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Circular RNAs (circRNAs) are stable and prevalent RNAs in eukaryotic cells that arise from back-splicing. Synthetic circRNAs and some endogenous circRNAs can encode proteins, raising the promise of circRNA as a platform for gene expression. In this study, we developed a systematic approach for rapid assembly and testing of features that affect protein production from synthetic circRNAs. To maximize circRNA translation, we optimized five elements: vector topology, 5′ and 3′ untranslated regions, internal ribosome entry sites and synthetic aptamers recruiting translation initiation machinery. Together, these design principles improve circRNA protein yields by several hundred-fold, provide increased translation over messenger RNA in vitro, provide more durable translation in vivo and are generalizable across multiple transgenes.

PDF file - 625K, NFAT1-deficient CD4+ T cells are resistant to soluble peptide-induced anergy in vivo

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We aimed to investigate whether combination assessment of phase angle (PhA) and skeletal muscle index (SMI), was a possible predictor of physical function at discharge from the hospital in patients with acute stroke.In this retrospective cohort study that was conducted from May 2020 and July 2021, we determined PhA and SMI using bioimpedance analysis (BIA) in patients with acute stroke. Patients were classified as normal, low PhA + SMI group, pre-sarcopenia (low SMI only), and dynapenia (low PhA only) using cut-off points (men: SMI7.0 kg/m2, PhA4.05 degrees; women: SMI5.7 kg/m2, PhA3.55 degrees). The main outcome was physical function based on functional independence measure motor (FIM-motor) score at discharge. Multiple regression analysis was used to determine the association between low PhA + SMI and FIM-motor score.We included 244 patients (161 men; mean age, 73.9 years). low PhA + SMI was found in 21 (8.6%) patients. Multiple regression analysis showed that low PhA + SMI was independently associated with the FIM-motor score at discharge (β= -0.099, 95%CI: -0.193,-0.005, p = 0.039). The PhA cutoff values for determining good functional results using receiver operating characteristic (ROC) curves were 5.36 for men (sensitivity = 0.769, specificity = 0.586, area under the curve [AUC] = 0.682), and 3.85 for women (sensitivity = It was 0.881, specificity = 0.481, AUC). Further, pearson correlation coefficient showed that PhA was significantly related to FIM-motor score in patients with mild or moderately severe stroke (mild: r = 0.472, p0.001; moderate: r = 0.524, p0.001).Combination of low PhA and SMI values at baseline, was an independent predictor of physical function at discharge in patients with acute stroke. The findings highlighted the importance of measuring PhA and SMI using BIA in patients with acute stroke.

International audience; The ground-state magnetic dipole moment of the neutron-rich O21 isotope has been measured via β-ray-detected nuclear magnetic resonance (β-NMR) spectroscopy by using a spin-polarized secondary beam of O21 produced from the Ne22 primary beam. From the present measurement, the g factor |gexp(Og.s.21)|=0.6036(14) has been determined. Based on the comparison of this value with Schmidt values, we unambiguously confirm the νd5/2 configuration with spin and parity assignments Iπ=5/2+ for the O21 ground state, suggested by previously reported studies. Consequently, the magnetic moment has been determined as μexp(Og.s.21)=(−)1.5090(35)μN. The obtained experimental magnetic moment is in good agreement with the predictions of the shell-model calculations using the USD, YSOX, and SDPF-M interactions as well as random phase approximation (RPA) calculations. This observation indicates that the O21 nucleus in its ground state does not manifest any anomalous structure and is not influenced by the proximity of the drip line.

Toxin-antitoxin (TA) systems are abundant genetic modules in bacterial chromosomes and on mobile elements. They are often patchily distributed and their physiological functions remain poorly understood. Here, we characterize a TA system inLegionella pneumophilathat is highly conserved acrossLegionellaspecies. This system is distantly related toEscherichia coliHipBST and we demonstrate that it is a functional tripartite TA system (denoted HipBSTLp). We identify HipBSTLphomologs in diverse taxa, yet in the Gammaproteobacteria these are almost exclusively found inLegionellaspecies. Notably, the toxin HipTLpwas previously reported to be a pathogenic effector protein that is translocated byL. pneumophilainto its eukaryotic hosts. Contrary to this, we find no signal of HipTLptranslocation beyond untranslocated control levels and make several observations consistent with a canonical role as a bacterial toxin. We present structural and biochemical insights into the regulation and neutralization of HipBSTLp, and identify key variations between this system and HipBSTEc. Finally, we show that the target of HipTLpis likely not conserved with any characterized HipA or HipT toxin. This work serves as a unique comparison of a TA system across bacterial species and illustrates the molecular diversity that exists within a single TA family.

The United States Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for the use of COVID-19 convalescent plasma (CCP) to treat hospitalized patients with COVID-19 in August 2020. However, by 4 February 2021, the FDA had revised the convalescent plasma EUA.

