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The appearance of new viruses and diseases has made the development of rapid and reliable diagnostic tests crucial. In light of it, we proposed a new method for assembling an electrochemical immunosensor, based on a one-step approach for selective layer formation. For this purpose, a mixture containing the immobilizing agent (polyxydroxybutyrate, PHB) and the recognition element (antibodies against SARS-CoV-2 nucleocapsid protein) was prepared and used to modify a screen-printed carbon electrode with electrodeposited graphene oxide, for the detection of SARS-CoV-2 nucleocapsid protein (N-protein). Under optimum conditions, N-protein was successfully detected in three different matrixes - saliva, serum, and nasal swab, with the lowest detectable values of 50 pg mL-1, 1.0 ng mL-1, and 50 pg mL-1, respectively. Selectivity was assessed against SARS-CoV-2 receptor-binding domain protein (RBD) and antibodies against yellow fever (YF), and no significant response was observed in presence of interferents, reinforcing the suitability of the proposed one-step approach for selective layer formation. The proposed biosensor was stable for up to 14 days, and the mixture was suitable for immunosensor preparation even after 60 days of preparation. The proposed assembly strategy reduces the cost, analysis time, and waste generation. This reduction is achieved through miniaturization, which results in the decreased use of reagents and sample volumes. Additionally, this approach enables healthcare diagnostics to be conducted in developing regions with limited resources. Therefore, the proposed one-step approach for selective layer formation is a suitable, simpler, and a reliable alternative for electrochemical immunosensing.

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The COVID-19 pandemic resulted in the collapse of healthcare systems and led to the development and application of several approaches of wastewater-based epidemiology to monitor infected populations. The main objective of this study was to carry out a SARS-CoV-2 wastewater based surveillance in Curitiba, Southern Brazil Sewage samples were collected weekly for 20 months at the entrance of five treatment plants representing the entire city and quantified by qPCR using the N1 marker. The viral loads were correlated with epidemiological data. The correlation by sampling points showed that the relationship between the viral loads and the number of reported cases was best described by a cross-correlation function, indicating a lag between 7 and 14 days amidst the variables, whereas the data for the entire city presented a higher correlation (0.84) with the number of positive tests at lag 0 (sampling day). The results also suggest that the Omicron VOC resulted in higher titers than the Delta VOC. Overall, our results showed that the approach used was robust as an early warning system, even with the use of different epidemiological indicators or changes in the virus variants in circulation. Therefore, it can contribute to public decision-makers and health interventions, especially in vulnerable and low-income regions with limited clinical testing capacity. Looking toward the future, this approach will contribute to a new look at environmental sanitation and should even induce an increase in sewage coverage rates in emerging countries.

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In the present work, we report an innovative approach for immunosensors construction. The experimental strategy is based on the anchoring of biological material at screen-printed carbon electrode (SPE) modified with electrodeposited Graphene Quantum Dots (GQD) and polyhydroxybutyric acid (PHB). It was used as functional substract basis for the recognition site receptor-binding domain (RBD) from coronavirus spike protein (SARS-CoV-2), for the detection of Anti-S antibodies (AbS). SEM images and EDS spectra suggest an interaction of the protein with GQD-PHB sites at the electrode surface. Differential pulse voltametric (DPV) measurements were performed before and after incubation, in presence of the target, shown a decrease in voltametric signal of an electrochemical probe ([Fe(CN)6]3/4-). Using the optimal experimental conditions, analytical curves were performed in PBS and human serum spiked with AbS showing a slight matrix effect and a relationship between voltametric signal and AbS concentration in the range of 100 ng mL-1 and 10 µg mL-1. The selectivity of the proposed sensor was tested against yellow fever antibodies (YF) and the selective layer on the electrode surface did not interact with these unspecific antibodies. Eight samples of blood serum were analyzed and 87.5% of these total investigated provided adequate results. In addition, the present approach showed better results against traditional EDC/NHS reaction with enhancements in time and the possibility to develop an immunosensor in a single drop, since the proteins can be anchored prior to the electrode modification step.

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Brazil was the epicenter of worldwide pandemics at the peak of its second wave. The genomic/proteomic perspective of the COVID-19 pandemic in Brazil could provide insights to understand the global pandemics behavior. In this study, we track SARS-CoV-2 molecular information in Brazil using real-time bioinformatics and data science strategies to provide a comparative and evolutive panorama of the lineages in the country. SWeeP vectors represented the Brazilian and worldwide genomic/proteomic data from Global Initiative on Sharing Avian Influenza Data (GISAID) between February 2020 and August 2021. Clusters were analyzed and compared with PANGO lineages. Hierarchical clustering provided phylogenetic and evolutionary analyses of the lineages, and we tracked the P.1 (Gamma) variant origin. The genomic diversity based on Chao's estimation allowed us to compare richness and coverage among Brazilian states and other representative countries. We found that epidemics in Brazil occurred in two moments with different genetic profiles. The P.1 lineages emerged in the second wave, which was more aggressive. We could not trace the origin of P.1 from the variants present in Brazil. Instead, we found evidence pointing to its external source and a possible recombinant event that may relate P.1 to a B.1.1.28 variant subset. We discussed the potential application of the pipeline for emerging variants detection and the PANGO terminology stability over time. The diversity analysis showed that the low coverage and unbalanced sequencing among states in Brazil could have allowed the silent entry and dissemination of P.1 and other dangerous variants. This study may help to understand the development and consequences of variants of concern (VOC) entry.

