TRPC3-Nox2 Protein Complex Formation Increases the Risk of SARS-CoV-2 Spike Protein-Induced Cardiomyocyte Dysfunction through ACE2 Upregulation.

Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of the key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells is initiated by binding with its receptor, angiotensin-converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we report that ibudilast, which we previously identified as a potent inhibitor of protein complex between transient receptor potential canonical (TRPC) 3 and NADPH oxidase (Nox) 2, attenuates the SARS-CoV-2 spike glycoprotein pseudovirus-evoked contractile and metabolic dysfunctions of neonatal rat cardiomyocytes (NRCMs). Epidemiologically reported risk factors of severe COVID-19, including cigarette sidestream smoke (CSS) and anti-cancer drug treatment, commonly upregulate ACE2 expression level, and these were suppressed by inhibiting TRPC3-Nox2 complex formation. Exposure of NRCMs to SARS-CoV-2 pseudovirus, as well as CSS and doxorubicin (Dox), induces ATP release through pannexin-1 hemi-channels, and this ATP release potentiates pseudovirus entry to NRCMs and human iPS cell-derived cardiomyocytes (hiPS-CMs). As the pseudovirus entry followed by production of reactive oxygen species was attenuated by inhibiting TRPC3-Nox2 complex in hiPS-CMs, we suggest that TRPC3-Nox2 complex formation triggered by panexin1-mediated ATP release participates in exacerbation of myocardial damage by amplifying ACE2-dependent SARS-CoV-2 entry.

Vaccine Breakthrough Cases in South Dakota: Impact of SARS-CoV-2 Variants in a Rural State.

BACKGROUND: Emergence of the SARS-CoV-2 Delta variant raised concern for greater transmissibility and severity of illness compared to the Alpha variant. Our objective was to compare SARS-CoV-2 vaccine breakthrough cases in South Dakota during the time periods where the Alpha and Delta variants of SARS-CoV-2 predominated. METHODS: Data were obtained from the South Dakota Department of Health's electronic disease surveillance system and South Dakota's Health Information Exchange. SARS-CoV-2 cases were matched with the immunization system data to verify vaccination status of vaccine breakthrough cases (VBC). The Alpha variant time-period (ATP) was defined as April 15-May 10, 2021 and the Delta variant time-period (DTP) as July 18-31, 2021. Case rates, demographics, risk factors, symptomology, and outcomes were compared for VBC during these periods. RESULTS: A total of 155 VBC were reported during the ATP and 153 during the DTP. The rate of SARS-CoV-2 VBC was 1.88 times higher for the DTP than the ATP. VBC during the ATP were more likely to present with no symptoms and during the DTP were more likely to present with subjective fever, cough, headache, loss or altered smell/taste, congestion, or postnasal drip. The average hospital length of stay was 6 days for the ATP and 4 days for the DTP. A total of 5 deaths were reported during the ATP compared to 1 death during the DTP. The non-statistically significant relation of the ATP and the DTP for hospital length of stay and number of deaths indicated a similar severity of disease. CONCLUSIONS: In fully vaccinated South Dakotans, the SARS-CoV-2 Delta variant was shown to cause 1.88 times higher breakthrough cases but resulted in similar severity of illness compared to the Alpha variant.

ATP and nucleic acids competitively modulate LLPS of the SARS-CoV2 nucleocapsid protein.

