RESUMEN
This study aimed to describe differences in prevalence and the long-term presence of nucleocapsid antibodies (N-antibodies) elicited by SARS-CoV-2 infection in a Swedish blood donor population not subjected to lockdown. We tested 20,651 blood donor samples for nucleocapsid antibodies from the beginning of March 2020 and 27 months onwards using the Roche Elecsys Anti-SARS-CoV-2 assay. The proportion of positive SARS-CoV-2 antibody samples was determined each week. After the exclusions of one-time donors and subjects with incomplete data, 19,726 samples from 4003 donors remained. Differences in antibody prevalences stratified for age, sex, and blood groups (ABO and RhD) were determined, as well as antibody loss and recovery. Lower antibody prevalence was seen for older donors, blood group AB, and RhD-negative subjects. A significant decrease in antibody titer between the first and the second antibody-positive donation was seen for the whole study group, females, older subjects, blood group O, AB, and RhD-positive subjects. The titer waned below the detection limit in 60 (3.0%) of 1983 N-antibody-positive donors, and for 18 of these donors, a second episode with antibodies was detected. We showed that N-antibodies persist for months or years and that surprisingly few antibody-positive donors lost their antibodies. We also conclude that antibody prevalence in a Swedish population never subject to lockdown did not apparently differ from populations that were subject to stricter regulations.
Asunto(s)
Anticuerpos Antivirales , Donantes de Sangre , COVID-19 , SARS-CoV-2 , Humanos , Donantes de Sangre/estadística & datos numéricos , Femenino , Suecia/epidemiología , Masculino , SARS-CoV-2/inmunología , Adulto , Anticuerpos Antivirales/sangre , COVID-19/inmunología , COVID-19/sangre , COVID-19/epidemiología , Persona de Mediana Edad , Factores de Edad , Factores Sexuales , Anciano , Estudios de Cohortes , Adulto Joven , Adolescente , Antígenos de Grupos Sanguíneos/inmunologíaRESUMEN
BACKGROUND: The recently launched high-throughput assays for detecting antibodies against SARS-CoV-2 has contributed to the managing strategies for the COVID-19 pandemic. This study aimed to investigate the performance of three high-throughput assays and one rapid lateral flow test relative to regulatory authorities' recommended criteria. METHODS: A total of 315 samples, including 150 pre-pandemic samples, 152 samples from SARS-CoV-2 RT-PCR positive individuals and 13 potentially cross-reactive samples were analysed with SARS-CoV-2 IgG (Abbott, Abbott Park, IL), Elecsys Anti-SARS-CoV-2 (Roche, Solna, Sweden), LIAISON SARS-CoV-2 S1/S2 IgG (DiaSorin, Saluggia, Italy) and 2019-nCOV IgG/IgM Rapid Test (Dynamiker Biotechnology Co., Tianjin, China). RESULTS: All assays performed with a high level of specificity ranging from 96.7% to 99.3%. Sensitivity differed more between the assays, Roche exhibiting the highest sensitivity of 98.7%. The corresponding figures for Abbott, DiaSorin and Dynamiker Biotechnology were 80.9%, 89.0% and 72.4%, respectively. CONCLUSIONS: The results of the evaluated SARS-CoV-2 assays vary considerably, as well as their ability to fulfil the performance criteria proposed by regulatory authorities. Introduction into clinical use in low-prevalent settings, should, therefore, be made with caution.
Asunto(s)
COVID-19 , SARS-CoV-2 , Anticuerpos Antivirales , Humanos , Inmunoensayo , Pandemias , Sensibilidad y EspecificidadRESUMEN
The Na(+)-coupled, high-affinity Pho89 plasma membrane phosphate transporter in Saccharomyces cerevisiae has so far been difficult to study because of its low activity and special properties. In this study, we have used a pho84Deltapho87Deltapho90Deltapho91Delta quadruple deletion strain of S. cerevisiae devoid of all transporter genes specific for inorganic phosphate, except for PHO89, to functionally characterize Pho89 under conditions where its expression is hyperstimulated. Under these conditions, the Pho89 protein is strongly upregulated and is the sole high-capacity phosphate transporter sustaining cellular acquisition of inorganic phosphate. Even if Pho89 is synthesized in cells grown at pH 4.5-8.0, the transporter is functionally active under alkaline conditions only, with a K(m) value reflecting high-affinity properties of the transporter and with a transport rate about 100-fold higher than that of the protein in a wild-type strain. Even under these hyperexpressive conditions, Pho89 is unable to sense and signal extracellular phosphate levels. In cells grown at pH 8.0, Pho89-mediated phosphate uptake at alkaline pH is cation-dependent with a strong activation by Na(+) ions and sensitivity to carbonyl cyanide m-chlorophenylhydrazone. The contribution of H(+)- and Na(+)-coupled phosphate transport systems in wild-type cells grown at different pH values was quantified. The contribution of the Na(+)-coupled transport system to the total cellular phosphate uptake activity increases progressively with increasing pH.