We study the diffuse background free-free emission induced by dark matter halos. Since dark matter halos host ionized thermal plasma, they could be an essential source of cosmological free-free emission. We evaluate the global background intensity and the anisotropy of this free-free emission. We show that the dominant contribution comes from dark matter halos with a mass close to the Jeans mass, $M_{\mathrm{halo}}\sim 10^{10} M_\odot$, around the redshift $z \sim 3$. Therefore, the intensity of the free-free emission is sensitive to the small-scale primordial curvature perturbations that form such small-mass dark matter halos. Although our obtained intensity of the global and anisotropic free-free emission is smaller than the $10\%$ level of the free-free emission observed in the high galactic region, we find that the free-free emission signal is modified by $\sim 20 \%$ even in the parameter set of the spectral index and the running, which is consistent with the recent Planck result. Therefore, the measurement of the cosmological free-free signals has the potential to provide more stringent constraints on the abundance of small-mass dark matter halos and the curvature perturbations including the spectral index and the running, while carefully removing the Galactic free-free emission is required through the multifrequency radio observation or the cross-correlation study with the galaxy surveys or 21-cm intensity map., Comment: 12 pages, 13 figures

Animals move their head and eyes as they explore and sample the visual scene. Previous studies have demonstrated neural correlates of head and eye movements in rodent primary visual cortex (V1), but the sources and computational roles of these signals are unclear. We addressed this by combining measurement of head and eye movements with high density neural recordings in freely moving mice. V1 neurons responded primarily to gaze shifts, where head movements are accompanied by saccadic eye movements, but not to head movements where compensatory eye movements stabilize gaze. A variety of activity patterns immediately followed gaze shifts, including units with positive, biphasic, or negative responses, and together these responses formed a temporal sequence following the gaze shift. These responses were greatly diminished in the dark for the vast majority of units, replaced by a uniform suppression of activity, and were similar to those evoked by sequentially flashed stimuli in head-fixed conditions, suggesting that gaze shift transients represent the temporal response to the rapid onset of new visual input. Notably, neurons responded in a sequence that matches their spatial frequency preference, from low to high spatial frequency tuning, consistent with coarse-to-fine processing of the visual scene following each gaze shift. Recordings in foveal V1 of freely gazing head-fixed marmosets revealed a similar sequence of temporal response following a saccade, as well as the progression of spatial frequency tuning. Together, our results demonstrate that active vision in both mice and marmosets consists of a dynamic temporal sequence of neural activity associated with visual sampling.HighlightsDuring free movement, neurons in mouse V1 respond to head movements that are accompanied by a gaze-shifting saccadic eye movement, but not a compensatory eye movement.Neurons respond to gaze shifts with diverse temporal dynamics that form a sequence across the population, from early positive responses to biphasic and negative responses.In darkness, most neurons show a uniform suppression following a gaze shift.Temporal dynamics of responses correspond to a neuron’s temporal and spatial frequency preferences, consistent with a coarse-to-fine processing sequence.A similar temporal sequence following saccades is observed in foveal V1 of freely gazing head-fixed marmosets, demonstrating shared aspects of active visual processing across species.

Kidney transplant recipients (KTRs) have a diminished response to SARS-CoV-2 vaccination compared with immunocompetent individuals. Deeper understanding of antibody responses in KTRs following third-dose vaccination would enable identification of those who remain unprotected against Omicron.We profiled antibody responses in KTRs pre- and at 1 and 3 mo post-third-dose SARS-CoV-2 mRNA-based vaccine. Binding antibody levels were determined by ELISA. Neutralization against wild type, Beta, Delta, and Omicron (BA.1) variants was determined using a SARS-CoV-2 spike-pseudotyped lentivirus assay.Forty-four KTRs were analyzed at 1 and 3 mo (n = 26) post-third dose. At 1 mo, the proportion of participants with a robust antibody response had increased significantly from baseline, but Omicron-specific neutralizing antibodies were detected in just 45% of KTRs. Median binding antibody levels declined at 3 mo, but the proportion of KTRs with a robust antibody response was unchanged; 38.5% KTRs maintained Omicron-specific neutralization at 3 mo. No clinical variables were significantly associated with Omicron-neutralizing antibodies, but antireceptor binding domain titers appeared to identify those with Omicron-specific neutralizing capacity.Over 50% of KTRs lack Omicron-specific neutralization capacity 1 mo post-third mRNA-vaccine dose. Antibody levels of responders were well preserved at 3 mo. Anti receptor binding domain antibody titers may identify patients with a detectable Omicron-neutralizing antibody response.