SARS-CoV-2 nucleocapsid (N) protein with very low mutation rates is the only structural protein which not only functions to package viral genomic RNA, but also manipulates host-cell machineries, thus representing a key target for drug development. Recent discovery of its liquid-liquid phase separation (LLPS) opens up a new direction for developing anti-SARS-CoV-2 strategies/drugs. However, so far the high-resolution mechanism of its LLPS still remains unknown. Here by DIC and NMR characterization, we have demonstrated: 1) nucleic acids modulate LLPS by dynamic and multivalent interactions over both folded NTD/CTD and Arg/Lys residues within IDRs; 2) ATP with concentrations > mM in all living cells but absent in viruses not only binds NTD/CTD, but also Arg residues within IDRs with a Kd of 2.8 mM; and 3) ATP dissolves nucleic-acid-induced LLPS by competitively displacing nucleic acid from binding the protein. Our study deciphers that the essential binding of N protein with nucleic acid and its LLPS are targetable by small molecules including ATP, which is emerging as a cellular factor controlling the host-SARS-CoV-2 interaction. Fundamentally, our results imply that the mechanisms of LLPS of IDR-containing proteins mediated by ATP and nucleic acids appear to be highly conserved from human to virus.

Discovering new potential inhibitors to SARS-CoV-2 RNA dependent RNA polymerase (RdRp) using high throughput virtual screening and molecular dynamics simulations.

RNA dependent RNA polymerase (RdRp), is an essential in the RNA replication within the life cycle of the severely acute respiratory coronavirus-2 (SARS-CoV-2), causing the deadly respiratory induced sickness COVID-19. Remdesivir is a prodrug that has seen some success in inhibiting this enzyme, however there is still the pressing need for effective alternatives. In this study, we present the discovery of four non-nucleoside small molecules that bind favorably to SARS-CoV-2 RdRp over the active form of the popular drug remdesivir (RTP) and adenosine triphosphate (ATP) by utilizing high-throughput virtual screening (HTVS) against the vast ZINC compound database coupled with extensive molecular dynamics (MD) simulations. After post-trajectory analysis, we found that the simulations of complexes containing both ATP and RTP remained stable for the duration of their trajectories. Additionally, it was revealed that the phosphate tail of RTP was stabilized by both the positive amino acid pocket and magnesium ions near the entry channel of RdRp which includes residues K551, R553, R555 and K621. It was also found that residues D623, D760, and N691 further stabilized the ribose portion of RTP with U10 on the template RNA strand forming hydrogen pairs with the adenosine motif. Using these models of RdRp, we employed them to screen the ZINC database of ~ 17 million molecules. Using docking and drug properties scoring, we narrowed down our selection to fourteen candidates. These were subjected to 200 ns simulations each underwent free energy calculations. We identified four hit compounds from the ZINC database that have similar binding poses to RTP while possessing lower overall binding free energies, with ZINC097971592 having a binding free energy two times lower than RTP.

MERS-CoV nsp1 regulates autophagic flux via mTOR signalling and dysfunctional lysosomes.

Autophagy, a cellular surveillance mechanism, plays an important role in combating invading pathogens. However, viruses have evolved various strategies to disrupt autophagy and even hijack it for replication and release. Here, we demonstrated that Middle East respiratory syndrome coronavirus (MERS-CoV) non-structural protein 1(nsp1) induces autophagy but inhibits autophagic activity. MERS-CoV nsp1 expression increased ROS and reduced ATP levels in cells, which activated AMPK and inhibited the mTOR signalling pathway, resulting in autophagy induction. Meanwhile, as an endonuclease, MERS-CoV nsp1 downregulated the mRNA of lysosome-related genes that were enriched in nsp1-located granules, which diminished lysosomal biogenesis and acidification, and inhibited autophagic flux. Importantly, MERS-CoV nsp1-induced autophagy can lead to cell death in vitro and in vivo. These findings clarify the mechanism by which MERS-CoV nsp1-mediated autophagy regulation, providing new insights for the prevention and treatment of the coronavirus.

COVID-19 Pandemic Effects on the Well-Being of Professional Pilots.

