipients of one or more doses of the COVID-19 vaccine in 2021. Those who had influenza vaccination in 2020 were also identified in the same cohort as a control. From remote monitoring data, the number of atrial high rate events (AHR) and % burden of AA in the three months post-vaccination was compared to the preceding three months using Wilcoxon signed rank test. Kruskal-Wallis test was used for group difference comparisons. New AF diagnosis was determined from ICD-10 diagnosis codes in Medicare claims. Result(s): First and 2nd doses of COVID vaccine (50% Pfizer, 47% Moderna, and 3% J&J) were administered to 7757 and 6579 individuals with a CIED (age 76.2 (+/-9.0) y, 49% males), respectively. In the same cohort, 4723 (61%) individuals received the influenza vaccine. A statistically significant increase in the number of AHR episodes and % burden of AA was noted in the three months post-vaccination compared to the preceding three months after the 1st and 2nd doses of the COVID-19 vaccine (Figure). No such association was noted following influenza vaccination. In subgroup analysis, AHR episodes increased significantly in age groups >70 and men. Post-vaccination increase in AHR episodes was more significant in those without a pre-vaccination history of AHR episodes (mean increase of AHR 6.9+/-88.4, p<0.001) and was non-significant in those with a preceding history of AHR (p=0.8). Among the 764 patients with no AF diagnosis in claims preceding the first COVID-19 vaccine, 87 (11.4%) developed a new AF diagnosis or AHR event in the first three months post-vaccination. Conclusion(s): We report a small but significant increase in the number of CIED-detected atrial arrhythmias following vaccination for COVID-19 but not influenza, specifically in men and age >70 years. Acknowledging the immense public health benefit of COVID-19 vaccines, our results should prompt increased awareness of evaluating for AF in this high-risk group following vaccination. [Formula presented]Copyright © 2023

First-in-class small molecule drugs in 2022

2022 is the third year of the global COVID-19 pandemic, and its troubles on new drug discovery are gradually apparent. 37 new drugs were approved by the FDA's Center for Drug Evaluation and Research (CDER) last year, down from the peak of 50 new drug approvals in 2021. Notably, first-in-class drugs still occupy a dominant position this year, with a total of 21 drugs. Among them, 7 are first-in-class small molecule drugs. Although the total number of new drug approvals in 2022 sharply decreased, some first-in-class small molecule drugs were regarded as significant, including mitapivat, the first oral activator targeting the pyruvate kinase (PK);mavacamten, the first selective allosteric inhibitor targeting the myocardial beta myosin ATPase;deucravacitinib, the first deuterated allosteric inhibitor targeting the tyrosine kinase 2 (TYK2);and lenacapavir, the first long-acting inhibitor targeting the HIV capsid. Generally, the research of first-in-class drugs needs to focus on difficult clinical problems and can treat some specific diseases through novel targets and biological mechanisms. There are tremendous challenges in the research processes of new drugs, including biological mechanism research, target selection, molecular screening, lead compound identification and druggability optimization. Therefore, the success of first-in-class drugs development has prominent guidance significance for new drug discovery. This review briefly describes the discovery background, research and development process and therapeutic application of 3 firstin- class small molecule drugs to provide research ideas and methods for more first-in-class drugs.Copyright © 2023, Chinese Pharmaceutical Association. All rights reserved.

Changes in the proteomics of exhaled breath condensate under the influence of inhaled hydrogen in patients with post-COVID syndrome.