Our group has previously used laboratory and commercially developed assays to understand the IgG responses to SARS-CoV-2 antigens, including nucleocapsid (N), spike (S), and receptor binding domain (RBD), in Canadian blood donors. In this current study, we analyzed 17,428 available and previously characterized retention samples collected from April 2020 to March 2021. The analysis compared the characteristics of the Abbott SARS-CoV-2 IgG II Quant assay (Abbott anti-spike [S], Abbott, Chicago, IL) against four other IgG assays. The Abbott anti-S assay has a qualitative threshold of 50 AU/mL. The four comparator assays were the Abbott anti-nucleocapsid (N) assay and three commonly used Canadian in-house IgG enzyme-linked immunosorbent assays (ELISAs) recognizing distinct recombinant viral antigens, full-length spike glycoprotein, glycoprotein RBD, and nucleocapsid. The strongest qualitative relationship was between Sinai RBD and the Abbott anti-S assay (kappa, 0.707; standard error [SE] of kappa, 0.018; 95% confidence interval, 0.671 to 0.743). We then scored each previously characterized specimen as positive when two anti-SARS-COV-2 assays identified anti-SARS-CoV-2 IgG in the specimen. Using this composite reference standard approach, the sensitivity of the Abbott anti-S assay was 95.96% (95% confidence interval [CI], 93.27 to 97.63%). The specificity of the Abbott anti-S assay was 99.35% (95% CI, 99.21 to 99.46%). Our study provides context on the use of commonly used SARS-CoV-2 serologies in Canada and identifies how these assays qualitatively compare to newer commercial assays. Our next steps are to assess how well the Abbott anti-S assays quantitatively detect wild-type and SARS-CoV-2 variants of concern.

Background: Kidney transplant recipients (KTR) have a diminished response to SARS-CoV-2 vaccination in comparison to immunocompetent individuals. Deeper understanding of the antibody response in KTRs following third-dose vaccination would enable identification of those who remain unprotected against Omicron and require additional treatment strategies. Methods: We profiled antibody responses in KTRs pre- and at one and three months post-third-dose SARS-CoV2 mRNA-based vaccine. Anti-spike and anti-RBD IgG levels were determined by ELISA. Neutralization against wild-type, Beta, Delta and Omicron (BA.1) variants was determined using a SARS-CoV-2 spike pseudotyped lentivirus assay. Results: 44 KTRs were analysed at 1 and 3 months (n=26) post-third-dose. At one month, the proportion of participants with a robust antibody response had increased significantly from baseline, but Omicron-specific neutralizing antibodies were detected in just 45% of KTRs. Median binding antibody levels declined at 3 months, but the proportion of KTRs with a robust antibody response was unchanged. 38.5% KTRs maintained Omicron-specific neutralization at 3 months. No clinical variables were significantly associated with detectable Omicron neutralizing antibodies, but anti-RBD titres appeared to identify those with Omicron-specific neutralizing capacity. Conclusion: Over 50% of KTRs lack an Omicron-specific neutralization response 1 month following a third mRNA-vaccine dose. Among responders, binding and neutralizing antibody responses were well preserved at 3 months. Anti-RBD antibody titres may be a useful identifier of patients with detectable Omicron neutralizing antibody response. Trial registration: Clinical Trials Ontario: ID 3604

BACKGROUND: Limited information is available on the impact of immunosuppressants on COVID-19 vaccination in patients with immune-mediated inflammatory diseases (IMID). METHODS: This observational cohort study examined the immunogenicity of SARS-CoV-2 mRNA vaccines in adult patients with inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, or psoriatic disease, with or without maintenance immunosuppressive therapies. Ab and T cell responses to SARS-CoV-2, including neutralization against SARS-CoV-2 variants, were determined before and after 1 and 2 vaccine doses. RESULTS: We prospectively followed 150 subjects, 26 healthy controls, 9 patients with IMID on no treatment, 44 on anti-TNF, 16 on anti-TNF with methotrexate/azathioprine (MTX/AZA), 10 on anti–IL-23, 28 on anti–IL-12/23, 9 on anti–IL-17, and 8 on MTX/AZA. Ab and T cell responses to SARS-CoV-2 were detected in all participants, increasing from dose 1 to dose 2 and declining 3 months later, with greater attrition in patients with IMID compared with healthy controls. Ab levels and neutralization efficacy against variants of concern were substantially lower in anti-TNF–treated patients than in healthy controls and were undetectable against Omicron by 3 months after dose 2. CONCLUSIONS: Our findings support the need for a third dose of the mRNA vaccine and for continued monitoring of immunity in these patient groups. FUNDING: Funded by a donation from Juan and Stefania Speck and by Canadian Institutes of Health (CIHR)/COVID-Immunity Task Force (CITF) grants VR-1 172711 and VS1-175545 (to THW and ACG), CIHR FDN-143250 (to THW), GA2-177716 (to VC, ACG, and THW), and GA1-177703 (to ACG) and the CIHR rapid response network to SARS-CoV-2 variants, CoVaRR-Net (to ACG).

A long-standing crucial question with atomic nuclei is whether or not α clustering occurs there. An α particle (helium-4 nucleus) comprises two protons and two neutrons, and may be the building block of some nuclei. This is a very beautiful and fascinating idea, and is indeed plausible because the α particle is particularly stable with a large binding energy. However, direct experimental evidence has never been provided. Here, we show whether and how α(-like) objects emerge in atomic nuclei, by means of state-of-the-art quantum many-body simulations formulated from first principles, utilizing supercomputers including K/Fugaku. The obtained physical quantities exhibit agreement with experimental data. The appearance and variation of the α clustering are shown by utilizing density profiles for the nuclei beryllium-8, -10 and carbon-12. With additional insight by statistical learning, an unexpected crossover picture is presented for the Hoyle state, a critical gateway to the birth of life.