OBJECTIVE: The aim was to evaluate the well-being of professional pilots using a survey that included the World Health Organization (WHO)-5 Well-Being Index.METHODS: An electronic survey was sent to pilots between June 17-August 2, 2021. Pilots self-categorized as: airline transport pilot (ATP), commercial pilot, or both. Chi-squared and Fisher's exact tests were used to evaluate differences between variables. Logistic regression was used to estimate the odds of impaired well-being.RESULTS: A total of 639 individuals returned the survey. The majority of respondents were ATPs and a majority identified as male. The average well-being score was 68.0 out of 100 possible, with 22% of respondents meeting the threshold definition of impaired well-being. The odds of having impaired well-being were not dependent on gender. In unadjusted analysis, the odds of impaired well-being were higher for those flying as commercial pilots as compared to airline transport pilots/both. Age was also strongly associated with impaired well-being, with younger pilots having greater odds of impaired well-being as compared to older pilots. Only a little over half of the surveyed pilots agreed or strongly agreed that pilot risk of occupational exposure to COVID-19 was appropriately controlled.CONCLUSION: This survey suggests an important connection between pilot age and impaired well-being scores during the COVID-19 pandemic. Future studies targeting the well-being of younger pilots will be of interest. Additionally, measures to reduce occupational risk of COVID-19 exposure may be helpful in view of the substantial fraction of pilots expressing concern regarding that risk.Stratton E, Haddon R, Murad MH, Petterson T, Nelson M, Cowl CT. COVID-19 pandemic effects on the well-being of professional pilots. Aerosp Med Hum Perform. 2022; 93(10):734-738.

SARS-CoV-2 mitochondriopathy in COVID-19 pneumonia exacerbates hypoxemia.

RATIONALE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 pneumonia. We hypothesize that SARS-CoV-2 causes alveolar injury and hypoxemia by damaging mitochondria in airway epithelial cells (AEC) and pulmonary artery smooth muscle cells (PASMC), triggering apoptosis and bioenergetic impairment, and impairing hypoxic pulmonary vasoconstriction (HPV), respectively. OBJECTIVES: We examined the effects of: A) human betacoronaviruses, SARS-CoV-2 and HCoV-OC43, and individual SARS-CoV-2 proteins on apoptosis, mitochondrial fission, and bioenergetics in AEC; and B) SARS-CoV-2 proteins and mouse hepatitis virus (MHV-1) infection on HPV. METHODS: We used transcriptomic data to identify temporal changes in mitochondrial-relevant gene ontology (GO) pathways post-SARS-CoV-2 infection. We also transduced AECs with SARS-CoV-2 proteins (M, Nsp7 or Nsp9) and determined effects on mitochondrial permeability transition pore (mPTP) activity, relative membrane potential, apoptosis, mitochondrial fission, and oxygen consumption rates (OCR). In human PASMC, we assessed the effects of SARS-CoV-2 proteins on hypoxic increases in cytosolic calcium, an HPV proxy. In MHV-1 pneumonia, we assessed HPV via cardiac catheterization and apoptosis using the TUNEL assay. RESULTS: SARS-CoV-2 regulated mitochondrial apoptosis, mitochondrial membrane permeabilization and electron transport chain (ETC) GO pathways within 2 hours of infection. SARS-CoV-2 downregulated ETC Complex I and ATP synthase genes, and upregulated apoptosis-inducing genes. SARS-CoV-2 and HCoV-OC43 upregulated and activated dynamin-related protein 1 (Drp1) and increased mitochondrial fission. SARS-CoV-2 and transduced SARS-CoV-2 proteins increased apoptosis inducing factor (AIF) expression and activated caspase 7, resulting in apoptosis. Coronaviruses also reduced OCR, decreased ETC Complex I activity and lowered ATP levels in AEC. M protein transduction also increased mPTP opening. In human PASMC, M and Nsp9 proteins inhibited HPV. In MHV-1 pneumonia, infected AEC displayed apoptosis and HPV was suppressed. BAY K8644, a calcium channel agonist, increased HPV and improved SpO2. CONCLUSIONS: Coronaviruses, including SARS-CoV-2, cause AEC apoptosis, mitochondrial fission, and bioenergetic impairment. SARS-CoV-2 also suppresses HPV by targeting mitochondria. This mitochondriopathy is replicated by transduction with SARS-CoV-2 proteins, indicating a mechanistic role for viral-host mitochondrial protein interactions. Mitochondriopathy is a conserved feature of coronaviral pneumonia that may exacerbate hypoxemia and constitutes a therapeutic target.

