Re-emergence of Gamma-like-II and emergence of Gamma-S:E661D SARS-CoV-2 lineages in the south of Brazil after the 2021 outbreak.

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.

Influence of donor age on the differentiation and division capacity of human adipose-derived stem cells.

BACKGROUND: Human adipose-derived stromal/stem cells (hASCs) are one of the most useful types of mesenchymal stromal/stem cells, which are adult multipotent cells with great therapeutic potential for the treatment of several diseases. However, for successful clinical application, it is critical that high-quality cells can be obtained. Diverse factors seem to be able to influence cell quality and performance, especially factors related to donors' intrinsic characteristics, such as age. Nevertheless, there is no consensus regarding this characteristic, and there is conflicting information in the literature. AIM: To investigate the growth kinetics and differentiation potential of adipose-derived stem cells isolated from the lipoaspirates of elderly and young donors. METHODS: hASCs were harvested from liposuctioned adipose tissue obtained from female donors (aged 20-70 years). Cells were distributed into two groups according to age range: old hASCs (oASCs, ≥ 55 years, n = 9) and young hASCs (yASCs, ≤ 35 years, n = 9). For each group, immunophenotypic characterization was performed by flow cytometry. Population doubling time was assessed over seven days. For adipogenic potential evaluation, lipid deposits were assessed after 7 d, 14 d and 21 d of adipogenic induction. Osteogenic potential was verified by analyzing cell mineralization after 14 d, 21 d and 28 d of osteogenic induction. mRNA expression of PPARγ2, CEBPA and Runx2 were detected by quantitative reverse transcription polymerase chain reaction. RESULTS: hASCs were successfully obtained, cultured, and grouped according to their age: yASCs (26.33 ± 4.66 years old) and oASCs (64.78 ± 4.58 years old). After maintenance of the cells in culture, there were no differences in morphology between cells from the young and old donors. Additionally, both groups showed classical immunophenotypic characteristics of mesenchymal stem/stromal cells. The average doubling time indicated that yASCs (4.09 ± 0.94 d) did not significantly differ from oASCs (4.19 ± 1.29 d). Concerning differentiation potential, after adipogenic and osteogenic induction, yASCs and oASCs were able to differentiate to greater levels than the noninduced control cells. However, no differences were found in the differentiation efficiency of yASCs and oASCs in adipogenesis or osteogenesis. Additionally, the mRNA expression of PPARγ2, CEBPA and Runx2 were similar in yASCs and oASCs. CONCLUSION: Our findings suggest that age does not seem to significantly affect the cell division or adipogenic or osteogenic differentiation ability of adipose-derived stem cells isolated from lipoaspirates.

TcNST2 encodes a Golgi-localized UDP-galactose transporter in Trypanosoma cruzi.

Survival and infectivity of trypanosomatids rely on cell-surface and secreted glycoconjugates, many of which contain a variable number of galactose residues. Incorporation of galactose to proteins and lipids occurs along the secretory pathway from UDP-galactose (UDP-Gal). Before being used in glycosylation reactions, however, this activated sugar donor must first be transported across the endoplasmic reticulum and Golgi membranes by a specific nucleotide sugar transporter (NST). In this study, we identified an UDP-Gal transporter (named TcNST2 and encoded by the TcCLB.504085.60 gene) from Trypanosoma cruzi, the etiological agent of Chagas disease. TcNST2 was identified by heterologous expression of selected putative nucleotide sugar transporters in a mutant Chinese Hamster Ovary cell line. TcNST2 mRNA levels were detected in all T. cruzi life-cycle forms, with an increase in expression in axenic amastigotes. Confocal microscope analysis indicated that the transporter is specifically localized to the Golgi apparatus. A three-dimensional model of TcNST2 suggested an overall structural conservation as compared with members of the metabolite transporter superfamily and also suggested specific features that could be related to its activity. The identification of this transporter is an important step toward a better understanding of glycoconjugate biosynthesis and the role NSTs play in this process in trypanosomatids.

Fluorescence-based assay for accurate measurement of transcriptional activity in trypanosomatid parasites.

Trypanosomatid parasites are causative agents of neglected human diseases. Their lineage diverged early from the common eukaryotic ancestor, and they evolved singular mechanisms of gene expression that are crucial for their survival. Studies on unusual and essential molecular pathways lead to new drug targets. In this respect, assays to analyze transcriptional activity will provide useful information to identify essential and specific factors. However, the current methods are laborious and do not provide global and accurate measures. For this purpose, a previously reported radiolabeling in vitro nascent mRNA methodology was used to establish an alternative fluorescent-based assay that is able to precisely quantify nascent mRNA using both flow cytometry and a high-content image system. The method allowed accurate and global measurements in Trypanosoma brucei, a representative species of trypanosomatid parasites. We obtained data demonstrating that approximately 70% of parasites from a population under normal growth conditions displayed mRNA transcriptional activity, whilst the treatment with α-amanitin (75 µg/ml) inhibited the polymerase II activity. The adaptation of the method also allowed the analyses of the transcriptional activity during the cell cycle. Therefore, the methodology described herein contributes to obtaining precise measurements of transcriptional rates using multiparametric analysis. This alternative method can facilitate investigations of genetic and biochemical processes in trypanosome parasites and consequently provide additional information related to new treatment or prophylaxis strategies involving these important human parasites.

Polysome profiling shows the identity of human adipose-derived stromal/stem cells in detail and clearly distinguishes them from dermal fibroblasts.

