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Abstract Background The Tpeak‐end(Tp‐e) has not been compared in all 12 ECG leads in healthy adults to determine if the Tp‐e varies across leads. If there is variation, it remains uncertain, which lead(s) are preferred for recording in order to capture the maximal Tp‐e value. Objective The purpose of the current study was to determine the optimal leads, if any, to capture the maximal Tp‐e interval in healthy young adults. Methods In 88 healthy adults (ages 21–38 years), including derivation (n = 21), validation (n = 20), and smoker/vaper (n = 47) cohorts, the Tp‐e was measured using commercial computer software (LabChart Pro 8 with ECG module, ADInstruments) in all 12 leads at rest and following a provocative maneuver, abrupt standing. Tp‐e was compared to determine which lead(s) most frequently captured the maximal Tp‐e interval. Results In the rest and abrupt standing positions, the Tp‐e was not uniform among the 12 leads; the maximal Tp‐e was most frequently captured in the precordial leads. At rest, grouping leads V2–V4 resulted in detection of the maximum Tp‐e in 85.7% of participants (CI 70.7, 99.9%) versus all other leads (p

The main protease of SARS-CoV-2 is an essential enzyme required for polyprotein cleavage during viral replication and thus is an excellent target for development of direct-acting antiviral compounds. Continued research efforts have elucidated several peptidic small molecules like GC376, boceprevir, and nirmatrelvir with potent anticoronaviral activity bearing optimized amino acid side chain residues. To reduce synthetic complexity and cost, we used simple chemical surrogates that were commercially readily available to develop new inhibitors that mimic the potency of these drug compounds. We synthesized and tested several analogue chimeras of GC376 and boceprevir that have surrogate residues at the P1 and/or P2 position in order to further improve target binding. Both P1 variants with either a nonpolar cyclobutyl or polar thiazol-4-yl alanine resulted in low-micromolar to submicromolar M pro inhibitors with strong antiviral activity in cell assays., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

Abstract Vector‐borne diseases constitute a major global health burden and are increasing in geographic range and prevalence. Mounting evidence has demonstrated that the vector microbiome can impact pathogen dynamics, making the microbiome a focal point in vector‐borne disease ecology. However, efforts to generalize preliminary findings across studies and systems and translate these findings into disease control strategies are hindered by a lack of fundamental understanding of the processes shaping the vector microbiome and the interactions therein. Here, we use 16S rRNA sequencing and apply a community ecology framework to analyze microbiome community assembly and interactions in Ixodes pacificus, the Lyme disease vector in the western United States. We find that vertical transmission routes drive population‐level patterns in I. pacificus microbial diversity and composition, but that microbial function and overall abundance do not vary over time or between clutches. Further, we find that the I. pacificus microbiome is not strongly structured based on competition but assembles nonrandomly, potentially due to vector‐specific filtering processes which largely eliminate all but the dominant endosymbiont, Rickettsia. At the scale of the individual I. pacificus, we find support for a highly limited internal microbial community, and hypothesize that the tick endosymbiont may be the most important component of the vector microbiome in influencing pathogen dynamics.