Modulation of ATP Production Influences Inorganic Polyphosphate Levels in Non-Athletes' Platelets at the Resting State.

Platelets produce inorganic polyphosphate (polyP) upon activation to stimulate blood coagulation. Some researchers have linked polyP metabolism to ATP production, although the metabolic linkage is yet to be elucidated. We found evidence for this possibility in our previous study on professional athletes (versus non-athletes), and proposed that the regulatory mechanism might be different for these two groups. To explore this aspect further, we investigated the effects of modulated ATP production on polyP levels. Blood samples were obtained from Japanese healthy, non-athletes in the presence of acid-citrate-dextrose. The platelets in the plasma were treated with oligomycin, rotenone, and GlutaMAX to modulate ATP production. PolyP level was quantified fluorometrically and visualized using 4',6-diamidino-2-phenylindole. Correlations between polyP and ATP or NADH were then calculated. Contrary to the hypothesis, inhibitors of ATP production increased polyP levels, whereas amino acid supplementation produced the opposite effect. In general, however, polyP levels were positively correlated with ATP levels and negatively correlated with NADH levels. Since platelets are metabolically active, they exhibit high levels of ATP turnover rate. Therefore, these findings suggest that ATP may be involved in polyP production in the resting platelets of non-athletes.

Hyperresponsive Platelets and a Reduced Platelet Granule Release Capacity Are Associated with Severity and Mortality in COVID-19 Patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is often associated with mild thrombocytopenia and increased platelet reactivity. OBJECTIVE: The aim of the current study was to investigate the adenosine triphosphate (ATP) release kinetics of platelets in hospitalized SARS-CoV-2-infected patients. METHODS: We studied time-dependent platelet activation in whole blood by monitoring the ATP release kinetics upon stimulation with a PAR1 receptor agonist in 41 hospitalized critically ill COVID-19 patients, 47 hospitalized noncritically ill COVID-19 patients, and 30 healthy controls. RESULTS: Our study demonstrated that platelets of critically ill COVID-19 patients were hyper-responsive with a shorter platelet response time (PRT) and a reduced platelet granule release capacity (GRC), probably due to chronic activation. The median PRT of COVID-19 patients admitted to the critical care unit was 10 and 7 seconds shorter than the median PRT in healthy controls and noncritical COVID-19 patients, respectively. Both PRT and GRC were also associated with D-dimer (Spearman r [r s] = -0.51, p < 0.0001 and r s = -0.23, p < 0.05), C-reactive protein (CRP) (r s = -0.59, p < 0.0001 and r s = -0.41, p < 0.01), and neutrophil-to-lymphocyte ratio (NLR) (r s = -0.42, p < 0.0001 and r s = -0.26, p < 0.05). Moreover, an increased PRT and a reduced GRC were associated with an increased mortality (odds ratio [OR]: 18.8, 95% confidence interval [CI]: 6.5-62.8, p < 0.0001 and OR: 4.0; 95% CI: 1.6-10.4, p < 0.01). These relationships remained significant after adjustment for age, sex, D-dimer, CRP, and NLR. CONCLUSION: Using an accessible agonist-induced platelet granule ATP release assay, we show that platelet hyper-responsiveness and reduced platelet GRC in COVID-19 patients were associated with critical illness and mortality.

Crystal structure of the Rho-associated coiled-coil kinase 2 inhibitor belumosudil bound to CK2α.