Although fibroblasts and multipotent stromal/stem cells, including adipose-derived stromal cells (ADSCs), have been extensively studied, they cannot be clearly distinguished from each other. We, therefore, investigated the cellular and molecular characteristics of ADSCs and fibroblasts. ADSCs and fibroblasts share several morphological similarities and surface markers, but were clearly found to be different types of cells. Contrary to previous reports, fibroblasts were not able to differentiate into adipocytes, osteoblasts, or chondrocytes. Polysome-bound mRNA profiling revealed that ∼ 1,547 genes were differentially expressed (DE) in the two cell types; the genes were related to cell adhesion, the extracellular matrix, differentiation, and proliferation. These findings were confirmed by functional analyses showing that ADSCs had a greater adhesion capacity than fibroblasts; the proliferation rate of fibroblasts was also higher than that of ADSCs. Importantly, 185 DE genes were integral to the plasma membrane and, thus, candidate markers for ADSC isolation and manipulation. We also observed that an established marker of fibroblasts and ADSCs, CD105, was overexpressed in ADSCs at both mRNA and protein levels. CD105 expression seemed to be related to differentiation capacity, at least for adipogenesis. This study shows that ADSCs and fibroblasts are distinct cell types. These findings should be taken into account when using these two cell types in basic and therapeutic studies.

Transplantation of SNAP-treated adipose tissue-derived stem cells improves cardiac function and induces neovascularization after myocardium infarct in rats.

Stem cell therapy has been considered a promise for damaged myocardial tissue. We have previously shown that S-nitroso-N-acetyl-D,L-penicillamine (SNAP) increases the expression of several muscular markers and VEGF in mesenchymal stem cells, indicating that transplantation of SNAP-treated cells could provide better functional outcomes. Here, we transplanted SNAP-treated adipose tissue-derived stem cells (ADSCs) in rat infarcted myocardium. After 30days, we observed a significant improvement of the ejection fraction in rats that received SNAP-treated ADSCs, compared with those that received untreated cells (p=0.008). Immunohistochemical reactions showed an increased expression of troponin T-C and von Willebrand factor, and organized vascular units in the infarcted area of tissue transplanted with treated ADSCs. SNAP exposure induced intracellular S-nitrosation, a decreased GSH/GSSG ratio, but did not increase cGMP levels. Collectively, these results indicate that SNAP alters the redox environment of ADSCs, possibly associated with a pre-differentiation state, which may improve cardiac function after transplantation.

Are purified or expanded cord blood-derived CD133+ cells better at improving cardiac function?

Endothelial progenitor cells (EPCs), which express the CD133 marker, can differentiate into mature endothelial cells (ECs) and create new blood vessels. Normal angiogenesis is unable to repair the injured tissues that result from myocardial infarction (MI). Patients who have high cardiovascular risks have fewer EPCs and their EPCs exhibit greater in vitro senescence. Human umbilical cord blood (HUCB)-derived EPCs could be an alternative to rescue impaired stem cell function in the sick and elderly. The aim of this study was to purify HUCB-derived CD133(+) cells, expand them in vitro and evaluate the efficacy of the purified and expanded cells in treating MI in rats. CD133(+) cells were selected for using CD133-coupled magnetic microbeads. Purified cells stained positive for EPC markers. The cells were expanded and differentiated in media supplemented with fetal calf serum and basic fibroblast growth factor, insulin-like growth factor-I and vascular endothelial growth factor (VEGF). Differentiation was confirmed by lack of staining for EPC markers. These expanded cells exhibited increased expression of mature EC markers and formed tubule-like structures in vitro. Only the expanded cells expressed VEGF mRNA. Cells were expanded up to 70-fold during 60 days of culture, and they retained their functional activity. Finally, we evaluated the therapeutic potential of purified and expanded CD133(+) cells in treating MI by intramyocardially injecting them into a rat model of MI. Rats were divided into three groups: A (purified CD133(+) cells-injected); B (expanded CD133(+) cells-injected) and C (saline buffer-injected). We observed a significant improvement in left ventricular ejection fraction for groups A and B. In summary, CD133(+) cells can be purified from HUCB, expanded in vitro without loosing their biological activity, and both purified and expanded cells show promising results for use in cellular cardiomyoplasty. However, further pre-clinical testing should be performed to determine whether expanded CD133(+) cells have any clinical advantages over purified CD133(+) cells.

Expression of cardiac function genes in adult stem cells is increased by treatment with nitric oxide agents.

Mesenchymal stem cells (MSCs) have received special attention for cardiomyoplasty because several studies have shown that they differentiate into cardiomyocytes both in vitro and in vivo. Nitric oxide (NO) is a free radical signaling molecule that regulates several differentiation processes including cardiomyogenesis. Here, we report an investigation of the effects of two NO agents (SNAP and DEA/NO), able to activate both cGMP-dependent and -independent pathways, on the cardiomyogenic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived stem cells (ADSCs). The cells were isolated, cultured and treated with NO agents. Cardiac- and muscle-specific gene expression was analyzed by indirect immunofluorescence, flow cytometry, RT-PCR and real-time PCR. We found that untreated (control) ADSCs and BM-MSCs expressed some muscle markers and NO-derived intermediates induce an increased expression of some cardiac function genes in BM-MSCs and ADSCs. Moreover, NO agents considerably increased the pro-angiogenic potential mostly of BM-MSCs as determined by VEGF mRNA levels.