A vitamina K é uma das quatro vitaminas lipossolúveis essenciais ao organismo. Sua principal função e a mais conhecida é atuar no processo de coagulação sanguínea. Estudos mais recentes, porém, têm demonstrado a relação entre os níveis inadequados de vitamina K no organismo com o desenvolvimento de diversas outras patologias como a aterosclerose, câncer, diabetes e doenças neurodegenerativas como Parkinson e Alzheimer. As fontes de vitamina K mais popularmente conhecidas são os vegetais de folhas verdes, porém, essas apresentam somente a vitamina K1. Atualmente, destaca-se o nattô, um alimento típico japonês à base de soja fermentada, como a principal fonte de vitamina K2, sua forma associada a efeitos além da coagulação sanguínea. A ingestão diária recomendada (IDR) de vitamina K tem sido muito discutida, pois atualmente estabelece-se apenas a IDR para a vitamina K, sem distinção entre as vitaminas K1 e K2. Tendo em vista os vários questionamentos em relação à vitamina K, o trabalho teve como objetivo apresentar uma atualização sobre esta importante vitamina, descrevendo um breve histórico sobre sua descoberta, suas principais fontes, as funções plenamente reconhecidas e as perspectivas sobre as novas evidências atribuídas especialmente à vitamina K2.

Ultracompact minihalos~(UCMHs) can form after the epoch of matter-radiation equality, if the density fluctuations of dark matter have significantly large amplitude on small scales. The constraint on the UCMH abundance allows us to access such small-scale fluctuations. In this paper, we present that, through the measurement of 21-cm fluctuations before the epoch of reionization~ we can obtain a constraint on the UCMH abundance. We calculate the 21-cm signal from UCMHs and show that UCMHs provide the enhancement of the 21-cm fluctuations. We also investigate the constraint on the UCMH abundance and small-scale curvature perturbations. Our results indicate that the upcoming 21-cm observation, the Square Kilometre Array (SKA), provides the constraint on amplitude of primordial curvature power spectrum, ${\cal A}_{\zeta} \lesssim 10^{-6}$ on $100~{\rm Mpc}^{-1} \lesssim k \lesssim 1000~{\rm Mpc}^{-1}$. Although it is not stronger than the one from the non-detection of gamma rays induced by dark matter annihilation in UCMHs, the constraint by the SKA will be important because this constraint is independent of the dark matter particle model., Comment: 8pages, 6 figures, published in MNRAS

We have previously used composite reference standards and latent class analysis (LCA) to evaluate the performance of laboratory assays in the presence of tarnished gold standards. Here, we apply these techniques to repeated, cross-sectional study of Canadian blood donors, whose sera underwent parallel testing with four separate SARS-CoV-2 antibody assays. We designed a repeated cross-sectional design with random cross-sectional sampling of all available retention samples (

This study attempted to understand the levels of neutralizing titers and the breadth of antibody protection against wild-type and variant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Canadian blood donors during the first 3 months of 2021. During this period, it is unlikely that many of the blood donors had received a second dose, since vaccine rollout had not yet ramped up, and less than 2% of the Canadian population had received a second dose of vaccine. A repeated cross-sectional design was used. A random cross-sectional sampling of all available Canadian Blood Services retention samples (

Vagus nerve stimulation (VNS) delivered during motor rehabilitation enhances recovery from a wide array of neurological injuries and was recently approved by the U.S. FDA for chronic stroke. The benefits of VNS result from precisely timed engagement of neuromodulatory networks during rehabilitative training, which promotes synaptic plasticity in networks activated by rehabilitation. Previous studies demonstrate that lesions that deplete these neuromodulatory networks block VNS-mediated plasticity and accompanying enhancement of recovery. There is a great deal of interest in determining whether commonly prescribed pharmacological interventions that influence these neuromodulatory networks would similarly impair VNS effects. Here, we sought to directly test the effects of three common pharmaceuticals at clinically relevant doses that target neuromodulatory pathways on VNS-mediated plasticity in rats. To do so, rats were trained on a behavioral task in which jaw movement during chewing was paired with VNS and received daily injections of either oxybutynin, a cholinergic antagonist, prazosin, an adrenergic antagonist, duloxetine, a serotonin-norepinephrine reuptake inhibitor, or saline. After the final behavioral session, intracortical microstimulation (ICMS) was used to evaluate reorganization of motor cortex representations, with area of cortex eliciting jaw movement as the primary outcome. In animals that received control saline injections, VNS paired with training significantly increased the movement representation of the jaw compared to naïve animals, consistent with previous studies. Similarly, none of the drugs tested blocked this VNS-dependent reorganization of motor cortex. The present results provide direct evidence that these common pharmaceuticals, when used at clinically relevant doses, are unlikely to adversely impact the efficacy of VNS therapy.