The small molecule belumosudil was initially identified as a selective inhibitor of Rho-associated coiled-coil kinase 2 (ROCK2) and has recently been approved for the treatment of graft-versus-host disease. However, recent studies have shown that many of the phenotypes displayed upon treatment with belumosudil were due to CK2α inhibition. CK2α is in itself a very promising therapeutic target for a range of conditions and has recently been put forward as a potential treatment for COVID-19. Belumosudil presents a promising starting point for the development of future CK2α inhibitors as it provides a safe, potent and orally bioavailable scaffold. Therefore, several of the major hurdles in drug development have already been overcome. Here, the crystal structure of belumosudil bound to the ATP site of CK2α is presented. This crystal structure combined with modelling studies further elucidates how belumosudil could be developed into a selective and potent CK2α or ROCK2 inhibitor.

The Australian Temperament Project Generation 3 study: a population-based multigenerational prospective cohort study of socioemotional health and development.

PURPOSE: The Australian Temperament Project Generation 3 Study (ATPG3) was established to examine the extent to which offspring social and emotional development is shaped in the decades prior to conception, in parent and grandparent histories of psychosocial adjustment (eg, emotional regulation, relationship quality and prosociality) and maladjustment (eg, depressive symptoms, substance use and antisociality). PARTICIPANTS: The Australian Temperament Project (ATP) commenced in 1983 as a population representative survey of the social and emotional health of 2443 young Australians (Generation 2: 4-8 months old) and their parents (Generation 1). Since then, families have been followed from infancy to young adulthood (16 waves). Between 2012 and 2018, the cohort was screened biannually for pregnancies (Generation 3), with assessments conducted in the third trimester of pregnancy, and at 8 weeks and 1 year postpartum. FINDINGS TO DATE: A total of 1167 offspring (607 female) born to 703 Generation 2 parents (400 mothers) were recruited into the ATPG3 Study. Findings to date highlight: (1) strong continuities in depressive symptoms and substance use from adolescence through to becoming a parent; (2) a role for persistent preconception mental health problems in risk for parent-child bonding difficulties, as well as infant emotional reactivity and behaviour problems; (3) the importance of secure attachments in adolescence in reducing long-term risk for postpartum mental health problems; and (4) the protective nature of perceived social support, both preconception and postpartum, in strengthening relationship quality and social support during the COVID-19 pandemic. FUTURE PLANS: Assessments of ATPG3 families in preschool and middle childhood are currently funded and underway. We intend to maintain the offspring cohort through childhood, adolescence, young adulthood and into parenthood. Data will be used to map preconception determinants of emotional health, and enhance approaches to population monitoring and targeted intervention over the life course and across generations.

Structural Insights into Binding of Remdesivir Triphosphate within the Replication-Transcription Complex of SARS-CoV-2.

Remdesivir is an adenosine analogue that has a cyano substitution in the C1' position of the ribosyl moiety and a modified base structure to stabilize the linkage of the base to the C1' atom with its strong electron-withdrawing cyano group. Within the replication-transcription complex (RTC) of SARS-CoV-2, the RNA-dependent RNA polymerase nsp12 selects remdesivir monophosphate (RMP) over adenosine monophosphate (AMP) for nucleotide incorporation but noticeably slows primer extension after the added RMP of the RNA duplex product is translocated by three base pairs. Cryo-EM structures have been determined for the RTC with RMP at the nucleotide-insertion (i) site or at the i + 1, i + 2, or i + 3 sites after product translocation to provide a structural basis for a delayed-inhibition mechanism by remdesivir. In this study, we applied molecular dynamics (MD) simulations to extend the resolution of structures to the measurable maximum that is intrinsically limited by MD properties of these complexes. Our MD simulations provide (i) a structural basis for nucleotide selectivity of the incoming substrates of remdesivir triphosphate over adenosine triphosphate and of ribonucleotide over deoxyribonucleotide, (ii) new detailed information on hydrogen atoms involved in H-bonding interactions between the enzyme and remdesivir, and (iii) direct information on the catalytically active complex that is not easily captured by experimental methods. Our improved resolution of interatomic interactions at the nucleotide-binding pocket between remedesivir and the polymerase could help to design a new class of anti-SARS-CoV-2 inhibitors.

Beneficial effect of polyphenols in COVID-19 and the ectopic F1 FO -ATP synthase: Is there a link?