OBJETIVO: Estudar a variação das taxas de mortalidade neonatal precoce, natimortalidade e de um conjunto de indicadores da rede de hospitais que prestaram atenção obstétrica ao Sistema Único de Saúde, visando o monitoramento das unidades hospitalares a partir do Sistema de Informações Hospitalares (SIH/SUS) e do Sistema de Nascidos Vivos (SINASC). MÉTODOS: Em 1997, 135 hospitais do Estado do Rio de Janeiro foram estudados por meio da análise estatística fatorial, pelo método de componentes principais. Estabeleceu-se a distribuição dos escores dos estabelecimentos nos dois primeiros componentes, o que permitiu classificar os hospitais segundo o perfil de risco materno das internações e os resultados da assistência. RESULTADOS: Observou-se que a rede obstétrica do Sistema Único de Saúde no Estado, responsável por cerca de 77,8% dos partos, possui 23% dos hospitais que realizam menos de 100 partos/ano. Entre os hospitais com perfil de internação de extremo risco materno e baixo desempenho encontram-se unidades consideradas referência para gestação de alto risco. Observou-se que 5% dos hospitais possuidores de estruturas de baixa complexidade apresentaram um perfil de risco materno alto e resultados da assistência questionáveis. CONCLUSÕES: O SIH/SUS mostrou ser uma importante fonte de dados para monitorar a natimortalidade e a mortalidade neonatal precoce hospitalares e para o planejamento das ações de vigilância das unidades hospitalares obstétricas e neonatais.OBJECTIVE: To analyze variations in early neonatal mortality, stillbirth rates, and a set of indicators collected from obstetric hospitals affiliated to the Brazilian National Unified Health System (SUS) for their monitoring through the Hospital Data System (SIH/SUS) and Live Births Data System (SINASC). METHODS: One-hundred and thirty five hospitals in the state of Rio de Janeiro were assessed in 1997. Factor analysis was conducted using principal components. Score distribution for the first two components were established, which allowed to classify hospitals according to maternal risk profile and care outcomes. RESULTS: Hospitals affiliated to SUS were responsible for 77.8% of all deliveries in the state of Rio de Janeiro and 23% of them performed fewer than 100 deliveries a year. Among hospitals of extreme high maternal risk and low performance, there were several units considered as referral centers for high-risk pregnancy. It was also observed that 5% of hospital units with low complexity infrastructures showed a profile of high maternal risk and questionable care outcomes. CONCLUSIONS: The Hospital Information Data System affiliated to the National Unified Health System has proven to be an important information source for monitoring hospital stillbirth and early neonatal mortality rates as well as for planning surveillance actions for health services providing obstetric and/or neonatal care.

OBJETIVO: Estudar a variação das taxas de mortalidade neonatal precoce, natimortalidade e de um conjunto de indicadores da rede de hospitais que prestaram atenção obstétrica ao Sistema Único de Saúde, visando o monitoramento das unidades hospitalares a partir do Sistema de Informações Hospitalares (SIH/SUS) e do Sistema de Nascidos Vivos (SINASC). MÉTODOS: Em 1997, 135 hospitais do Estado do Rio de Janeiro foram estudados por meio da análise estatística fatorial, pelo método de componentes principais. Estabeleceu-se a distribuição dos escores dos estabelecimentos nos dois primeiros componentes, o que permitiu classificar os hospitais segundo o perfil de risco materno das internações e os resultados da assistência. RESULTADOS: Observou-se que a rede obstétrica do Sistema Único de Saúde no Estado, responsável por cerca de 77,8% dos partos, possui 23% dos hospitais que realizam menos de 100 partos/ano. Entre os hospitais com perfil de internação de extremo risco materno e baixo desempenho encontram-se unidades consideradas referência para gestação de alto risco. Observou-se que 5% dos hospitais possuidores de estruturas de baixa complexidade apresentaram um perfil de risco materno alto e resultados da assistência questionáveis. CONCLUSÕES: O SIH/SUS mostrou ser uma importante fonte de dados para monitorar a natimortalidade e a mortalidade neonatal precoce hospitalares e para o planejamento das ações de vigilância das unidades hospitalares obstétricas e neonatais.