Asunto(s)
Dosificación de Gen , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Eliminación de Gen , Concentración de Iones de Hidrógeno , Cinética , Proteínas de Transporte de Fosfato/genética , Fosfatos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Sodio/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/genética , Desacopladores/farmacologíaRESUMEN
In Saccharomyces cerevisiae, the Pho84 high-affinity transport system is the major phosphate transporter activated when the cells experience a limitation in external phosphate. In this study, we have compared the phosphate-responsive mechanism of cells expressing PHO84 with a Deltapho84 strain by use of a phosphate analogue, methylphosphonate, which was judged to be suitable for assessment of phosphate homeostasis in the cells. Intracellular levels of the analogue, which in several respects mimicks phosphate, were monitored by (31)P NMR spectroscopy. Results show that methylphosphonate is a nonhydrolyzable and nonutilizable analogue that cannot be used to replenish phosphate or polyphosphate in yeast cells grown under conditions of phosphate limitation. However, the presence of methylphosphonate under such conditions represses the Pho5 acidic phosphatase activity of PHO84 cells, a finding that implies a direct role of the analogue in the regulation of phosphate-responsive genes and/or proteins. Likewise, accumulation of the Pho84 protein at the plasma membrane of the same cells is inhibited by methylphosphonate, although the derepressive expression of the PHO84 gene is unperturbed. Thus, a post-transcriptional regulation is suggested. Supportive of this suggestion is the fact that addition of methylphosphonate to cells with abundant and active Pho84 at the plasma membrane causes enhanced internalization of the Pho84 protein. Altogether, these observations suggest that the Pho84 transporter is regulated not only at the transcriptional level but also by a direct molecule-sensing mechanism at the protein level.
Asunto(s)
Compuestos Organofosforados/química , Fosfatos/metabolismo , Simportadores de Protón-Fosfato/biosíntesis , Simportadores de Protón-Fosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/metabolismo , Fosfatasa Ácida/antagonistas & inhibidores , Fosfatasa Ácida/metabolismo , Transporte Biológico/genética , Regulación Fúngica de la Expresión Génica , Genes myc , Concentración de Iones de Hidrógeno , Compuestos Organofosforados/metabolismo , Simportadores de Protón-Fosfato/antagonistas & inhibidores , Simportadores de Protón-Fosfato/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/genética , Supresión GenéticaRESUMEN
Membrane transport systems active in cellular inorganic phosphate (P(i)) acquisition play a key role in maintaining cellular P(i) homeostasis, independent of whether the cell is a unicellular microorganism or is contained in the tissue of a higher eukaryotic organism. Since unicellular eukaryotes such as yeast interact directly with the nutritious environment, regulation of P(i) transport is maintained solely by transduction of nutrient signals across the plasma membrane. The individual yeast cell thus recognizes nutrients that can act as both signals and sustenance. The present review provides an overview of P(i) acquisition via the plasma membrane P(i) transporters of Saccharomyces cerevisiae and the regulation of internal P(i) stores under the prevailing P(i) status.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Fosfatos/metabolismo , Saccharomyces cerevisiae/metabolismo , Algoritmos , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Datos de Secuencia Molecular , Fosfatos/química , Polifosfatos/química , Estructura Secundaria de Proteína , Simportadores de Protón-Fosfato/química , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
A widely accepted mechanism for selective degradation of plasma membrane proteins is via ubiquitination and/or phosphorylation events. Such a regulated degradation has previously been suggested to rely on the presence of a specific SINNDAKSS sequence within the protein. Modification of a partly conserved SINNDAKSS-like sequence in the C-terminal tail of the Pho84 phosphate transporter, in combination with C-terminal fusion of green fluorescent protein or a MYC epitope, were used to evaluate the presence of this sequence and its role in the regulated degradation. The functional Pho84 mutants in which this SINNDAKSS-like sequence was altered or truncated were subjected to degradation like that of the wild type, suggesting that degradation of the Pho84 protein is regulated by factors other than properties of this sequence.