COVID-19 has been proposed to be an endothelial disease, as endothelial damage and oxidative stress contribute to its systemic inflammatory and thrombotic events. Polyphenols, natural antioxidant compounds appear as promising agents to prevent and treat COVID-19. Polyphenols bind and inhibit the F1 Fo -ATP synthase rotary catalysis. An early target of polyphenols may be the ectopic F1 Fo -ATP synthase expressed on the endothelial plasma membrane. Among the pleiotropic beneficial action of polyphenols in COVID-19, modulation of the ecto-F1 Fo -ATP synthase, lowering the oxidative stress produced by the electron transfer chain coupled to it, would not be negligible.

State-of-the-Art Molecular Dynamics Simulation Studies of RNA-Dependent RNA Polymerase of SARS-CoV-2.

Molecular dynamics (MD) simulations are powerful theoretical methods that can reveal biomolecular properties, such as structure, fluctuations, and ligand binding, at the level of atomic detail. In this review article, recent MD simulation studies on these biomolecular properties of the RNA-dependent RNA polymerase (RdRp), which is a multidomain protein, of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are presented. Although the tertiary structures of RdRps in SARS-CoV-2 and SARS-CoV are almost identical, the RNA synthesis activity of RdRp of SARS-CoV is higher than SARS-CoV-2. Recent MD simulations observed a difference in the dynamic properties of the two RdRps, which may cause activity differences. RdRp is also a drug target for Coronavirus disease 2019 (COVID-19). Nucleotide analogs, such as remdesivir and favipiravir, are considered to be taken up by RdRp and inhibit RNA replication. Recent MD simulations revealed the recognition mechanism of RdRp for these drug molecules and adenosine triphosphate (ATP). The ligand-recognition ability of RdRp decreases in the order of remdesivir, favipiravir, and ATP. As a typical recognition process, it was found that several lysine residues of RdRp transfer these ligand molecules to the binding site such as a "bucket brigade." This finding will contribute to understanding the mechanism of the efficient ligand recognition by RdRp. In addition, various simulation studies on the complexes of SARS-CoV-2 RdRp with several nucleotide analogs are reviewed, and the molecular mechanisms by which these compounds inhibit the function of RdRp are discussed. The simulation studies presented in this review will provide useful insights into how nucleotide analogs are recognized by RdRp and inhibit the RNA replication.

Smartphones as an Ecological Niche of Microorganisms: Microbial Activities, Assembly, and Opportunistic Pathogens.

Smartphone usage and contact frequency are unprecedentedly high in this era, and they affect humans mentally and physically. However, the characteristics of the microorganisms associated with smartphones and smartphone hygiene habits remain unclear. In this study, using various culture-independent techniques, including high-throughput sequencing, real-time quantitative PCR (RT-qPCR), the ATP bioluminescence system, and electron microscopy, we investigated the structure, assembly, quantity, and dynamic metabolic activity of the bacterial community on smartphone surfaces and the user's dominant and nondominant hands. We found that smartphone microbiotas are more similar to the nondominant hand microbiotas than the dominant hand microbiotas and show significantly decreased phylogenetic diversity and stronger deterministic processes than the hand microbiota. Significant interindividual microbiota differences were observed, contributing to an average owner identification accuracy of 70.6% using smartphone microbiota. Furthermore, it is estimated that approximately 1.75 × 106 bacteria (2.24 × 104/cm2) exist on the touchscreen of a single smartphone, and microbial activities remain stable for at least 48 h. Scanning electron microscopy detected large fragments harboring microorganisms, suggesting that smartphone microbiotas live on the secreta or other substances, e.g., human cell debris and food debris. Fortunately, simple smartphone cleaning/hygiene could significantly reduce the bacterial load. Taken together, our results demonstrate that smartphone surfaces not only are a reservoir of microbes but also provide an ecological niche in which microbiotas, particularly opportunistic pathogens, can survive, be active, and even grow. IMPORTANCE Currently, people spend an average of 4.2 h per day on their smartphones. Due to the COVID-19 pandemic, this figure may still be increasing. The high frequency of smartphone usage may allow microbes, particularly pathogens, to attach to-and even survive on-phone surfaces, potentially causing adverse effects on humans. We employed various culture-independent techniques in this study to evaluate the microbiological features and hygiene of smartphones, including community assembly, bacterial load, and activity. Our data showed that deterministic processes drive smartphone microbiota assembly and that approximately 1.75 × 106 bacteria exist on a single smartphone touchscreen, with activities being stable for at least 48 h. Fortunately, simple smartphone cleaning/hygiene could significantly reduce the bacterial load. This work expands our understanding of the microbial ecology of smartphone surfaces and might facilitate the development of electronic device cleaning/hygiene guidelines to support public health.