Aim. To study the effect of inhalation therapy with an active hydrogen (AH) on the protein composition of exhaled breath condensate (EBC) in patients with post-COVID syndrome (PCS). Material and methods. This randomized controlled parallel prospective study included 60 patients after coronavirus disease 2019 (COVID-19) with PCS during the recovery period and clinical manifestations of chronic fatigue syndrome who received standard therapy according to the protocol for managing patients with chronic fatigue syndrome (CFS). The patients were divided into 2 groups: group 1 (main) - 30 people who received standard therapy and AH inhalations (SUISONIA, Japan) for 10 days, and group 2 (control) - 30 medical workers who received only standard therapy. Patients in both groups were comparable in sex and mean age. All participants in the study were sampled with EBC on days 1 and 10. Samples were subjected to tryptic digestion and high-performance liquid chromatography combined with tandem mass spectrometry analysis using a nanoflow chromatograph (Dionex 3000) in tandem with a high-resolution time-of-flight mass spectrometer (timsTOF Pro). Results. A total of 478 proteins and 1350 peptides were identified using high resolution mass spectrometry. The number of proteins in samples after AH therapy, on average, is 12% more than before treatment. An analysis of the distribution of proteins in different groups of patients showed that only half of these proteins (112) are common for all groups of samples and are detected in EBC before, after, and regardless of hydrogen therapy. In addition to the qualitative difference in the EBC protein compositions in different groups, quantitative changes in the concentration of 36 proteins (mainly structural and protective) were also revealed, which together made it possible to reliably distinguish between subgroups before and after treatment. It is worth noting that among these proteins there are participants of blood coagulation (alpha-1-antitrypsin), chemokine- and cytokine-mediated inflammation, and a number of signaling pathways (cytoplasmic actin 2), response to oxidative stress (thioredoxin), glycolysis (glyceraldehyde-3- phosphate dehydrogenase), etc. Conclusion. The use of hydrogen therapy can contribute to the switching of a number of physiological processes, which may affect the success of recovery in PCS patients. In particular, the obtained results indicate the activation of aerobic synthesis of adenosine triphosphate in mitochondria by hydrogen therapy, which correlates well with the decrease in the blood lactate level detected by laboratory studies. At the same time, this therapy can inhibit pro-inflammatory activity, negatively affecting the coagulation and signaling pathways of integrins and apoptosis, and, in addition, activate protective pathways, tricarboxylic acid cycle, FAS signaling, and purine metabolism, which may be essential for effective recovery after COVID-19.Copyright © 2023 Vserossiiskoe Obshchestvo Kardiologov. All rights reserved.

Targeting virus-induced vulnerabilities using synthetic lethality as a new class of host-based antivirals

RNA viruses are the major class of human pathogens responsible for many global health crises, including the COVID-19 pandemic. However, the current repertoire of U.S. Food and Drug Administration (FDA)-approved antivirals is limited to only nine out of the known 214 human-infecting RNAviruses, and almost all these antivirals target viral proteins. Traditional antiviral development generally proceeds in a virus-centric fashion, and successful therapies tend to be only marginally effective as monotherapies, due to dose-limiting toxicity and the rapid emergence of drug resistance. Host-based antivirals have potential to alleviate these shortcomings, but do not typically discriminate between infected and uninfected cells, thus eliciting unintended effects. In infected cells where host proteins are repurposed by a virus, normal host protein functions are compromised;a situation analogous to a loss-of-function mutation, and cells harboring the hypomorph have unique vulnerabilities. As well-established in model systems and in cancer therapeutics, these uniquely vulnerable cells can be selectively killed by a drug that inhibits a functionally redundant protein. This is the foundation of synthetic lethality (SL). To test if viral induced vulnerabilities can be exploited for viral therapeutics, we selectively targeted synthetic lethal partners of GBF1, a Golgi membrane protein and a critical host factor for many RNA viruses including poliovirus, Coxsackievirus, Dengue, Hepatitis C and E virus, and Ebola virus. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. A genome-wide chemogenomic CRISPR screen identified synthetic lethal partners of GBF1 and revealed ARF1 as the top hit. Disruption of ARF1, selectively killed cells that synthesize poliovirus 3A alone or in the context of a poliovirus replicon. Combining 3A expression with sub-lethal amounts of GCA - a specific inhibitor of GBF1 further exacerbated the GBF1-ARF1 SL effect. Together our data demonstrate proof of concept for host-based SL targeting of viral infection. We are currently testing all druggable synthetic lethal partners of GBF1 from our chemogenomic CRISPR-screen, in the context of dengue virus infection for their abilities to selectively kill infected cells and inhibit viral replication and infection. Importantly, these SL gene partners of viral-induced hypomorphs only become essential in infected cells and in principle, targeting them will have minimal effects on uninfected cells. Our strategy to target SL interactions of the viral-induced hypomorph has the potential to change the current paradigm for host-based therapeutics that can lead to broad-spectrum antivirals and can be applied to other intracellular pathogens. This work is supported by National Institutes of Health grants R01 GM112108 and P41 GM109824, R21 AI151344 and foundation grant FDN-167277 from the Canadian Institutes of Health Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