An approach to assess severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (and past infection) was developed. For virus detection, the SARS-CoV-2 virus nucleocapsid protein (NP) was targeted. To detect the NP, antibodies were immobilized on magnetic beads to capture the NPs, which were subsequently detected using rabbit anti-SARS-CoV-2 nucleocapsid antibodies and alkaline phosphatase (AP)-conjugated anti-rabbit antibodies. A similar approach was used to assess SARS-CoV-2-neutralizing antibody levels by capturing spike receptor-binding domain (RBD)-specific antibodies utilizing RBD protein-modified magnetic beads and detecting them using AP-conjugated anti-human IgG antibodies. The sensing mechanism for both assays is based on cysteamine etching-induced fluorescence quenching of bovine serum albumin-protected gold nanoclusters where cysteamine is generated in proportion to the amount of either SARS-CoV-2 virus or anti-SARS-CoV-2 receptor-binding domain-specific immunoglobulin antibodies (anti-RBD IgG antibodies). High sensitivity can be achieved in 5 h 15 min for the anti-RBD IgG antibody detection and 6 h 15 min for virus detection, although the assay can be run in "rapid" mode, which takes 1 h 45 min for the anti-RBD IgG antibody detection and 3 h 15 min for the virus. By spiking the anti-RBD IgG antibodies and virus in serum and saliva, we demonstrate that the assay can detect the anti-RBD IgG antibodies with a limit of detection (LOD) of 4.0 and 2.0 ng/mL in serum and saliva, respectively. For the virus, we can achieve an LOD of 8.5 × 10 5 RNA copies/mL and 8.8 × 10 5 RNA copies/mL in serum and saliva, respectively. Interestingly, this assay can be easily modified to detect myriad analytes of interest.

A sensor capable of quantifying both anti-SARS-CoV-2 spike receptor-binding domain (RBD) antibody levels and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in saliva and serum was developed. This was accomplished by exploiting the enzymatic reaction of maltose and orthophosphate (PO 4 3- ) in the presence of maltose phosphorylase to generate an equivalent amount of glucose that was detected using a commercial glucometer test strip and a potentiostat. Important for this approach is the ability to generate PO 4 3- in an amount that is directly related to the concentration of the analytes. RBD-modified magnetic microparticles were used to capture anti-SARS-CoV-2 spike RBD antibodies, while particles modified with anti-SARS-CoV-2 nucleocapsid antibodies were used to capture SARS-CoV-2 nucleocapsid protein from inactivated virus samples. A magnet was used to isolate and purify the magnetic microparticles (with analyte attached), and alkaline phosphatase-conjugated secondary antibodies were bound to the analytes attached to the respective magnetic microparticles. Finally, through enzymatic reactions, specific amounts of PO 4 3- (and subsequently glucose) were generated in proportion to the analyte concentration, which was then quantified using a commercial glucometer test strip. Utilizing glucose test strips makes the sensor relatively inexpensive, with a cost per test of ∼US $7 and ∼US $12 for quantifying anti-SARS-CoV-2 spike RBD antibody and SARS-CoV-2, respectively. Our sensor exhibited a limit of detection of 0.42 ng/mL for anti-SARS-CoV-2 spike RBD antibody, which is sensitive enough to quantify typical concentrations of antibodies in COVID-19-infected or vaccinated individuals (>1 μg/mL). The limit of detection for the SARS-CoV-2 virus is 300 pfu/mL (5.4 × 10 6 RNA copies/mL), which exceeds the performance recommended by the WHO (500 pfu/mL). In addition, the sensor exhibited good selectivity when challenged with competing analytes and could be used to quantify analytes in saliva and serum matrices with an accuracy of >94% compared to RT-qPCR.

Interferon regulatory factors (IRFs) are key elements of antiviral innate responses that regulate the transcription of interferons (IFNs) and IFN-stimulated genes (ISGs). While the sensitivity of human coronaviruses to IFNs has been characterized, antiviral roles of IRFs during human coronavirus infection are not fully understood. Type I or II IFN treatment protected MRC5 cells from human coronavirus 229E infection, but not OC43. Cells infected with 229E or OC43 upregulated ISGs, indicating that antiviral transcription is not suppressed. Antiviral IRFs, IRF1, IRF3 and IRF7, were activated in cells infected with 229E, OC43 or severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2). RNAi knockdown and overexpression of IRFs demonstrated that IRF1 and IRF3 have antiviral properties against OC43, while IRF3 and IRF7 are effective in restricting 229E infection. IRF3 activation effectively promotes transcription of antiviral genes during OC43 or 229E infection. Our study suggests that IRFs may be effective antiviral regulators against human coronavirus infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Duncan, Xu, Licursi, Joyce, Saffran, Liu, Gohda, Tyrrell, Kawaguchi and Hirasawa.)