Circular RNAs (circRNAs) are stable and prevalent RNAs in eukaryotic cells that arise from back-splicing. Synthetic circRNAs and some endogenous circRNAs can encode proteins, raising the promise of circRNA as a platform for gene expression. In this study, we developed a systematic approach for rapid assembly and testing of features that affect protein production from synthetic circRNAs. To maximize circRNA translation, we optimized five elements: vector topology, 5' and 3' untranslated regions, internal ribosome entry sites and synthetic aptamers recruiting translation initiation machinery. Together, these design principles improve circRNA protein yields by several hundred-fold, provide increased translation over messenger RNA in vitro, provide more durable translation in vivo and are generalizable across multiple transgenes.

BackgroundLimited information is available on the impact of immunosuppressants on COVID-19 vaccination in patients with immune-mediated inflammatory diseases (IMID).MethodsThis observational cohort study examined the immunogenicity of SARS-CoV-2 mRNA vaccines in adult patients with inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, or psoriatic disease, with or without maintenance immunosuppressive therapies. Antibody and T cell responses to SARS-COV-2, including neutralization against SARS-CoV-2 variants were determined before and after 1 and 2 vaccine doses.ResultsWe prospectively followed 150 subjects, 26 healthy controls, 9 IMID patients on no treatment, 44 on anti-TNF, 16 on anti-TNF with methotrexate/azathioprine (MTX/AZA), 10 on anti-IL-23, 28 on anti-IL-12/23, 9 on anti-IL-17, and 8 on MTX/AZA. Antibody and T cell responses to SARS-CoV-2 were detected in all participants, increasing from dose 1 to dose 2 and declining 3 months later, with greater attrition in IMID patients compared to healthy controls. Antibody levels and neutralization efficacy against variants of concern were substantially lower in anti-TNF treated patients than in healthy controls and were undetectable against Omicron by 3 months after dose 2.ConclusionsOur findings support the need for a third dose of mRNA vaccine and for continued monitoring of immunity in these patient groups.FundingFunded by a donation from Juan and Stefania Speck and by Canadian Institutes of Health (CIHR) /COVID-Immunity Task Force (CITF) grants VR-1 172711 and VS1-175545 (T.H.W. and A.C.G); CIHR FDN-143250 (T.H.W.), GA2-177716 (V.C., A.C.G., T.W.), GA1-177703 (A.C.G.) and the CIHR rapid response network to SARS-CoV-2 variants, CoVaRR-Net (to A.C.G.).

Primordial black holes (PBHs) whose masses are in $\sim[10^{-15}M_\odot,10^{-11}M_{\odot}]$ have been extensively studied as a candidate of whole dark matter (DM). One of the probes to test such a PBH-DM scenario is scalar-induced stochastic gravitational waves (GWs) accompanied with the enhanced primordial fluctuations to form the PBHs with frequency peaked in the mHz band being targeted by the LISA mission. In order to utilize the stochastic GWs for checking the PBH-DM scenario, it needs to exactly relate the PBH abundance and the amplitude of the GWs spectrum. Recently in Kitajima et al., the impact of the non-Gaussianity of the enhanced primordial curvature perturbations on the PBH abundance has been investigated based on the peak theory, and they found that a specific non-Gaussian feature called the exponential tail significantly increases the PBH abundance compared with the Gaussian case. In this work, we investigate the spectrum of the induced stochastic GWs associated with PBH DM in the exponential-tail case. In order to take into account the non-Gaussianity properly, we employ the diagrammatic approach for the calculation of the spectrum. We find that the amplitude of the stochastic GW spectrum is slightly lower than the one for the Gaussian case, but it can still be detectable with the LISA sensitivity. We also find that the non-Gaussian contribution can appear on the high-frequency side through their complicated momentum configurations. Although this feature emerges under the LISA sensitivity, it might be possible to obtain information about the non-Gaussianity from GW observation with a deeper sensitivity such as the DECIGO mission., Comment: 33 pages, 19 figures

Misturas para bolo são preparações constituídas basicamente de açúcar, farinha de trigo, amidos ou féculas, gordura e ingredientes que conferem cor, sabor e aroma, dentre outros aditivos, sendo, portanto, considerado um alimento ultraprocessado. Seu consumo tem aumentado nos últimos anos devido à facilidade e rapidez no preparo e por apresentar um bom custo-benefício, o que acarretou também no aumento de novos produtos no mercado. Assim, o trabalho teve como objetivo realizar análises quantitativas e qualitativas em doze amostras de misturas para bolo colhidas pela Vigilância Sanitária do Estado de São Paulo para fins de análise de controle. As análises de rotulagem foram realizadas de acordo com as legislações brasileiras e as determinações de carboidratos totais e pesquisa de corantes artificiais, seguindo os procedimentos descritos em Métodos Físico-químicos para Análise de Alimentos do Instituto Adolfo Lutz. Do total de amostras analisadas, 50% estavam em desacordo com a legislação em relação aos dizeres de rotulagem, sendo o uso de imagens que podem levar o consumidor a erro ou engano, a principal inconformidade. A pesquisa de corantes artificiais estava de acordo com o declarado na lista de ingredientes e a determinação de carboidratos totais estava de acordo com os valores declarados na informação nutricional nas amostras avaliadas. Há necessidade de monitoramento constante desse tipo de alimento, tendo em vista as inconformidades observadas na rotulagem e especialmente em relação à sua composição visando o atendimento à meta de redução de açúcares do Ministério da Saúde.