SARS-CoV-2 Nsp13 Catalytic Efficiency is Regulated by ATP: Mg2+ Stoichiometry and Functional Cooperativity Among Nsp13 Molecules

Coronavirus disease 19 (COVID-19) is a highly contagious and lethal disease caused by the SARS-CoV-2 positive-strand RNA virus. Nonstructural protein 13 (Nsp13) is the highly conserved ATPase/helicase required for replication of the SARS-CoV-2 genome which allows for the infection and transmission of COVID-19. We biochemically characterized the purified recombinant SARS-CoV-2 Nsp13 helicase protein expressed using a eukaryotic cell-based system and characterized its catalytic functions, focusing on optimization of its reaction conditions and assessment of functional cooperativity among Nsp13 molecules during unwinding of duplex RNA substrates. These studies allowed us to carefully determine the optimal reaction conditions for binding and unwinding various nucleic acid substrates. Previously, ATP concentration was suggested to be an important factor for optimal helicase activity by recombinant SARS-CoV-1 Nsp13. Apart from a single study conducted using fixed concentrations of ATP, the importance of the essential divalent cation for Nsp13 helicase activity had not been examined. Given the importance of the divalent metal ion cofactor for ATP hydrolysis and helicase activity, we assessed if the molar ratio of ATP to Mg2+ was important for optimal SARS-CoV-2 Nsp13 RNA helicase activity. We determined that Nsp13 RNA helicase activity was dependent on ATP and Mg2+ concentrations with an optimum of 1 mM Mg2+ and 2 mM ATP. Next, we examined Nsp13 helicase activity as a function of equimolar ATP:Mg2+ ratio and determined that helicase activity decreased as the equimolar concentration increased, especially above 5 mM. We determined that Nsp13 catalytic functions are sensitive to Mg2+ concentration suggesting a regulatory mechanism for ATP hydrolysis, duplex unwinding, and protein remodeling, processes that are implicated in SARS-CoV-2 replication and proofreading to ensure RNA synthesis fidelity. Evidence is presented that excess Mg2+ impairs Nsp13 helicase activity by dual mechanisms involving both allostery and ionic strength. In addition, using single-turnover reaction conditions, Nsp13 unwound partial duplex RNA substrates of increasing doublestranded regions (16-30 base pairs) with similar kinetic efficiency, suggesting the enzyme unwinds processively in this range under optimal reaction conditions. Furthermore, we determined that Nsp13 displayed sigmoidal behavior for helicase activity as a function of enzyme concentration, suggesting that functional cooperativity and oligomerization are important for optimal activity. The observed functional cooperativity of Nsp13 protomers suggests the essential coronavirus RNA helicase has roles in RNA processing events beyond its currently understood involvement in the SARS-CoV-2 replication-transcription complex (RTC), in which it was suggested that only one of the two Nsp13 subunits has a catalytic function, whereas the other has only a structural role in complex stability. Altogether, the intimate regulation of Nsp13 RNA helicase by divalent cation and protein oligomerization suggests drug targets for modulation of enzymatic activity that may prove useful for the development of novel anti-coronavirus therapeutic strategies. This work was supported by the Intramural Training Program, National Institute on Aging (NIA), NIH, and a Special COVID-19 Grant from the Office of the Scientific Director, NIA, NIH.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Predictors of poor outcomes in patients with pandemic viral infections (influenza AH1N1pdm09, COVID-19) on a ventilator.