Enhancing CRISPR-Cas-Mediated Detection of Nucleic Acid and Non-nucleic Acid Targets Using Enzyme-Labeled Reporters.

We introduce a new method to generate an amplified signal in CRISPR-Cas-based detection. Target recognition activates a CRISPR-Cas complex, leading to catalytic cleavage of horseradish peroxidase (HRP)-labeled oligonucleotides from the surface of microbeads. We show that the HRP released into solution can be monitored through colorimetric, fluorometric, or luminescent approaches, yielding up to ∼75-fold turn-on signal and limits of detection (LODs) as low as ∼10 fM. Compared to Cas-based detection with a conventional fluorophore/quencher reporter, this strategy improves the LOD by ∼30-fold. As a proof-of-concept, we show the rapid (<1 h), PCR-free, and room temperature (25 °C) detection of a nucleic acid marker for the SARS-CoV-2 virus with the naked eye at clinically relevant concentrations. We further show that the probe set can be programmed to be recognized and activated in the presence of non-nucleic acid targets. Specifically, we show adenosine triphosphate (ATP) binding to an aptamer can activate CRISPR-Cas and trigger a colorimetric readout, enabling the analysis of ATP in human serum samples with sensitivity on par with that of several commercially available kits. Taken together, the strategy reported herein offers a simple and sensitive platform to detect analytes where target amplification is either inconvenient (e.g., PCR under point-of-care settings) or impossible.

SARS-CoV-2 infection and replication kinetics in different human cell types: The role of autophagy, cellular metabolism and ACE2 expression.

AIMS: This study evaluated SARS-CoV-2 replication in human cell lines derived from various tissues and investigated molecular mechanisms related to viral infection susceptibility and replication. MAIN METHODS: SARS-CoV-2 replication in BEAS-2B and A549 (respiratory tract), HEK-293 T (kidney), HuH7 (liver), SH-SY5Y (brain), MCF7 (breast), Huvec (endothelial) and Caco-2 (intestine) was evaluated by RT-qPCR. Concomitantly, expression levels of ACE2 (Angiotensin Converting Enzyme) and TMPRSS2 were assessed through RT-qPCR and western blot. Proteins related to autophagy and mitochondrial metabolism were monitored in uninfected cells to characterize the cellular metabolism of each cell line. The effect of ACE2 overexpression on viral replication in pulmonary cells was also investigated. KEY FINDINGS: Our data show that HuH7, Caco-2 and MCF7 presented a higher viral load compared to the other cell lines. The increased susceptibility to SARS-CoV-2 infection seems to be associated not only with the differential levels of proteins intrinsically related to energetic metabolism, such as ATP synthase, citrate synthase, COX and NDUFS2 but also with the considerably higher TMPRSS2 mRNA expression. The two least susceptible cell types, BEAS-2B and A549, showed drastically increased SARS-CoV-2 replication capacity when ACE2 was overexpressed. These modified cell lines are relevant for studying SARS-CoV-2 replication in vitro. SIGNIFICANCE: Our data not only reinforce that TMPRSS2 expression and cellular energy metabolism are important molecular mechanisms for SARS-CoV-2 infection and replication, but also indicate that HuH7, MCF7 and Caco-2 are suitable models for mechanistic studies of COVID-19. Moreover, pulmonary cells overexpressing ACE2 can be used to understand mechanisms associated with SARS-CoV-2 replication.

