Scaling up a chemically-defined aggregate-based suspension culture system for neural commitment of human pluripotent stem cells

The demand of high cell numbers for applications in cellular therapies and drug screening requires the development of scalable platforms capable to generating highly pure populations of tissue-specific cells from human pluripotent stem cells. This work describes the scaling-up of an aggregate-based culture system for neural induction of human induced pluripotent stem cells (hiPSCs) under chemically-defined conditions. Since initial cell density and aggregate size have an important impact in the expansion and commitment of these cells into a particular lineage, a combination of non-enzymatic dissociation and rotary agitation was successfully used to produce homogeneous populations of hiPSC aggregates with an optimal (140 µm) and narrow distribution of diameters (coefficient of variation of 21.6%). Scalable neural commitment of hiPSCs as 3D aggregates was then performed in 50 mL spinner flasks, and process optimization using a factorial design approach was developed involving parameters such as agitation rate and seeding density. We were able to produce neural progenitor cell cultures, that at the end of a 6-day neural induction process contained less than 3% of Oct4-positive cells and that, after replating, retained more than 60% of Pax6-positive neural cells. Furthermore, after scalable differentiation, hiPSC-derived neural progenitors still retained their multipotent potential, being able to give rise to neuronal and glial cells. The results presented in this work should set the stage for the future generation of a clinically relevant number of human neural progenitors for transplantation and other biomedical applications using totally controlled, automated and reproducible large-scale bioreactor culture systems

The transcription factor Ndt80 is a repressor of candida parapsilosis virulence attributes

Candida parapsilosis is an emergent opportunistic yeast among hospital settings that affects mainly neonates and immunocompromised patients. Its most remarkable virulence traits are the ability to adhere to prosthetic materials, as well as the formation of biofilm on abiotic surfaces. The Ndt80 transcription factor was identified as one of the regulators of biofilm formation by C. parapsilosis; however, its function in this process was not yet clarified. By knocking out NDT80 (CPAR2-213640) gene, or even just one single copy of the gene, we observed substantial alterations of virulence attributes, including morphogenetic changes, adhesion and biofilm growth profiles. Both ndt80Δ and ndt80ΔΔ mutants changed colony and cell morphologies from smooth, yeast-shaped to crepe and pseudohyphal elongated forms, exhibiting promoted adherence to polystyrene microspheres and notably, forming a higher amount of biofilm compared to wild-type strain. Interestingly, we identified transcription factors Ume6, Cph2, Cwh41, Ace2, Bcr1, protein kinase Mkc1 and adhesin Als7 to be under Ndt80 negative regulation, partially explaining the phenotypes displayed by the ndt80ΔΔ mutant. Furthermore, ndt80ΔΔ pseudohyphae adhered more rapidly and were more resistant to murine macrophage attack, becoming deleterious to such cells after phagocytosis. Unexpectedly, our findings provide the first evidence for a direct role of Ndt80 as a repressor of C. parapsilosis virulence attributes. This finding shows that C. parapsilosis Ndt80 functionally diverges from its homolog in the close related fungal pathogen C. albicans.info:eu-repo/semantics/publishedVersio

Estudo comparativo entre o tecido ósseo criopreservado e o conservado em glicerol a 98%