Considering the commonality of the pathogenetic links of the critical forms of COVID-19 and influenza AH1N1pdm09 (cytokine over-release syndrome), the question arises: will the predictors of an unfavorable outcome in patients on mechanical ventilation and, accordingly, the universal tactics of respiratory support in these diseases be identical? Objective. In a comparative aspect, to characterize patients with influenza AH1N1pdm09 and COVID-19 who were on mechanical ventilation, to identify additional clinical and laboratory risk factors for death, to determine the degree of influence of respiratory support (RP) tactics on an unfavorable outcome in the studied category of patients. Patients and methods. Patients treated on the basis of resuscitation and intensive care departments of the State Budgetary Healthcare Institution "SKIB" in Krasnodar and the State Budgetary Healthcare Institution "IB No 2" in Sochi were studied: group 1 - 31 people with influenza AH1N1pdm09 (21 people died - subgroup 1A;10 people survived - subgroup 1B) and group 2 - 50 people with COVID-19 (29 patients died - subgroup 2A;21 people survived - subgroup 2B). All patients developed hypoxemic ARF. All patients received step-by-step tactics of respiratory support, starting with oxygen therapy and ending with the use of "traditional" mechanical ventilation. Continuous variables were compared in subgroups of deceased and surviving patients for both nosologies at the stages: hospital admission;registration of hypoxemia and the use of various methods of respiratory therapy;development of multiple organ dysfunctions. With regard to the criteria for which a statistically significant difference was found (p < 0.05), we calculated a simple correlation, the relative risk of an event (RR [CI 25-75%]), the cut-off point, which corresponded to the best combination of sensitivity and specificity. Results. Risk factors for death of patients with influenza AH1N1pdm09 on mechanical ventilation: admission to the hospital later than the 8th day of illness;the fact of transfer from another hospital;leukocytosis >=10.0 x 109/l, granulocytosis >=5.5 x 109/l and LDH level >=700.0 U/l at admission;transfer of patients to mechanical ventilation on the 9th day of illness and later;SOFA score >=8;the need for pressor amines and replacement of kidney function. Predictors of poor outcome in ventilated COVID-19 patients: platelet count <=210 x 109/L on admission;the duration of oxygen therapy for more than 4.5 days;the use of HPNO and NIV as the 2nd step of RP for more than 2 days;transfer of patients to mechanical ventilation on the 14th day of illness and later;oxygenation index <=80;the need for pressors;SOFA score >=8. Conclusion. When comparing the identified predictors of death for patients with influenza and COVID-19 who needed mechanical ventilation, there are both some commonality and differences due to the peculiarities of the course of the disease. A step-by-step approach to the application of respiratory support methods is effective both in the case of patients with influenza AH1N1pdm09 and patients with COVID-19, provided that the respiratory support method used is consistent with the current state of the patient and his respiratory system, timely identification of markers of ineffectiveness of the respiratory support stage being carried out and determining the optimal moment escalation of respiratory therapy.Copyright © 2022, Dynasty Publishing House. All rights reserved.