Identification of missing hierarchical relations in the vaccine ontology using acquired term pairs.

BACKGROUND: The Vaccine Ontology (VO) is a biomedical ontology that standardizes vaccine annotation. Errors in VO will affect a multitude of applications that it is being used in. Quality assurance of VO is imperative to ensure that it provides accurate domain knowledge to these downstream tasks. Manual review to identify and fix quality issues (such as missing hierarchical is-a relations) is challenging given the complexity of the ontology. Automated approaches are highly desirable to facilitate the quality assurance of VO. METHODS: We developed an automated lexical approach that identifies potentially missing is-a relations in VO. First, we construct two types of VO concept-pairs: (1) linked; and (2) unlinked. Each concept-pair further derives an Acquired Term Pair (ATP) based on their lexical features. If the same ATP is obtained by a linked concept-pair and an unlinked concept-pair, this is considered to indicate a potentially missing is-a relation between the unlinked pair of concepts. RESULTS: Applying this approach on the 1.1.192 version of VO, we were able to identify 232 potentially missing is-a relations. A manual review by a VO domain expert on a random sample of 70 potentially missing is-a relations revealed that 65 of the cases were valid missing is-a relations in VO (a precision of 92.86%). CONCLUSIONS: The results indicate that our approach is highly effective in identifying missing is-a relation in VO.

Assessment of Community Behavior and COVID-19 Transmission during Festivities in India: A Qualitative Synthesis through a Media Scanning Technique.

In India during the first wave of COVID-19 infection, the authorities were concerned about the advent of the festive season, which could lead to a surge in cases of SARS-CoV-2 infection. The present study attempted to assess the socio-behavioral aspects of COVID-appropriate behavior (CAB) at individual and community levels, and their impact on the transmission of COVID-19 during festivities in India. Media scanning was conducted to qualitatively assess CAB by analyzing 284 news reports from across India; quantitative data on COVID-19 daily cases from March 2020 to December 2020 were used to determine the trends of the adjusted test positivity (ATP) ratio for six cities. Peaks in ATP were observed in Chandigarh, Delhi-NCR in North India during and after Dussehra and Deepavali, and in Mumbai, in the west, after Navratri. Additionally, a surge in ATP was observed in Trivandrum after Onam and in Chennai around Deepavali in the south; meanwhile, in the eastern city of Kolkata, cases increased following Durga Puja. The major challenges were adherence to CAB viz. social distancing, hygiene, and compliance with the mask mandate. Microlevel CAB indicated relatively higher laxity in maintaining hand hygiene in all cities. Observations from the current study indicate that innovative community-driven initiatives during festivals in each geographical zone are key to the large-scale implementation of disease prevention measures.

Discovery and Mechanistic Study of Mycobacterium tuberculosis PafA Inhibitors.

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis (Mtb) and is ranked as the second killer infectious disease after COVID-19. Proteasome accessory factor A (PafA) is considered an attractive target because of its low sequence conservation in humans and its role in virulence. In this study, we designed a mutant of Mtb PafA that enabled large-scale purification of active PafA. Using a devised high-throughput screening assay, two PafA inhibitors were discovered. ST1926 inhibited Mtb PafA by binding in the Pup binding groove, but it was less active against Corynebacterium glutamicum PafA because the ST1926-binding residues are not conserved. Bithionol bound to the conserved ATP-binding pocket, thereby, inhibits PafA in an ATP-competitive manner. Both ST1926 and bithionol inhibited the growth of an attenuated Mtb strain (H37Ra) at micromolar concentrations. Our work thus provides new tools for tuberculosis research and a foundation for future PafA-targeted drug development for treating tuberculosis.