The targeted screening and sequencing approaches for COVID-19 surveillance need to be adjusted to fit the evolving surveillance objectives which necessarily change over time. We present the development of variant screening assays that can be applied to new targets in a timely manner and enable multiplexing of targets for efficient implementation in the laboratory. By targeting the HV69/70 deletion for Alpha, K417N for Beta, K417T for Gamma, and HV69/70 deletion plus K417N for sub-variants BA.1, BA.3, BA.4, and BA.5 of Omicron, we achieved simultaneous detection and differentiation of Alpha, Beta, Gamma, and Omicron in a single assay. Targeting both T478K and P681R mutations enabled specific detection of the Delta variant. The multiplex assays used in combination, targeting K417N and T478K, specifically detected the Omicron sub-variant BA.2. The limits of detection for the five variants of concern were 4-16 copies of the viral RNA per reaction. Both assays achieved 100% clinical sensitivity and 100% specificity. Analyses of 377 clinical samples and 24 wastewater samples revealed the Delta variant in 100 clinical samples (nasopharyngeal and throat swab) collected in November 2021. Omicron BA.1 was detected in 79 nasopharyngeal swab samples collected in January 2022. Alpha, Beta, and Gamma variants were detected in 24 wastewater samples collected in May-June 2021 from two major cities of Alberta (Canada), and the results were consistent with the clinical cases of multiple variants reported in the community., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

OBJETIVO: Apesar da reconhecida importância em acompanhar a evolução temporal da mortalidade infantil precoce, a deficiência das estatísticas vitais no Brasil ainda permanece na agenda atual dos problemas que impedem o seu acompanhamento espaço-temporal. Realizou-se estudo com o objetivo de investigar o Sistema de Informações Hospitalares (SIH/SUS) como fonte de informações, para estimar a natimortalidade e a mortalidade neonatal. MÉTODOS: Propõe-se um método para estimar a natimortalidade e a mortalidade neonatal, o qual foi aplicado para todos os Estados das regiões Nordeste, Sul e Sudeste e para o Pará, no ano de 1995. Para fins comparativos, o Sistema de Informações sobre Mortalidade (SIM/MS) foi utilizado para estimar as taxas sob estudo, após a correção do número de nascidos vivos por um método demográfico. RESULTADOS: O SIH/SUS forneceu mais óbitos fetais e neonatais precoces do que o SIM/MS em grande parte das unidades federadas da região Nordeste. Adicionalmente para os Estados localizados nas regiões Sul e Sudeste, que apresentam, em geral, boa cobertura do registro de óbitos, as taxas calculadas pelos dois sistemas de informação tiveram valores semelhantes. CONCLUSÕES: Considerando a cobertura incompleta das estatísticas vitais no Brasil e a agilidade do SIH/SUS em disponibilizar as informações em meio magnético, conclui-se que o uso do SIH/SUS poderá trazer inúmeras contribuições para análise do comportamento espaço-temporal do componente neonatal da mortalidade infantil no território brasileiro, em anos recentes.