The SARS-CoV-2 Nsp13 Helicase Required for Coronavirus Replication Interacts with Structured Nucleic Acids in a Strand-Specific Manner and Catalyzes a Novel Protein-RNA Remodeling Activity Implicated in the Proofreading Mechanism of the Replication-Transc

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded, positive-sense RNA virus responsible for COVID-19, requires a set of virally encoded nonstructural proteins that compose a replication-transcription complex (RTC) to replicate its 30 kilobase genome. One such nonstructural protein within the RTC is Nsp13, a highly conserved molecular motor ATPase/helicase. Upon purification of the recombinant SARS-CoV-2 Nsp13 protein expressed using a eukaryotic cell-based system, we biochemically characterized the enzyme by examining its catalytic functions, nucleic acid substrate specificity, and putative protein-nucleic acid remodeling activity. We determined that Nsp13 preferentially interacts with single-stranded (ss) DNA compared to ssRNA during loading to unwind with greater efficiency a partial duplex helicase substrate. The binding affinity of Nsp13 to nucleic acid was confirmed through electrophoretic mobility shift assays (EMSA) by determining that Nsp13 binds to DNA substrates with significantly greater efficiency than RNA. These results demonstrate strand-specific interactions of SARS-CoV-2 Nsp13 that dictate its ability to load and unwind structured nucleic acid substrates. We next determined that Nsp13 catalyzed unwinding of double-stranded (ds) RNA forked duplexes on substrates containing a backbone disruption (neutrally charged polyglycol linker (PGL)) was strongly inhibited when the PGL was positioned in the 5' ssRNA overhang, suggesting an unwinding mechanism in which Nsp13 is strictly sensitive to perturbation of the translocating strand sugar-phosphate backbone integrity. Furthermore, we demonstrated for the first time the ability of the coronavirus Nsp13 helicase to disrupt a high-affinity nucleic acid-protein interaction, i.e., a streptavidin tetramer bound to biotinylated RNA or DNA substrate, in a uni-directional manner and with a preferential displacement of the streptavidin complex from biotinylated ssDNA versus ssRNA. In contrast to the poorly hydrolysable ATP-gamma-S or non-hydrolysable AMP-PNP, ATP supports Nsp13-catalyzed disruption of the nucleic acidprotein complex, suggesting that nucleotide binding by Nsp13 is not sufficient for protein-RNA disruption and the chemical energy of nucleoside triphosphate hydrolysis is required to fuel remodeling of protein bound to RNA or DNA. Our results build upon structural studies of the SARS-CoV-2 RTC in which it was suggested that Nsp13 pushes the RNA polymerase (Nsp12) backward on the template RNA strand. Experimental evidence from our studies demonstrate that Nsp13 helicase efficiently remodels a large high affinity protein-RNA complex in a manner dependent on its intrinsic ATP hydrolysis function. We proposed that this novel biochemical activity of Nsp13 is relevant to its role in SARS-CoV-2 RNA processing functions and replication. It was proposed that Nsp13 facilitates proofreading during coronavirus replication when a mismatched base is inadvertently incorporated into the SARS-CoV-2 genome during replication to reposition the RTC so that the proofreading nuclease complex (Nsp14-Nsp10) can gain access and remove the nascently synthesized nucleotide to ensure polymerase fidelity. Our findings implicate a direct catalytic role of Nsp13 in protein-RNA remodeling during coronavirus genome replication beyond its duplex strand separation or structural stabilization of the RTC, yielding new insight into the proofreading mechanism. This work was supported by the Intramural Training Program, National Institute on Aging (NIA), NIH, and a Special COVID-19 Grant from the Office of the Scientific Director, NIA, NIH.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Mitochondrial Dysfunction Is Associated with Post-Acute Sequelae of Covid-19