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BACKGROUND: We report a genomic surveillance of SARS-CoV-2 lineages circulating in Paraná, southern Brazil, from March 2020 to April 2021. Our analysis, based on 333 genomes, revealed that the first variants detected in the state of Paraná in March 2020 were the B.1.1.33 and B.1.1.28 variants. The variants B.1.1.28 and B.1.1.33 were predominant throughout 2020 until the introduction of the variant P.2 in August 2020 and a variant of concern (VOC), Gamma (P.1), in January 2021. The VOC Gamma, a ramification of the B.1.1.28 lineage first detected in Manaus (northern Brazil), has grown rapidly since December 2020 and was thought to be responsible for the deadly second wave of COVID-19 throughout Brazil. METHODS: The 333 genomic sequences of SARS-CoV-2 from March 2020 to April 2021 were generated as part of the genomic surveillance carried out by Fiocruz in Brazil Genomahcov Fiocruz. SARS-CoV-2 sequencing was performed using representative samples from all geographic areas of Paraná. Phylogenetic analyses were performed using the 333 genomes also included other SARS-CoV-2 genomes from the state of Paraná and other states in Brazil that were deposited in the GISAID. In addition, the time-scaled phylogenetic tree was constructed with up to 3 random sequences of the Gamma variant from each state in Brazil in each month of 2021. In this analysis we also added the sequences identified as the B.1.1.28 lineage of the Amazonas state and and the Gamma-like-II (P.1-like-II) lineage identified in different regions of Brazil. RESULTS: Phylogenetic analyses of the SARS-CoV-2 genomes that were previously classified as the VOC Gamma lineage by WHO/PANGO showed that some genomes from February to April 2021 branched in a monophyletic clade and that these samples grouped together with genomes recently described with the lineage Gamma-like-II. Additionally, a new mutation (E661D) in the spike (S) protein has been identified in nearly 10% of the genomes classified as the VOC Gamma from Paraná in March and April 2021.Finally, we analyzed the correlation between the lineage and the Gamma variant frequency, age group (patients younger or older than 60 years old) and the clinical data of 86 cases from the state of Paraná. CONCLUSIONS: Our results provided a reliable picture of the evolution of the SARS-CoV-2 pandemic in the state of Paraná characterized by the dominance of the Gamma strain, as well as a high frequencies of the Gamma-like-II lineage and the S:E661D mutation. Epidemiological and genomic surveillance efforts should be continued to unveil the biological relevance of the novel mutations detected in the VOC Gamma in Paraná.

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Breast cancer (BC) is the most frequently diagnosed female cancer and second leading cause of death. Despite the discovery of many antineoplastic drugs for BC, the current therapy is not totally efficient. In this study, we investigated the potential of repurposing the well-known diabetes type II drug liraglutide to modulate epigenetic modifications in BC cells lines in vitro and in vivo via Ehrlich mice tumors models. The in vitro results revealed a significant reduction on cell viability, migration, DNMT activity and displayed lower levels of global DNA methylation in BC cell lines after liraglutide treatment. The interaction between liraglutide and the DNMT enzymes resulted in a decrease profile of DNA methylation for the CDH1, ESR1 and ADAM33 gene promoter regions and, consequently, increased their gene and protein expression levels. To elucidate the possible interaction between liraglutide and the DNMT1 protein, we performed an in silico study that indicates liraglutide binding in the catalytic cleft via hydrogen bonds and salt bridges with the interdomain contacts and disturbs the overall enzyme conformation. The in vivo study was also able to reveal that liraglutide and the combined treatment of liraglutide and paclitaxel or methotrexate were effective in reducing tumor growth. Moreover, the modulation of CDH1 and ADAM33 mouse gene expression by DNA demethylation suggests a role for liraglutide in DNMT activity in vivo. Altogether, these results indicate that liraglutide may be further analysed as a new adjuvant treatment for BC.

