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The current Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) pandemic is a public health emergency of international concern. Sensitive and precise diagnostic tools are urgently needed. In this study, we developed a SARS-CoV-2 spike (S1) protein enzyme-linked immunosorbent assay (ELISA) to detect SARS-CoV-2-specific antibodies. The SARS-CoV-2 S1 ELISA was found to be specific [97.8% (95% CI, 96.7% - 98.5%)], reproducible and precise (intra-assay coefficient of variability (CV) 5.3%, inter-assay CV 7.9%). A standard curve and the interpolation of arbitrary ELISA units per milliliter served to reduce the variability between different tests and operators. Cross-reactivity to other human coronaviruses was addressed by using sera positive for MERS-CoV- and hCoV HKU1-specific antibodies. Monitoring antibody development in various samples of twenty-three and single samples of twenty-nine coronavirus disease 2019 (COVID−19) patients revealed seroconversion and neutralizing antibodies against authentic SARS-CoV-2 in all cases. The comparison of the SARS-CoV-2 (S1) ELISA with a commercially available assay showed a better sensitivity for the in-house ELISA. The results demonstrate a high reproducibility, specificity and sensitivity of the newly developed ELISA, which is suitable for the detection of SARS-CoV-2 S1 protein-specific antibody responses., Highlights • A highly sensitive and specific SARS-CoV-2 S1 ELISA was developed. • A standard curve is included to reduce variability between assays and operators. • Excellent intra-assay coefficient of variability (CV) 5.3%, inter-assay CV 7.9%. • Good correlation to the virus neutralization test VNT100.

DNA analysis can yield information usable to identify individuals or to associate suspects, victims, and crime scene to one another but does not support a contextualization of trace material. Knowing the composition of biological material from a crime scene, e.g. whether a stain contains saliva or semen, is complementary to DNA-based identification and can be essential in reconstructing the course of events in criminal investigations. Research in the forensic applications of RNA analysis has surged considerably during the last two decades and provided recent advancements for forensic casework. Therefore, as a proof of concept, different commercially available DNA extraction kits were compared to assess their potential to simultaneously co-isolate messengerRNA and microRNA in DNA eluates of forensically relevant materials. The results showed that only one extraction kit elutes enough RNA molecules to perform messengerRNA body fluid identification whereas for microRNA-specific expression two of the tested kits yielded sufficient RNA concentrations.

Lassa mammarenavirus (LASV) is a rodent-borne arenavirus endemic to several West African countries. It is the causative agent of human Lassa fever, an acute viral hemorrhagic fever disease. To date, no therapeutics or vaccines against LASV have obtained regulatory approval. Polyclonal neutralizing antibodies derived from hyperimmunized animals may offer a useful strategy for prophylactic and therapeutic intervention to combat human LASV infections. The LASV envelope surface glycoprotein complex (GP) is the major target for neutralizing antibodies, and it is the main viral antigen used for the design of an LASV vaccine. Here, we assessed the immunogenic potential of mammalian cell-derived virus-like particles (VLPs) expressing GP from the prototypic LASV strain Josiah in a native-like conformation as the sole viral antigen. We demonstrate that an adjuvanted prime-boost immunization regimen with GP-derived VLPs elicited neutralizing antibody responses in rabbits, suggesting that effective antigenic epitopes of GP were displayed. Notably, these antibodies exhibited broad reactivity across five genetic lineages of LASV. VLP-based immunization strategies may represent a powerful approach for generating polyclonal sera containing cross-reactive neutralizing antibodies against LASV.