Background: Mitochondrial (mt) dysfunction has been described in acute severe SARS-CoV2 infection. It remains unclear whether the disturbances in mt are also present in post-acute sequelae of COVID-19 (PASC). Method(s): We analyzed cross-sectional data from participants without history of COVID and SARS-CoV2 antibody negative (COVID-), with documented prior COVID and full recovery (COVID+ PASC-), and with prior COVID with PASC as defined by the CDC (COVID+PASC+). Mt respiration was measured from peripheral blood mononuclear cells utilizing the Seahorse XFe96 analyzer. Generalized linear regression was used to compare estimates of mt and non-mt respirations, and unadjusted odds ratios using multinomial logistic regression to assess if mt respiration were associated with PASC. Result(s): For this analysis, 59 participants were enrolled, 71.19% (n=42) had a confirmed COVID-19 diagnosis. The overall mean age was 47.47 +/- 14.86 years, 69.49% (n=41) were females and 33.90% (n=20) were non-white race. There was no difference in demographics between participants with and without COVID (p>=0.72). Amongst all COVID+ participants, 19% (n=11) had hypertension and 8% (n=5) had diabetes. Among all COVID+, the median time between COVID diagnosis and study evaluation was 210 (IQR: 119, 453) days, and 50% (n=21) of COVID+ experienced persistent symptoms consistent with PASC. PASC participants had the highest observed values in non-mt respiration (21.57 +/- 10.77 pmol/min), basal respiration (38.95 +/- 17.58 pmol/min), proton leak (10.41 +/- 3.1), maximal respiration (103.91 +/- 58.63 pmol/min), spare respiratory capacity (64.96 +/- 41.82 pmol/min), and ATP production (28.55 +/-14.85 pmol/min). Basal respiration, ATP production, maximal respiration, and non-mt respiration were highest in PASC compared to COVID- (p<=0.02). There was marginal evidence (p=0.05) of a mean difference (8.09 pmol/min) in ATP production between COVID+PASC+ and COVID+PASC-, without differences in proton leak (p=0.23) or spare respiration capacity (p=0.07). Every unit increase in non-mt respiration, basal respiration, maximal respiration, and ATP production increased the predicted odds of PASC by 10.99, 5.6, 1.6 and 6.2%, respectively (Figure). Conclusion(s): Individuals with PASC are consuming more oxygen and producing more ATP in the PBMCs compared to controls. There also appears to be increased PBMC ATP production between PASC and COVID+. We hypothesize that this may reflect a crucial pathogenic mechanism in PASC that may be associated with ongoing inflammation. (Figure Presented).

Adipose tissue, systematic inflammation, and neurodegenerative diseases

Obesity is associated with several diseases, including mental health. Adipose tissue is distributed around the internal organs, acting in the regulation of metabolism by storing and releasing fatty acids and adipokine in the tissues. Excessive nutritional intake results in hypertrophy and proliferation of adipocytes, leading to local hypoxia in adipose tissue and changes in these adipokine releases. This leads to the recruitment of immune cells to adipose tissue and the release of pro-inflammatory cytokines. The presence of high levels of free fatty acids and inflammatory molecules interfere with intracellular insulin signaling, which can generate a neuroinflammatory process. In this review, we provide an up-to-date discussion of how excessive obesity can lead to possible cognitive dysfunction. We also address the idea that obesity-associated systemic inflammation leads to neuroinflammation in the brain, particularly the hypothalamus and hippocampus, and that this is partially responsible for these negative cognitive outcomes. In addition, we discuss some clinical models and animal studies for obesity and clarify the mechanism of action of anti-obesity drugs in the central nervous system.Copyright © 2023 Wolters Kluwer Medknow Publications. All rights reserved.

Dysfunctional purinergic signaling correlates with disease severity in COVID-19 patients.

Ectonucleotidases modulate inflammatory responses by balancing extracellular ATP and adenosine (ADO) and might be involved in COVID-19 immunopathogenesis. Here, we explored the contribution of extracellular nucleotide metabolism to COVID-19 severity in mild and severe cases of the disease. We verified that the gene expression of ectonucleotidases is reduced in the whole blood of patients with COVID-19 and is negatively correlated to levels of CRP, an inflammatory marker of disease severity. In line with these findings, COVID-19 patients present higher ATP levels in plasma and reduced levels of ADO when compared to healthy controls. Cell type-specific analysis revealed higher frequencies of CD39+ T cells in severely ill patients, while CD4+ and CD8+ expressing CD73 are reduced in this same group. The frequency of B cells CD39+CD73+ is also decreased during acute COVID-19. Interestingly, B cells from COVID-19 patients showed a reduced capacity to hydrolyze ATP into ADP and ADO. Furthermore, impaired expression of ADO receptors and a compromised activation of its signaling pathway is observed in COVID-19 patients. The presence of ADO in vitro, however, suppressed inflammatory responses triggered in patients' cells. In summary, our findings support the idea that alterations in the metabolism of extracellular purines contribute to immune dysregulation during COVID-19, possibly favoring disease severity, and suggest that ADO may be a therapeutic approach for the disease.