OBJETIVO: Apesar da reconhecida importância em acompanhar a evolução temporal da mortalidade infantil precoce, a deficiência das estatísticas vitais no Brasil ainda permanece na agenda atual dos problemas que impedem o seu acompanhamento espaço-temporal. Realizou-se estudo com o objetivo de investigar o Sistema de Informações Hospitalares (SIH/SUS) como fonte de informações, para estimar a natimortalidade e a mortalidade neonatal. MÉTODOS: Propõe-se um método para estimar a natimortalidade e a mortalidade neonatal, o qual foi aplicado para todos os Estados das regiões Nordeste, Sul e Sudeste e para o Pará, no ano de 1995. Para fins comparativos, o Sistema de Informações sobre Mortalidade (SIM/MS) foi utilizado para estimar as taxas sob estudo, após a correção do número de nascidos vivos por um método demográfico. RESULTADOS: O SIH/SUS forneceu mais óbitos fetais e neonatais precoces do que o SIM/MS em grande parte das unidades federadas da região Nordeste. Adicionalmente para os Estados localizados nas regiões Sul e Sudeste, que apresentam, em geral, boa cobertura do registro de óbitos, as taxas calculadas pelos dois sistemas de informação tiveram valores semelhantes. CONCLUSÕES: Considerando a cobertura incompleta das estatísticas vitais no Brasil e a agilidade do SIH/SUS em disponibilizar as informações em meio magnético, conclui-se que o uso do SIH/SUS poderá trazer inúmeras contribuições para análise do comportamento espaço-temporal do componente neonatal da mortalidade infantil no território brasileiro, em anos recentes.

This study attempts to explore the anorexia nervosa (AN) patient's subjective experience in family therapy by employing a qualitative inquiry. The data collection process required the participant to review videotapes of her family sessions and then to write down her thoughts in response to a core research question: "What was your experience of the family therapy sessions?" We tried our best to minimize any possible influence from the research setting. Unexpectedly, a core idea about the anorexic body emerged from the patient's writings. This core idea was divided into four themes: (1) the development of the anorexic body; (2) the anorexic body and body weight; (3) the anorexic body and clothing; (4) maintaining the anorexic body. This study suggests that a qualitative client-driven approach can reveal the AN patient's perceptions of her body. Most importantly, this paper ends by providing recommendations for qualitative researchers: adopting a not-knowing position and being open to learn that knowledge can be found accidentally. URN: urn:nbn:de:0114-fqs030117

Coastal ecosystems such as sand dunes, mangrove forests, and salt marshes provide natural storm protection for vulnerable shorelines. At the same time, storms erode and redistribute biological materials among coastal systems via wrack. Yet how such cross-ecosystem subsidies affect post-storm recovery is not well understood. Here, we report an experimental investigation into the effect of storm wrack on eco-geomorphological recovery of a coastal embryo dune in north-eastern Florida, USA, following hurricane Irma. We contrasted replicated 100-m2 wrack-removal and unmanipulated (control) plots, measuring vegetation and geomorphological responses over 21 months. Relative to controls, grass cover was reduced 4-fold where diverse storm wrack, including seagrass rhizomes, seaweed, and wood, was removed. Wrack removal was also associated with a reduction in mean elevation, which persisted until the end of the experiment when removal plots had a 14% lower mean elevation than control plots. These results suggest that subsides of wrack re-distributed from other ecosystem types (e.g. seagrasses, macroalgae, uplands): i) enhances the growth of certain dune-building grasses; and ii) boosts the geomorphological recovery of coastal dunes. Our study also indicates that the practice of post-storm beach cleaning to remove wrack-a practice widespread outside of protected areas-may undermine the resilience of coastal dunes and their services., Competing Interests: The authors have declared that no competing interests exist.

The SARS-CoV-2 pandemic is an ongoing threat to global health, and wide-scale vaccination is an efficient method to reduce morbidity and mortality. We designed and evaluated two DNA plasmid vaccines, based on the pIDV-II system, expressing the SARS-CoV-2 spike gene, with or without an immunogenic peptide, in mice, and in a Syrian hamster model of infection. Both vaccines demonstrated robust immunogenicity in BALB/c and C57BL/6 mice. Additionally, the shedding of infectious virus and the viral burden in the lungs was reduced in immunized hamsters. Moreover, high-titers of neutralizing antibodies with activity against multiple SARS-CoV-2 variants were generated in immunized animals. Vaccination also protected animals from weight loss during infection. Additionally, both vaccines were effective at reducing both pulmonary and extrapulmonary pathology in vaccinated animals. These data show the potential of a DNA vaccine for SARS-CoV-2 and suggest further investigation in large animal and human studies could be pursued., (© 2022. The Author(s).)