OBJECTIVE: To compare the bone graft cryopreservation method (at -80ºC) with a preservation method using a 98% glycerol solution at room temperature (10ºC-35ºC), by testing the antibacterial and fungal effects of 98% glycerol and comparatively analyzing the observed histological changes resulting from the use of both methods. METHOD: This study was of 30 samples of trabecular bone tissue from 10 patients undergoing total hip arthroplasty. Each femoral head provided 3 samples that were randomized into 3 groups, namely, the control group, the cryopreserved group, and the group preserved in a 98% glycerol at room temperature for 1 year. The samples were submitted to histomorphologic, cell feasibility, and microbiologic analyses. The results were statistically analyzed using the McNemar test, with a statistical significance index of 0.05. RESULTS: Values obtained using the McNemar test to compare probability distributions of histomorphologic variables (mature or lamellar bone, immature bone, and necrosis) and cell feasibility (osteoblasts and osteoclasts) indicated that there is no difference between the distributions of variables under the 3 experimental conditions. Microbiological analysis of the 98% glycerol solution and bone fragments from samples stored for 1 year at room temperature did not show bacterial or fungal growth. The histological and microbiological investigation were performed at 2 different time points: immediately after the sample processing and after 1 year. CONCLUSION: The method used to preserve bone grafts kept in 98% glycerol at room temperature (10ºC-35ºC) was similar to cryopreservation in terms of bone matrix preservation; no bacteria or fungi were found in the samples.OBJETIVO: Comparar o método da criopreservação de enxertos ósseos (- 80º C) com o da conservação em glicerol a 98% em temperatura ambiente (10º C a 35º C), testando os efeitos antibacterianos e antifúngicos do glicerol a 98% e analisando comparativamente as alterações histológicas verificadas e decorrentes do emprego dos dois métodos. MÉTODO: Este estudo foi constituído de 30 amostras de tecido ósseo trabecular provenientes de 10 pacientes, submetidos a Artroplastia Total do Quadril. Cada cabeça femoral forneceu 3 amostras e estas foram divididas aleatoriamente em 3 grupos, a saber: controle, criopreservado e conservado em glicerol a 98% à temperatura ambiente durante um ano. As amostras foram encaminhadas à Anatomia Patológica para estudo histomorfologico, de viabilidade celular, e microbiológico. Os resultados foram analisados estatisticamente pelo método de McNemar, com índice de significância de 0,05. RESULTADOS: A análise dos valores obtidos no teste de McNemar na comparação das distribuições de probabilidades das variáveis da histomorfologia (osso maduro ou lamelar, osso imaturo e necrose) e da viabilidade celular (osteoblastos e osteoclastos) indica não haver diferença entre as distribuições das variáveis nas três condições experimentais. A análise microbiológica da solução de glicerol a 98% e dos fragmentos ósseos das amostras armazenadas durante um ano em temperatura ambiente não apresentou crescimento bacteriano ou de fungos. As espécimens do grupo controle foram analisadas histológica e microbiologicamente logo após a coleta das mesmas. CONCLUSÃO: O método de conservação de enxertos ósseos mantidos no glicerol a 98% em temperatura ambiente (10ºC a 35ºC) foi similar ao da criopreservação quanto à preservação da matriz óssea e à ausência de crescimento de bactérias ou fungos

Genome-wide analysis of expansin superfamily in wild Arachis discloses a stress-responsive expansin-like B gene

Expansins are plant cell wall-loosening proteins involved in adaptive responses to environmental stimuli and various developmental processes. The first genome-wide analysis of the expansin superfamily in the Arachis genus identified 40 members in A. duranensis and 44 in A. ipaënsis, the wild progenitors of cultivated peanut (A. hypogaea). These expansins were further characterized regarding their subfamily classification, distribution along the genomes, duplication events, molecular structure, and phylogeny. A RNA-seq expression analysis in different Arachis species showed that the majority of these expansins are modulated in response to diverse stresses such as water deficit, rootknot nematode (RKN) infection, and UV exposure, with an expansin-like B gene (AraEXLB8) displaying a highly distinct stress-responsive expression profile. Further analysis of the AraEXLB8 coding sequences showed high conservation across the Arachis genotypes, with eight haplotypes identified. The modulation of AraEXLB8 expression in response to the aforementioned stresses was confirmed by qRT-PCR analysis in distinct Arachis genotypes, whilst in situ hybridization revealed transcripts in different root tissues according to the stress imposed. The overexpression of AraEXLB8 in soybean (Glycine max) composite plants remarkably decreased the number of galls in transformed hairy roots inoculated with RKN. This study improves the current understanding of the molecular evolution, divergence, and gene expression of expansins in Arachis, and provides molecular and functional insights into the role of expansin-like B, the less-studied plant expansin subfamily

Using Recombinant Proteins from Lutzomyia longipalpis Saliva to Estimate Human Vector Exposure in Visceral Leishmaniasis Endemic Areas

During the blood meal, female sand flies (insects that transmit the parasite Leishmania) inject saliva containing a large variety of molecules with different pharmacological activities that facilitate the acquisition of blood. These molecules can induce the production of anti-saliva antibodies, which can then be used as markers for insect (vector) biting or exposure. Epidemiological studies using sand fly salivary gland sonicate as antigens are hampered by the difficulty of obtaining large amounts of salivary glands. In the present study, we have investigated the use of two salivary recombinant proteins from the sand fly Lutzomyia longipalpis, considered the main vector of visceral leishmaniasis, as an alternative method for screening of exposure to the sand fly. We primarily tested the suitability of using the recombinant proteins to estimate positive anti-saliva ELISA test in small sets of serum samples. Further, we validated the assay in a large sample of 1,077 individuals from an epidemiological survey in a second area endemic for visceral leishmaniasis. Our findings indicate that these proteins represent a promising epidemiological tool that can aid in implementing control measures against leishmaniasis