Size-classified monitoring of ATP bioluminescence for rapid assessment of biological distribution in airborne particulates.

The COVID-19 pandemic ignited massive research into the rapid detection of bioaerosols. In particular, nanotechnology-based detection strategies are proposed as alternatives because of issues in bioaerosol enrichment and lead time for molecular diagnostics; however, the practical implementation of such techniques is still unclear due to obstacles regarding the large research and development effort and investment for the validation. The use of adenosine triphosphate (ATP) bioluminescence (expressed as relative luminescence unit (RLU) per unit volume of air) of airborne particulate matter (PM) to determine the bacterial population as a representative of the total bioaerosols (viruses, bacteria, and fungi) has been raised frequently because of the high reponse speed, resolution, and compatibility with culture-based bioaerosol monitoring. On the other hand, additional engineering attempts are required to confer significance because of the size-classified (bioluminescence for different PM sizes) and specific (bioluminescence per unit PM mass) biological risks of air for providing proper interventions in the case of airborne transmission. In this study, disc-type impactors to cut-off aerosols larger than 1 µm, 2.5 µm, and 10 µm were designed and constructed to collect PM1, PM2.5, and PM10 on sampling swabs. This engineering enabled reliable size-classified bioluminescence signals using a commercial ATP luminometer after just 5 min of air intake. The simultaneous operations of a six-stage Andersen impactor and optical PM spectrometers were conducted to determine the correlations between the resulting RLU and colony forming unit (CFU; from the Andersen impactor) or PM mass concentration (deriving specific bioluminescence).

Web-based pediatric visual acuity testing at home

Introduction: Pediatric eye care access, particularly in rural areas, has been an ongoing concern. The COVID-19 pandemic has led to a growing appreciation and acceptance of a role for telemedicine in pediatric eye care. However, many at-home visual acuity (VA) charts and apps have poor test design or inaccurate optotype sizes, and may passively provide misinformation for clinical decision making.1-3 We evaluated the new M&S EyeSimplify At-Home Visual Acuity Tests, which include web-based versions of the ATS-HOTV and E-ETDRS tests commonly used in clinical trials. Method(s): Children with and without VA deficits were enrolled. In-office VA was tested with the M&S Smart System ATS-HOTV (ages 3-6;N = 34;68 eyes) or E-ETDRS (ages 7-12;N = 31;62 eyes) protocol. The child was registered on the EyeSimplify web-based portal and the parent was emailed a link to the at-home VA test. The portal notified us when at-home testing was completed and provided us on-line access to VA results. Equivalence of the two test settings was evaluated by mean difference and 95% limits of agreement (LOA) using Bland-Altmann analysis. Result(s): The mean difference between in-office and at-home was small for both ATS-HOTV (0.01 + 0.08 logMAR) and E-ETDRS 0.04 + 0.08 logMAR;95% LOA = -0.15 to 0.17 and -0.11 to 0.19, respectively, comparable to test-retest agreement in an office setting. Conclusion/Relevance: The M&S EyeSimplify At-Home Visual Acuity Tests provided VA equivalent to in-office testing. If the burden of travel is significant, at-home testing may provide the information needed to continue care via telemedicine consultation when it might otherwise be discontinued or delayed.Copyright © 2022

Prognostic value of brain natriuretic peptide in COVID-19 with or without acute heart failure