RESUMO: A presente pesquisa objetivou avaliar a qualidade fisiológica de sementes de pimenta (Capsicum chinense Jacq.) var. Bode Vermelha obtidas de frutos em diferentes estádios de maturação. Os frutos de C. chinense foram separados em cinco estádios de maturação, baseando-se na coloração do pericarpo (frutos verdes - estádio 1, frutos alaranjados - estádio 2, frutos vermelhos-claros - estádio 3, frutos vermelhos - estádio 4 e frutos vermelho-carmim - estádio 5). Para a avaliação da maturação e da qualidade fisiológica das sementes foram determinadas as características biométricas dos frutos e sementes, massa de mil sementes, teor de água das sementes, condutividade elétrica, teste padrão de germinação, tempo médio de germinação, porcentagem de plântulas normais, velocidade de formação de plântulas, além dos testes de envelhecimento acelerado, e comprimento de plântulas. Os caracteres comprimento, diâmetro, massa e número de sementes dos frutos e comprimento, largura, espessura e massa das sementes foram analisados por meio de parâmetros estimados utilizando-se estatística descritiva. Para as demais variáveis foi utilizado delineamento experimental inteiramente casualizado. Os resultados foram submetidos à análise de variância e a comparação de médias pelo teste Scott-Knott, ao nível de 5% de probabilidade. O estádio de maturação influencia na qualidade fisiológica das sementes de Capsicum chinense Jacq. var. Bode Vermelho, sendo as sementes oriundas de frutos de coloração vermelho (estádio 4) a vermelho-carmim (estádio 5) as que apresentaram os melhores desempenhos e, portanto, estão fisiologicamente maduras, sendo os mais indicados para obtenção das sementes. ABSTRACT: This research aimed to evaluate the physiological quality of pepper (Capsicum chinense Jacq.) var. Bode Vermelha seeds, obtained from fruits at different ripening stages. The fruits of C. Chinese were separated according to the ripening stages, based on pericarp color (green fruits - stage 1, orange fruits - stage 2, light red fruits - stage 3, red fruits - stage 4, and carmine-red fruits - stage 5). To evaluate the ripeness and the physiological quality of the seeds were determined the biometric characteristics of the fruits and seeds, mass of thousand seeds, seeds water content, electrical conductivity, standard germinations test, speed of germination index, percentage of normal seedlings, speed of seedlings formation, accelerated aging, and seedling length. The characters length, diameter, weight, and number of seeds of the fruits and length, width, thickness, and mass of the seeds were analyzed by the estimated parameters using descriptive statistics. For the other variables was used the completely randomized design. The results were submitted to variance analysis and comparison of means by the Scott-Knott test at 5% probability. The ripeness stage affects the physiological quality of C. chinense Jacq. var. Bode Red seeds, being the seeds from red (stage 4) to carmine-red (stage 5) fruits those with the best performance thus, they are physiologically ripe, being the most indicated to seeds obtaining.

We have developed a microfluidic chip-based qualitative assay for sensitive (10 RNA copies) detection of multiple transcripts in single cells. We determined the expression patterns of 17 developmentally important genes and isoforms in individual mouse preimplantation embryos from superovulated matings and blastomeres. The ubiquitously expressed histone variant H3f3a and the transcription factor Pou5f1 generated mRNA-derived products in all analyzed (1-cell, 2-cell, 4-cell, and morula stage) embryos and in all analyzed blastomeres from 16-cell embryos, indicating a uniform reactivation of pluripotency gene expression during mouse preimplantation development. In contrast, mRNA expression of different methyltransferases for DNA methylation, methylcytosine-binding proteins for chromatin modification, and base excision repair enzymes, which may provide a mechanism for active demethylation, varied considerably between individual cells from the same embryo and even more dramatically between cells from different embryos. We conclude that at a given point in time the transcriptome encoding the reprogramming machinery and, by extrapolation, genome reprogramming differs between blastomeres. By studying the cell-to-cell variability in gene expression, we can distinguish the following two classes: mouse 16-cell embryos in which most cells express the reprogramming machinery and embryos in which most cells do not contain detectable mRNA levels of DNA and chromatin modification genes. Immunolocalization of DNMT3A, MBD3, APEX1, and LIG3 in most or all nuclei of 40-60-cell embryos is a good indicator of functional activity of genes that are activated by the 16-cell stage.

The nature of mitochondrial DNA heteroplasmy is still unclear. It could either be caused by two mitochondrial DNA (mtDNA) haplotypes coexisting within a single cell or by an admixture of homoplasmic cells, each of which contains only one type of mtDNA molecule. To address this question, single lymphocytes were separated by flow cytometry assisted cell sorting and analyzed by cycle sequencing or minisequencing. To attain the required PCR sensitivity, the reactions were carried out on the surface of chemically structured glass slides in a reaction volume of 1-2 microl. In this study, blood samples from two healthy donors showing mitochondrial point heteroplasmy in direct sequencing (195Y and 234R, respectively) were analyzed. Nearly 96% of single lymphocytes tested were found to be in a homoplasmic state, but heteroplasmic cells were also detected. These results suggest that mitochondrial point heteroplasmy in blood may well be mainly due to the mixture of homoplasmic cells.

Phasins play an important role in the formation of poly(3-hydroxybutyrate) [poly(3HB)] granules and affect their size. Recently, three homologues of the phasin protein PhaP1 were identified inRalstonia eutrophastrain H16. The functions of PhaP2, PhaP3 and PhaP4 were examined by analysis ofR. eutrophaH16 deletion strains (ΔphaP1, ΔphaP2, ΔphaP3, ΔphaP4, ΔphaP12, ΔphaP123and ΔphaP1234). When cells were grown under conditions permissive for poly(3HB) accumulation, the wild-type strain and all single-phasin negative mutants (ΔphaP2, ΔphaP3and ΔphaP4), with the exception of ΔphaP1, showed similar growth and poly(3HB) accumulation behaviour, and also the size and number of the granules were identical. The single ΔphaP1mutant and the ΔphaP12, ΔphaP123and ΔphaP1234mutants showed an almost identical growth behaviour; however, they accumulated poly(3HB) at a significantly lower level than wild-type and the single ΔphaP2, ΔphaP3or ΔphaP4mutants. Gel-mobility-shift assays and DNaseI footprinting experiments demonstrated the capability of the transcriptional repressor PhaR to bind to a DNA region +36 to +46 bp downstream of thephaP3start codon. The protected sequence exhibited high similarity to the binding sites of PhaR upstream ofphaP1, which were identified recently. In contrast, PhaR did not bind to the upstream or intergenic regions ofphaP2andphaP4, thus indicating that the expression of these two phasins is regulated in a different way. Our current model for the regulation of phasins inR. eutrophastrain H16 was extended and confirmed.