Serological screening in a large-scale municipal survey in Cascais, Portugal, during the first waves of the COVID-19 pandemic: lessons for future pandemic preparedness efforts

BackgroundSerological surveys for SARS-CoV-2 were used early in the COVID-19 pandemic to assess epidemiological scenarios. In the municipality of Cascais (Portugal), serological testing combined with a comprehensive socio-demographic, clinical and behavioral questionnaire was offered to residents between May 2020 and beginning of 2021. In this study, we analyze the factors associated with adherence to this municipal initiative, as well as the sociodemographic profile and chronic diseases clinical correlates associated to seropositivity. We aim to contribute with relevant information for future pandemic preparedness efforts.MethodsThis was a cross-sectional study with non-probabilistic sampling. Citizens residing in Cascais Municipality went voluntarily to blood collection centers to participate in the serological survey. The proportion of participants, stratified by socio-demographic variables, was compared to the census proportions to identify the groups with lower levels of adherence to the survey. Univariate and multivariate logistic regression were used to identify socio-demographic, clinical and behavioral factors associated with seropositivity.ResultsFrom May 2020 to February 2021, 19,608 participants (9.2% of the residents of Cascais) were included in the study. Based on the comparison to census data, groups with lower adherence to this survey were men, the youngest and the oldest age groups, individuals with lower levels of education and unemployed/inactive. Significant predictors of a reactive (positive) serological test were younger age, being employed or a student, and living in larger households. Individuals with chronic diseases generally showed lower seroprevalence.ConclusionThe groups with low adherence to this voluntary study, as well as the socio-economic contexts identified as more at risk of viral transmission, may be targeted in future pandemic situations. We also found that the individuals with chronic diseases, perceiving higher risk of serious illness, adopted protective behaviors that limited infection rates, revealing that health education on preventive measures was effective for these patients

Enhanced Leishmania braziliensis Infection Following Pre-Exposure to Sandfly Saliva

Parasites of the genus Leishmania cause a variety of diseases known as leishmaniasis, that are transmitted by bites of female sand flies that, during blood-feeding, inject humans with parasites and saliva. It was shown that, in mice, immunity to sand-fly saliva is able to protect against the development of leishmaniasis. We have investigated, in the present study, whether this finding extends the sand fly species Lutzomyia intermedia, which is responsible for transmission of Leishmania braziliensis, a parasite species able to cause destructive skin lesions that can be fatal if left untreated. We observed that mice injected with sand fly saliva develop a specific immune response against salivary proteins. Most importantly, however, this immune response was unable to protect mice against a challenge infection with L. braziliensis, indicating that exposure to this sand fly saliva is harmful to the host. Indeed, subjects with cutaneous leishmaniasis have a higher immune response against L. intermedia saliva. These findings indicate that the anti-saliva immune response to sand fly saliva plays an important role in the outcome of leishmaniasis caused by L. braziliensis, in both mice and humans, and emphasize possible hurdles in the development of vaccines based on sand fly saliva

Effects of 3,4-Methylenedioxymethamphetamine Administration on Retinal Physiology in the Rat

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) is known to produce euphoric states, but may also cause adverse consequences in humans, such as hyperthermia and neurocognitive deficits. Although MDMA consumption has been associated with visual problems, the effects of this recreational drug in retinal physiology have not been addressed hitherto. In this work, we evaluated the effect of a single MDMA administration in the rat electroretinogram (ERG). Wistar rats were administered MDMA (15 mg/kg) or saline and ERGs were recorded before (Baseline ERG), and 3 h, 24 h, and 7 days after treatment. A high temperature (HT) saline-treated control group was also included. Overall, significantly augmented and shorter latency ERG responses were found in MDMA and HT groups 3 h after treatment when compared to Baseline. Twenty-four hours after treatment some of the alterations found at 3 h, mainly characterized by shorter latency, tended to return to Baseline values. However, MDMA-treated animals still presented increased scotopic a-wave and b-wave amplitudes compared to Baseline ERGs, which were independent of temperature elevation though the latter might underlie the acute ERG alterations observed 3 h after MDMA administration. Seven days after MDMA administration recovery from these effects had occurred. The effects seem to stem from specific changes observed at the a-wave level, which indicates that MDMA affects subacutely (at 24 h) retinal physiology at the outer retinal (photoreceptor/bipolar) layers. In conclusion, we have found direct evidence that MDMA causes subacute enhancement of the outer retinal responses (most prominent in the a-wave), though ERG alterations resume within one week. These changes in photoreceptor/bipolar cell physiology may have implications for the understanding of the subacute visual manifestations induced by MDMA in humans