Background: Although Brain Natriuretic Peptide (BNP) provides strong prognostic information of an unfavorable outcome in patients with acute heart failure (AHF), there is little information of its relevance as a biomarker for outcomes in COVID-19 and its complications Purpose: To evaluate the association of increased BNP levels with complications and in-hospital mortality in a cohort of hospitalized COVID-19 patients. Method(s): The study included COVID-19 patients with data on BNP levels included in the ISACS COVID-19 registry. The population was categorized according to the presence of peak BNP levels >=100 pg/mL during hospitalization. Primary outcomes included in-hospital mortality, AHF or acute respiratory failure (ARF, defined as PiO2/FiO2<300 mmHg or need for mechanical ventilation). Calculations were conducted using age and sex-adjusted multivariable logistic regression analyses. Results were also stratified according to presence or absence of cardiovascular disease (CVD) history. Differences between subgroups were verified for statistical significance using test for interaction. Result(s): Of the 1152 patients included in the study, 615 (53.4%) had elevated BNP levels. These subjects were older (69.9+/-13.8 vs 59.1+/-16.8, p-value<0.001), had higher rates of cardiovascular risk factors (82.9% vs 57.7%, p-value<0.001) and presented more frequently with a prior history of CVD (either ischemic heart disease, cerebrovascular disease, venous thromboembolism, atrial fibrillation or a history of revascularization) (50.1% vs 27.5%, p-value<0.001). No sex differences were observed. When considering outcomes, BNP levels >=100 pg/mL were associated with increased rates of in-hospital mortality (32.9% vs 4.9%, p-value<0.001), even after adjustment for demographic characteristics (OR: 7.35;95% CI: 4.75-11.40;p-value<0.001). High BNP levels were also strongly associated with an increased risk of AHF (OR 19.9;95% CI 8.6-45.9;pvalue< 0.001), a correlation that persisted both in patients with and without a prior CVD history (p for interaction=0.29). Of note, patients with elevated BNP also had a higher likelihood of developing ARF (OR 2.7;95% CI 2.1- 3.6;p-value<0.001), even in absence of AHF (OR 3.00;95% CI 2.20-4.1;p-value<0.001). Conclusion(s): In COVID-19, blood BNP level not only appears to be predictor of in-hospital mortality and AHF but was also independently associated with an increased risk of ARF. This finding supports the routine use of BNP in all patients admitted to hospital for COVID-19, regardless of a prior history of CVD.

Post-translational modifications: Host defence mechanism, pathogenic weapon, and emerged target of anti-infective drugs

Post-translational modifications are changes introduced to proteins after their translation. They are the means to generate molecular diversity, expand protein function, control catalytic activity and trigger quick responses to a wide range of stimuli. Moreover, they regulate numerous biological processes, including pathogen invasion and host defence mechanisms. It is well established that bacteria and viruses utilize post-translational modifications on their own or their host's proteins to advance their pathogenicity. Doing so, they evade immune responses, target signaling pathways and manipulate host cytoskeleton to achieve survival, replication and propagation. Many bacterial species secrete virulence factors into the host and mediate hostpathogen interactions by inducing post-translational modifications that subvert fundamental cellular processes. Viral pathogens also utilize post translational modifications in order to overcome the host defence mechanisms and hijack its cellular machinery for their replication and propagation. For example, many coronavirus proteins are modified to achieve host invasion, evasion of immune responses and utilization of the host translational machinery. PTMs are also considered potential targets for the development of novel therapeutics from natural products with antibiotic properties, like lasso peptides and lantibiotics. The last decade, significant progress was made in understanding the mechanisms that govern PTMs and mediate regulation of protein structure and function. This urges the identification of relevant molecular targets, the design of specific drugs and the discovery of PTM-based medicine. Therefore, PTMs emerge as a highly promising field for the investigation and discovery of new therapeutics for many infectious diseases.Copyright © 2021 Bentham Science Publishers.