Recurring coronavirus outbreaks, such as the current COVID-19 pandemic, establish a necessity to develop direct-acting antivirals that can be readily administered and are active against a broad spectrum of coronaviruses. Described in this Article are novel α-acyloxymethylketone warhead peptidomimetic compounds with a six-membered lactam glutamine mimic in P1. Compounds with potent SARS-CoV-2 3CL protease and in vitro viral replication inhibition were identified with low cytotoxicity and good plasma and glutathione stability. Compounds 15e , 15h , and 15l displayed selectivity for SARS-CoV-2 3CL protease over CatB and CatS and superior in vitro SARS-CoV-2 antiviral replication inhibition compared with the reported peptidomimetic inhibitors with other warheads. The cocrystallization of 15l with SARS-CoV-2 3CL protease confirmed the formation of a covalent adduct. α-Acyloxymethylketone compounds also exhibited antiviral activity against an alphacoronavirus and non-SARS betacoronavirus strains with similar potency and a better selectivity index than remdesivir. These findings demonstrate the potential of the substituted heteroaromatic and aliphatic α-acyloxymethylketone warheads as coronavirus inhibitors, and the described results provide a basis for further optimization.

Samples of nasopharyngeal swabs (NPS) are commonly used for the detection of SARS-CoV-2 and diagnosis of COVID-19. As an alternative, self-collection of saliva and gargle samples minimizes transmission to healthcare workers and relieves the pressure of resources and healthcare personnel during the pandemic. This study aimed to develop an enhanced method enabling simultaneous viral inactivation and RNA preservation during on-site self-collection of saliva and gargle samples. Our method involves the addition of saliva or gargle samples to a newly formulated viral inactivation and RNA preservation (VIP) buffer, concentration of the viral RNA on magnetic beads, and detection of SARS-CoV-2 using reverse transcription quantitative polymerase chain reaction directly from the magnetic beads. This method has a limit of detection of 25 RNA copies per 200 μL of gargle or saliva sample and 9-111 times higher sensitivity than the viral RNA preparation kit recommended by the United States Centers for Disease Control and Prevention. The integrated method was successfully used to analyze more than 200 gargle and saliva samples, including the detection of SARS-CoV-2 in 123 gargle and saliva samples collected daily from two NPS-confirmed positive SARS-CoV-2 patients throughout the course of their infection and recovery. The VIP buffer is stable at room temperature for at least 6 months. SARS-CoV-2 RNA (65 copies/200 μL sample) is stable in the VIP buffer at room temperature for at least 3 weeks. The on-site inactivation of SARS-CoV-2 and preservation of the viral RNA enables self-collection of samples, reduces risks associated with SARS-CoV-2 transmission, and maintains the stability of the target analyte., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Replication of SARS-CoV-2, the coronavirus causing COVID-19, requires a main protease (M pro ) to cleave viral proteins. Consequently, M pro is a target for antiviral agents. We and others previously demonstrated that GC376, a bisulfite prodrug with efficacy as an anti-coronaviral agent in animals, is an effective inhibitor of M pro in SARS-CoV-2. Here, we report structure-activity studies of improved GC376 derivatives with nanomolar affinities and therapeutic indices >200. Crystallographic structures of inhibitor-M pro complexes reveal that an alternative binding pocket in M pro , S4, accommodates the P3 position. Alternative binding is induced by polar P3 groups or a nearby methyl. NMR and solubility studies with GC376 show that it exists as a mixture of stereoisomers and forms colloids in aqueous media at higher concentrations, a property not previously reported. Replacement of its Na + counter ion with choline greatly increases solubility. The physical, biochemical, crystallographic, and cellular data reveal new avenues for M pro inhibitor design., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