Acute lung injury (ALI) still poses a major challenge in critical care medicine. Neutrophils, platelets, and chemokines are all considered key components in the development of ALI. The Duffy antigen receptor for chemokines (DARC ) is thought to be involved in scavenging, transendothelial transport, and presentation of neutrophil-specific chemokines. DARC is expressed on endothelial cells and erythrocytes but not on leukocytes. Here, we show that DARC is crucial for chemokine-mediated leukocyte recruitment in vivo. However, we also demonstrate that changes in chemokine and chemokine receptor homeostasis, associated with Darc gene deficiency, exert strong anti-inflammatory effects. Neutrophils from Darc gene-deficient ( Darc−/ −) mice display a more prolonged downregulation of CXCR2 during severe inflammation than neutrophils from wild-type mice. In a CXCR2-dependent model of acid-induced ALI, Darc gene deficiency prevents ALI. Darc−/ −mice demonstrate fully preserved oxygenation, only a small increase in vascular permeability, and a complete lack of pulmonary neutrophil recruitment. Further analysis reveals that only neutrophils but neither endothelial cells nor erythrocytes from Darc−/ −mice confer protection from ALI. The protection appears to be due to abolished pulmonary recruitment of neutrophils from Darc−/ −mice. The generation of neutrophil-platelet aggregates, a key mechanism in both pulmonary neutrophil recruitment and thrombus formation, is also affected by altered CXCR2 homeostasis in Darc−/ −mice. CXCR2 blockade enhances the formation of platelet-neutrophil aggregates and thereby corrects a formerly unknown bleeding defect in Darc−/ −mice. In summary, our study suggests that chemokine/chemokine receptor homeostasis plays a previously unrecognized and crucial role in severe ALI.

Review on PSGL-1 effects on signaling in myeloid leukocytes activates following selectin engagement. Cell-cell interactions mediating leukocyte recruitment and inflammation are crucial for host defense. Leukocyte recruitment into injured tissue proceeds in a multistep process. The first contact of leukocytes with endothelial cells (“capturing” or “tethering”) is mediated by selectins and their counter-receptor P-selectin glyco-protein ligand (PSGL)-1. During capture and rolling, leukocytes collect different inflammatory signals, which can activate various pathways. Integration of these signals leads to leukocyte activation, integrin-mediated arrest, cytoskeleton rearrangement, polarization, and transmigration. PSGL-1 on leukocytes also binds to activated platelets, where P-selectin is expressed at locally high site densities following α-granule fusion with the plasma membrane. Here, we review the signaling functions of PSGL-1 and speculate how the different known signaling events might relate to different phases of leukocyte recruitment.

Analysis of the genome sequence of the polyhydroxyalkanoate- (PHA) accumulating bacteriumRalstonia eutrophastrain H16 revealed three homologues (PhaP2, PhaP3 and PhaP4) of the phasin protein PhaP1. PhaP1 is known to constitute the major component of the layer at the surface of poly(3-hydroxybutyrate), poly(3HB), granules. PhaP2, PhaP3 and PhaP4 exhibited 42, 49 and 45 % identity or 61, 62 and 63 % similarity to PhaP1, respectively. The calculated molecular masses of PhaP1, PhaP2, PhaP3 and PhaP4 were 20·0, 20·2, 19·6 and 20·2 kDa, respectively. RT-PCR analysis showed thatphaP2,phaP3andphaP4were transcribed under conditions permissive for accumulation of poly(3HB). 2D PAGE of the poly(3HB) granule proteome and analysis of the detected proteins by MALDI-TOF clearly demonstrated that PhaP1, PhaP3 and PhaP4 are bound to the poly(3HB) granules in the cells. PhaP3 was expressed at a significantly higher level in PhaP1-negative mutants. Occurrence of an unknown protein with an N-terminal amino-acid sequence identical to that of PhaP2 in crude cellular extracts ofR. eutrophahad previously been shown by others. Although PhaP2 could not be localizedin vivoon poly(3HB) granules,in vitroexperiments clearly demonstrated binding of PhaP2 to these granules. Further analysis of complete or partial genomes of other poly(3HB)-accumulating bacteria revealed the existence of multiple phasin homologues inRalstonia solanacearum,Burkholderia fungorumandAzotobacter vinelandii. These new and unexpected findings should affect our current models of PHA-granule structure and may also have a considerable impact on the establishment of heterologous production systems for PHAs.