Antibody testing against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in detecting previous exposures and analyzing vaccine-elicited immune responses. Here, we describe a scalable solution to detect and quantify SARS-CoV-2 antibodies, discriminate between natural infection- and vaccination-induced responses, and assess antibody-mediated inhibition of the spike-angiotensin converting enzyme 2 (ACE2) interaction.We developed methods and reagents to detect SARS-CoV-2 antibodies by enzyme-linked immunosorbent assay (ELISA). The main assays focus on the parallel detection of immunoglobulin (Ig)Gs against the spike trimer, its receptor binding domain (RBD) and nucleocapsid (N). We automated a surrogate neutralisation (sn)ELISA that measures inhibition of ACE2-spike or -RBD interactions by antibodies. The assays were calibrated to a World Health Organization reference standard.Our single-point IgG-based ELISAs accurately distinguished non-infected and infected individuals. For seroprevalence assessment (in a non-vaccinated cohort), classifying a sample as positive if antibodies were detected for ≥ 2 of the 3 antigens provided the highest specificity. In vaccinated cohorts, increases in anti-spike and -RBD (but not -N) antibodies are observed. We present detailed protocols for serum/plasma or dried blood spots analysis performed manually and on automated platforms. The snELISA can be performed automatically at single points, increasing its scalability.Measuring antibodies to three viral antigens and identify neutralising antibodies capable of disrupting spike-ACE2 interactions in high-throughput enables large-scale analyses of humoral immune responses to SARS-CoV-2 infection and vaccination. The reagents are available to enable scaling up of standardised serological assays, permitting inter-laboratory data comparison and aggregation.

Background: People living with chronic kidney disease (CKD) have been disproportionately affected by the coronavirus disease 2019 (COVID-19) pandemic, including higher rates of infection, hospitalization, and death. Data on responsiveness to COVID-19 vaccination strategies and immunogenicity are limited, yet required to inform vaccination strategies in this at-risk population. Objective: The objective of this study is to characterize the longitudinal serologic response to COVID-19 vaccination. Design: This is a prospective observational cohort study. Setting: Participating outpatient kidney programs within Ontario and British Columbia. Patients: Up to 2500 participants with CKD G3b-5D receiving COVID-19 vaccination, including participants receiving dialysis and kidney transplant recipients (CKD G1T-5T). Measurements: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG antibodies (anti-spike, anti-receptor binding domain, anti-nucleocapsid) will be detected by ELISA (enzyme-linked immunosorbent assay) from serum or dried blood spot testing. In a subset of participants, neutralizing antibodies against novel variants of concern will be evaluated. Peripheral blood mononuclear cells will be collected for exploratory immune profiling of SARS-CoV-2 specific cellular immunity. Methods: Participants will be recruited prior to or following any COVID-19 vaccine dose and have blood sampled for serological testing at multiple timepoints: 1, 3, 6, 9, and 12 months post vaccination. When possible, samples will be collected prior to a dose or booster. Participants will remain in the study for at least 1 year following their last COVID-19 vaccine dose. Strengths and limitations: The adaptive design of this study allows for planned modification based on emerging evidence or rapid changes in public health policy surrounding vaccination. Limitations include incomplete earlier timepoints for blood collection due to rapid vaccination of the population. Conclusions: This large multicenter serologic study of participants living with kidney disease will generate data on the kinetics of SARS-CoV-2 immune response to vaccination across the spectrum of CKD, providing insights into the amplitude and duration of immunity conferred by COVID-19 vaccination and allowing for characterization of factors associated with immune response. The results of this study may be used to inform immunization guidelines and public health recommendations for the 4 million Canadians living with CKD.