SARS-CoV-2 is the etiological agent of COVID19. There are currently several licensed vaccines approved for human use and most of them target the spike protein in the virion envelope to induce protective immunity. Recently, variants that spread more quickly have emerged. There is evidence that some of these variants are less sensitive to neutralization in vitro, but it is not clear whether they can evade vaccine induced protection. In this study, we tested SARS-CoV-2 spike RBD as a vaccine antigen and explored the effect of formulation with Alum/MPLA or AddaS03 adjuvants. Our results show that RBD induces high titers of neutralizing antibodies and activates strong cellular immune responses. There is also significant cross-neutralization of variants B.1.1.7 and B.1.351 and to a lesser extent, SARS-CoV-1. These results indicate that recombinant RBD can be a viable candidate as a stand-alone vaccine or as a booster shot to diversify our strategy for COVID19 protection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

CRISPR-Cas systems integrated with nucleic acid amplification techniques improve both analytical specificity and sensitivity. We describe here issues and solutions for the successful integration of reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube under an isothermal condition (40 °C). Specific detection of a few copies of a viral DNA sequence was achieved in less than 20 min. However, the sensitivity was orders of magnitude lower for the detection of viral RNA due to the slow initiation of RPA when the complementary DNA (cDNA) template remained hybridized to RNA. During the delay of RPA, the crRNA-Cas12a ribonucleoprotein (RNP) gradually lost its activity in the RPA solution, and nonspecific amplification reactions consumed the RPA reagents. We overcame these problems by taking advantage of the endoribonuclease function of RNase H to remove RNA from the RNA-cDNA hybrids and free the cDNA as template for the RPA reaction. As a consequence, we significantly enhanced the overall reaction rate of an integrated assay using RT-RPA and CRISPR-Cas12a for the detection of RNA. We showed successful detection of 200 or more copies of the S gene sequence of SARS-CoV-2 RNA within 5-30 min. We applied our one-tube assay to 46 upper respiratory swab samples for COVID-19 diagnosis, and the results from both fluorescence intensity measurements and end-point visualization were consistent with those of RT-qPCR analysis. The strategy and technique improve the sensitivity and speed of RT-RPA and CRISPR-Cas12a assays, potentially useful for both semi-quantitative and point-of-care analyses of RNA molecules.

Tragically, the death toll from the COVID-19 pandemic continues to rise, and with variants being observed around the globe new therapeutics, particularly direct-acting antivirals that are easily administered, are desperately needed. Studies targeting the SARS-CoV-2 3CL protease, which is critical for viral replication, with different peptidomimetics and warheads is an active area of research for development of potential drugs. To date, however, only a few publications have evaluated the nitrile warhead as a viral 3CL protease inhibitor, with only modest activity reported. This article describes our investigation of P3 4-methoxyindole peptidomimetic analogs with select P1 and P2 groups with a nitrile warhead that are potent inhibitors of SARS-CoV-2 3CL protease and demonstrate in vitro SARS-CoV-2 antiviral activity. A selectivity for SARS-CoV-2 3CL protease over human cathepsins B, S and L was also observed with the nitrile warhead, which was superior to that with the aldehyde warhead. A co-crystal structure with SARS-CoV-2 3CL protease and a reversibility study indicate that a reversible, thioimidate adduct is formed when the catalytic sulfur forms a covalent bond with the carbon of the nitrile. This effort also identified efflux as a property limiting antiviral activity of these compounds, and together with the positive attributes described these results provide insight for further drug development of novel nitrile peptidomimetics targeting SARS-CoV-2 3CL protease., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Single-cell RNA sequencing (scRNA-seq) provides rich transcriptomic information for studying molecular events and cell heterogeneity at the single-cell level. However, it is challenging to obtain sequence information from rare or low-abundance genes in the presence of other highly abundant genes. We report here a CRISPR-Cas9 technique for the depletion of high-abundance transcripts, resulting in preferential enrichment of rare transcripts. We demonstrate an application of this CRISPR-mediated enrichment technique to scRNA-seq of liver cells infected with hepatitis B virus (HBV). Direct sequencing without the CRISPR-mediated enrichment detected HBV RNA in only 0.6% of the cells. The CRISPR-mediated depletion of the three most abundant transcripts resulted in selective enrichment of the HBV transcript and successful sequencing of HBV RNA in more than 74% of the cells. The improvement enabled a study of HBV infection and interferon treatment of a liver cell model. Gene clusters between the control and HBV-infected Huh7.5-NTCP cells were similar, suggesting that HBV infection did not significantly alter gene expression of the host cells. The treatment with interferon alpha dramatically changed the gene expression of Huh7.5-NTCP cells. These results from the single cell RNA-seq analysis of 7370 cells are consistent with those of bulk experiments, suggesting that HBV is a "stealth virus".