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Two mixed-valence octadecavanadates, (NH4)2(Me4N)5[VIV12VV6O42I]·Me4NI·5H2O (V18I) and [{K6(OH2)12VIV11VV7O41(PO4)·4H2O}n] (V18P), were synthesized and characterized by single-crystal X-ray diffraction analysis and FTIR, Raman, 51V NMR, EPR and UV/Vis/NIR spectroscopies. The chemoprotective activity of V18I and V18P towards the alkylating agent diethyl sulfate was assessed in E. coli cultures. The complex V18I was nontoxic in concentrations up to 5.0 mmol L-1, while V18P presented moderate toxicity in the concentration range 0.10 - 10 mmol L-1. Conversely, a ca. 35% enhancement in culture growth as compared to cells treated only with diethyl sulfate was observed upon addition of V18I (0.10 to 2.5 mmol L-1), while the combination of diethyl sulfate with V18P increased the cytotoxicity presented by diethyl sulfate alone. 51V NMR and EPR speciation studies showed that V18I is stable in solution, while V18P suffers partial breakage to give low nuclearity oxidometalates of vanadium(V) and (IV). According to the results, the chemoprotective effect depends strongly on the direct reactivity of the polyoxidovanadates (POV) towards the alkylating agent. The reaction of diethyl sulfate with V18I apparently produces a new, rearranged POV instead of poorly-reactive breakage products, while V18P shows the formation and subsequent consumption of low-nuclearity species. The correlation of this chemistry with that of other mixed-valence polyoxidovanadates, [H6VIV2VV12O38PO4]5- (V14) and [VIV8VV7O36Cl]6- (V15), suggests a relationship between stability in solution and chemoprotective performance.

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Aeromonas are bacteria broadly spread in the environment, particularly in aquatic habitats and can induce human infections. Several virulence factors have been described associated with bacterial pathogenicity, such as the Type VI Secretion System (T6SS). This system translocates effector proteins into target cells through a bacteriophage-like contractile structure encoded by tss genes. Here, a total of 446 Aeromonas genome sequences were screened for T6SS and the proteins subjected to in silico analysis. The T6SS-encoding locus was detected in 243 genomes and its genes are encoded in a cluster containing 13 core and 5 accessory genes, in highly conserved synteny. The amino acid residues identity of T6SS proteins ranges from 78 to 98.8%. In most strains, a pair of tssD and tssI is located upstream the cluster (tssD-2, tssI-2) and another pair was detected distant from the cluster (tssD-1, tssI-1). Significant variability was seen in TssI (VgrG) C-terminal region, which was sorted in four groups based on its sequence length and protein domains. TssI containing ADP-ribosyltransferase domain are associated exclusively with TssI-1, while genes coding proteins carrying DUF4123 (a conserved domain of unknown function) were observed downstream tssI-1 or tssI-2 and escort of possible effector proteins. Genes coding proteins containing DUF1910 and DUF1911 domains were located only downstream tssI-2 and might represent a pair of toxin/immunity proteins. Nearly all strains display downstream tssI-3, that codes for a lysozyme family domain protein. These data reveal that Aeromonas T6SS cluster synteny is conserved and the low identity observed for some genes might be due to species heterogeneity or its niche/functionality.

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Immunological methods to detect SARS-CoV-2 seroconversion in humans are important to track COVID-19 cases and the humoral response to SARS-CoV-2 infections and immunization to future vaccines. The aim of this work was to develop a simple chromogenic magnetic bead-based immunoassay which allows rapid, inexpensive, and quantitative detection of human antibodies against SARS-CoV-2 in serum, plasma, or blood. Recombinant 6xHis-tagged SARS-CoV-2 Nucleocapsid protein was mobilized on the surface of Ni2+ magnetic beads and challenged with serum or blood samples obtained from controls or COVID-19 cases. The beads were washed, incubated with anti-human IgG-HPR conjugate, and immersed into a solution containing a chromogenic HPR substrate. Bead transfer and homogenization between solutions was aided by a simple low-cost device. The method was validated by two independent laboratories, and the performance to detect SARS-CoV-2 seroconversion in humans was in the same range as obtained using the gold standard immunoassays ELISA and Luminex, though requiring only a fraction of consumables, instrumentation, time to deliver results, and volume of sample. Furthermore, the results obtained with the method described can be visually interpreted without compromising accuracy as demonstrated by validation at a point-of-care unit. The magnetic bead immunoassay throughput can be customized on demand and is readily adapted to be used with any other 6xHis tagged protein or peptide as antigen to track other diseases.

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Azospirillum brasilense is a diazotrophic microorganism capable of associating with roots of important grasses and cereals, promoting plant growth and increasing crop yields. Nitrogen levels and the Ntr regulatory system control the nitrogen metabolism in A. brasilense. This system comprises the nitrogen regulatory proteins GlnD, which is capable of adding uridylyl groups to the PII proteins, GlnB (PII-1) and GlnZ (PII-2), under limiting nitrogen levels. Under such conditions, the histidine kinase NtrB (nitrogen regulatory protein B) cannot interact with GlnB and phosphorylate NtrC (nitrogen regulatory protein C). The phosphorylated form of NtrC acts as a transcriptional activator of genes involved in the metabolism of alternative nitrogen sources. Considering the key role of NtrC in nitrogen metabolism in A. brasilense, in this work we evaluated the proteomic and metabolomic profiles of the wild-type FP2 strain and its mutant ntrC grown under high and low nitrogen. Analysis of the integrated data identifies novel NtrC targets, including proteins involved in the response against oxidative stress (i.e., glutathione S-transferase and hydroperoxide resistance protein), underlining the importance of NtrC to bacterial survival under oxidative stress conditions.

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