The evaluation of humoral protective immunity against SARS-CoV-2 remains crucial in understanding both natural immunity and protective immunity conferred by the several vaccines implemented in the fight against COVID-19. The reference standard for the quantification of antibodies capable of neutralizing SARS-CoV-2 is the plaque-reduction neutralization test (PRNT). However, given that it is a laboratory-developed assay, validation is crucial in order to ensure sufficient specificity and intra- and interassay precision. In addition, a multitude of other serological assays have been developed, including enzyme-linked immunosorbent assay (ELISA), flow cytometry-based assays, luciferase-based lentiviral pseudotype assays, and commercially available human ACE2 receptor-blocking antibody tests, which offer practical advantages in the evaluation of the protective humoral response against SARS-CoV-2. In this study, we validated a SARS-CoV-2 PRNT to assess both 50% and 90% neutralization of SARS-CoV-2 according to guidelines outlined by the World Health Organization. Upon validation, the reference-standard PRNT demonstrated excellent specificity and both intra- and interassay precision. Using the validated assay as a reference standard, we characterized the neutralizing antibody response in specimens from patients with laboratory-confirmed COVID-19. Finally, we conducted a small-scale multilaboratory comparison of alternate SARS-CoV-2 PRNTs and surrogate neutralization tests. These assays demonstrated substantial to perfect interrater agreement with the reference-standard PRNT and offer useful alternatives to assess humoral immunity against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the causal agent of COVID-19, has infected over 246 million people and led to over 5 million deaths as of October 2021. With the approval of several efficacious COVID-19 vaccines, methods to evaluate protective immune responses will be crucial for the understanding of long-term immunity in the rapidly growing vaccinated population. The PRNT, which quantifies SARS-CoV-2-neutralizing antibodies, is used widely as a reference standard to validate new platforms but has not undergone substantial validation to ensure excellent inter- and intraassay precision and specificity. Our work is significant, as it describes the thorough validation of a PRNT, which we then used as a reference standard for the comparison of several alternative serological methods to measure SARS-CoV-2-neutralizing antibodies. These assays demonstrated excellent agreement with the reference-standard PRNT and include high-throughput platforms, which can greatly enhance capacity to assess both natural and vaccine-induced protective immunity against SARS-CoV-2.

BACKGROUND: Testing for antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in detecting previous exposures and analyzing vaccine-elicited immune responses. Here, we describe a scalable "Made-in-Canada" solution that can detect and quantify SARS-CoV-2 antibodies, discriminate between natural infection- and vaccination-induced responses, and assess antibody-mediated inhibition of the spike-angiotensin converting enzyme 2 (ACE2) interaction. METHODS: We developed a set of methods and reagents to detect SARS-CoV-2 antibodies by enzyme-linked immunosorbent assay (ELISA). The main assays focus on the parallel detection of immunoglobulin (Ig)Gs against the spike trimer, its receptor binding domain (RBD), and the nucleocapsid (N) protein. These antigens are complemented by a detection antibody (human anti-IgG fused to horseradish peroxidase (HRP)) and a positive control reference antibody (recombinant IgG against the RBD), permitting intra- and inter-laboratory comparisons. Using this toolkit and commercial reagents, we optimized automated ELISAs on two different high throughput platforms to measure antibody responses to SARS-CoV-2 antigens. The assays were calibrated to a reference standard from the World Health Organization. We also automated a surrogate neutralization (sn)ELISA that measures inhibition of ACE2-Spike or -RBD interactions by antibodies using biotinylated ACE2. RESULTS: Our individual IgG-based ELISAs measure antibody levels in single-point measurements in reference to a standard antibody curve to accurately distinguish non-infected and infected individuals (area under the curve > 0.96 for each assay). Positivity thresholds can be established in individual assays using precision-recall analysis (e.g., by fixing the false positive rate), or more stringently, by scoring against the distribution of the means of negative samples across multiple assays performed over several months. For seroprevalence assessment (in a non-vaccinated cohort), classifying a sample as positive if antibodies were detected for at least 2 of the 3 antigens provided the highest specificity. In vaccinated cohorts, increases in anti-spike and -RBD (but not -N) antibodies are observed. Here, we present detailed protocols to perform these assays using either serum/plasma or dried blood spots both manually and on two automated platforms, and to express the results in international units to facilitate data harmonization and inter-study comparisons. We also demonstrate that the snELISA can be performed automatically at single points, increasing the scalability of this functional assay for large seroprevalence studies. INTERPRETATION: The ability to measure antibodies to three viral antigens and identify neutralizing antibodies capable of disrupting spike-ACE2 interactions in high-throughput assays enables large-scale analyses of humoral immune responses to SARS-CoV-2 infection and vaccination. The "Made-in-Canada" set of protein reagents, produced at the National Research Council of Canada are publicly available to enable the up-scaling of standardized serological assays, permitting nationwide data comparison and aggregation.

Background While some studies have found an association between built environments and physical function, it is unclear whether living in a hilly area is associated with poor physical function among older adults, and whether this association varies by car driving status. This cross-sectional study investigated the association between hilliness and physical function in community-dwelling older adults and whether it varied according to their car driving status. Methods Data were collected from 633 participants aged 60 years and older living in Okinoshima Town, Shimane prefecture, Japan, in 2018. Physical function was objectively assessed; < 1.0 m/s was defined as poor walking speed, and hilliness was measured by mean land slope within a 1000-meter network buffer zone around each participant's home using geographic information system. Logistic regression examined whether hilliness was associated with poor walking speed adjusted for sex, age, body mass index, current smoking, current alcohol consumption, exercise habits, current chronic disease, and living arrangements. A stratified analysis by car driving status was also conducted. Results A total of 159 (25.1%) participants had poor walking speed. After adjusting for all confounders, hilliness was positively associated with poor walking speed (OR = 1.07, 95% CI, 1.02-1.12). The stratified analysis by car driving status showed that living in a hilly area was positively associated with poor walking speed among non-drivers (OR = 1.10, 95% CI, 1.03-1.17), though there were no associations among drivers (OR = 1.00, 95% CI 0.92-1.08). Conclusions Our study found that a hilly environment was associated with poor walking speed in community-dwelling older adults in Japan. Moreover, car driving status potentially modified the relationship between living in a hilly environment and poor walking speed. Key messages Older adults living in a hilly environment were more likely to have poor walking speed, compared to those living in less hilly area. The stratified analysis by car driving status showed that non-driving older adults living in a rural hilly environment may be at high risk for poor physical function.