Objectives: A major COVID-19 vaccine strategy is to induce antibodies that prevent interaction between the Spike protein's receptor-binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2). These vaccines will also induce T-cell responses. However, concerns were raised that aberrant vaccine-induced immune responses may exacerbate disease. We aimed to identify minimal epitopes on the RBD that would induce antibody responses that block the interaction of the RBD and ACE2 as a strategy leading to an effective vaccine with reduced risk of inducing immunopathology., Methods: We procured a series of overlapping 20-amino acid peptides spanning the RBD and asked which were recognised by plasma from COVID-19 convalescent patients. Identified epitopes were conjugated to diphtheria-toxoid and used to vaccinate mice. Immune sera were tested for binding to the RBD and for their ability to block the interaction of the RBD and ACE2., Results: Seven putative vaccine epitopes were identified. Memory B-cells (MBCs) specific for one of the epitopes were identified in the blood of convalescent patients. When used to vaccinate mice, six induced antibodies that bound recRBD and three induced antibodies that could partially block the interaction of the RBD and ACE2. However, when the sera were combined in pairs, we observed significantly enhanced inhibition of binding of RBD to ACE2. Two of the peptides were located in the main regions of the RBD known to contact ACE2. Of significant importance to vaccine development, two of the peptides were in regions that are invariant in the UK and South African strains., Conclusion: COVID-19 convalescent patients have SARS-CoV-2-specific antibodies and MBCs, the specificities of which can be defined with short peptides. Epitope-specific antibodies synergistically block RBD-ACE2 interaction., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

An estimated two billion people worldwide have been infected with hepatitis B virus (HBV). Despite the high infectivity of HBV in vivo, a lack of easily infectable in vitro culture systems hinders studies of HBV. Overexpression of the sodium taurocholate co-transporting polypeptide (NTCP) bile acid transporter in hepatoma cells improved infection efficiency. We report here a hepatoma cell culture system that does not require dimethyl sulfoxide (DMSO) for HBV infection. We overexpressed NTCP in Huh7.5 cells and allowed these cells to differentiate in a medium supplemented with human serum (HS) instead of fetal bovine serum (FBS). We show that human serum culture enhanced HBV infection in Huh7.5-NTCP cells, e.g., in HS cultures, HBV pgRNA levels were increased by as much as 200-fold in comparison with FBS cultures and 19-fold in comparison with FBS+DMSO cultures. Human serum culture increased levels of hepatocyte differentiation markers, such as albumin secretion, in Huh7.5-NTCP cells to similar levels found in primary human hepatocytes. N-glycosylation of NTCP induced by culture in human serum may contribute to viral entry. Our study demonstrates an in vitro HBV infection of Huh7.5-NTCP cells without the use of potentially toxic DMSO.