Introduction The recent advances in the treatment for heart failure with reduced ejection fraction (HFrEF) have been remarkable, while no therapy has convincingly improved the prognosis in HF patients with preserved (HFpEF) and mid-range (HFmrEF) ejection fraction. Frequent decompensations of HF lead to progressive deterioration of cardiac and renal function, and quality of life. Hence, prior studies have reported that the mortality of HFrEF patients increases as hospitalization for HF repeats. However, it is still unclear whether this trend applies for HFpEF and HFmrEF patients. Purpose We aimed to compare the prognostic impact of re-hospitalization due to HF on cardiovascular death (CVD) among HFrEF, HFmrEF and HFpEF patients. Methods This observational study included 1,930 consecutive patients who were hospitalized for worsening of HF and discharged alive. Of them, patients who have never or have not been hospitalized for HF at least last 2 years, were finally analyzed. Patients were consisted of the population with HFrEF (EF50%, n=291). Patients in each EF-classified population were divide into 2 subgroups based on whether patients were re-hospitalized for HF during the observational period, respectively. The primary endpoint of this study was CVD. Results During the observation period, Kaplan-Meier analysis showed that patients who were re-hospitalized for HF had higher event rate of CVD in HFrEF group (Log-rank p=0.008, Figure). Even after adjusting with multivariate covariates including age, sex, EF, brain natriuretic peptide and estimated glomerular filtration rate, re-hospitalization for HF was an independent predictor for CVD in HFrEF group (HR: 1.95, 95% CI: 1.11–2.86, p=0.029). However, in HFmrEF group and HFpEF group, there was no significant difference in the rates of CVD between 2 subgroups divided whether patients were re-hospitalized for HF or not (p=0.91, p=0.34, respectively). Conclusion Re-hospitalization for HF affected the CVD rate in HFrEF group, but not in HFmrEF and HFpEF groups. The prevention of re-hospitalization for HF is important particularly in HFrEF patients in order to improve cardiovascular mortality. Funding Acknowledgement Type of funding sources: None.

Background Hypochloremia is associated with a poor prognosis of heart failure (HF) patients. This phenomenon is sustained even in HF with preserved ejection fraction (HFpEF). Serum chloride level is known to be affected by serum renin secretion; however, this relationship is one of the least investigated field in HF patients. Renin-angiotensin system (RAS) inhibitor is recommended as a first-line medication for HF patients with reduced left ventricular ejection fraction, but no prior studies of RAS inhibitors have achieved to improve the prognosis of HFpEF patients. Purpose We investigated the relationship between baseline serum chloride level and the prognostic impact of RAS inhibitor in HFpEF patients. Methods This is an observational study including 1,913 consecutive patients who admitted to hospital due to worsening of HF and discharged alive in a single university hospital. After excluding patients who received regular hemodialysis and whose left ventricular ejection fraction were under 50%, 506 HFpEF patients were ultimately analyzed. They were categorized into tertiles by serum chloride levels at discharge (T1: −100 mEq/L, T2: 101–104 mEq/L, T3: 105- mEq/L), and patients in each category were further divided into subgroups depending on the prescription of RAS inhibitor at discharge (RAS inhibitor group and Non-RAS inhibitor group). The primary endpoint of this study was death from any cause. Results During the observation period with 479 days of median follow-up, 77 (15.2%) died. Patients in the RAS inhibitor group had significantly better prognosis than those in the Non-RAS inhibitor group in T1 category (Log-rank: p=0.003, Figure). In contrast, there was no statistical difference in the mortality between the RAS inhibitor group and Non-RAS inhibitor group in T2 and T3 categories (Log-rank: p=0.15, p=0.81, respectively, Figure). Multivariate Cox regression analysis in T1 category revealed that taking RAS inhibitor at discharge was independently associated with a lower mortality rate, even after the adjustment of diverse covariates (hazard ratio: 0.40, 95% confidence interval: 0.20–0.80). Conclusion In this observational study, the administration of RAS inhibitor was associated with an improved prognosis of HFpEF patients only in low serum chloride level at discharge. Therapeutic strategy focusing on the chloride level may be one of the promising options to find the light on a unintervenable prognosis of HFpEF. Funding Acknowledgement Type of funding sources: None.