Karen Steponavicius Cruz Borbely

Concerns over the replicability and reproducibility of published research have grown in many research fields, but empirical data to inform policies are still scarce. Biomedical research in Brazil expanded rapidly over the last three decades, with no systematic assessment of the replicability of its findings. With this in mind, we set up the Brazilian Reproducibility Initiative, a multicenter replication of published experiments from Brazilian science using three common experimental methods: the MTT assay, the reverse transcription polymerase chain reaction (RT-PCR) and the elevated plus maze (EPM). A total of 56 laboratories performed 143 replications of 56 experiments; of these, 90 replications of 45 experiments were considered valid by an independent committee. Replication rates for these experiments varied between 20 and 44% according to five predefined criteria. In median terms, ratios between group means were 58% lower in replications than in original experiments, while coefficients of variation were 82% higher. Effect size decrease was smaller for MTT experiments, original results with less variability and those considered more challenging to replicate, while t values for replications were positively correlated with researcher predictions about replicability, and negatively correlated with the rate of publications by the original article’s last author. Deviations from preregistered protocols were very common in replications, most frequently due to reasons inherent to the experimental model or related to infrastructure and logistics. Our results highlight factors that limit the replicability of results published by researchers in Brazil and suggest ways by which this scenario can be improved.

The aim was to investigate the long-term effects of metformin ingestion on high-intensity interval training on performance, glycogen concentration (GC), GLUT-4 content, and metabolomics outcomes in rats. Fifty male Wistar rats were randomly divided into baseline, metformin (500 mg daily), and control groups. Training consisted of 4 sets of 10 jumps with 30 s of passive recovery per day, 5 days/week for 8 weeks. The intensity equivalent was 50% of body mass (BM) in the first four weeks and 70% of BM in the last four weeks. The animals were submitted to a weekly jump test until exhaustion at 50% of BM. Serum and tissues were collected at baseline and after 4 and 8 weeks for biochemical and metabolomics analysis. The number of jumps increased in the Control group without a significant difference between groups at 4 and 8 weeks. GLUT4 was lower in the gastrocnemius muscle in the Metformin at the fourth week compared to Control (P=0.03) and compared to Metformin (P=0.02) and Control (P=0.01) at eight weeks. Hepatic and soleus GC were not altered by metformin. Gastrocnemius GC was lower after 8 weeks in the Metformin group compared to Control (P=0.01). Significantly lower levels of pyruvate and phenylalanine and higher levels of ethanol, formate, betaine, very low-density lipoprotein, low-density lipoprotein, and creatine were found in the Metformin compared to the Control. Although chronic administration of metformin decreased food intake and negatively influenced the synthesis of muscle glycogen, it did not significantly change physical performance compared to the Control.

Background: Age-related impairments in macrophage functions have important consequences for the health of the elderly population. The aging process is also accompanied by a reduction in several hormones, including growth hormone (GH). Previous studies have shown that this hormone can affect macrophage activity in young individuals; however, the biological effects of GH stimulation on macrophages during aging have not yet been elucidated. Objective: The aim of this work was to investigate the in vitro effects of GH on peritoneal macrophages from aged mice. Methods: Peritoneal macrophages isolated from young (4 months-old) and old (12-15 months-old) mice were treated in vitro with 100 ng/mL of GH for 24 hours. After treatment, cells were analysed for cell morphology, reactive oxygen species (ROS) production, expression of integrins, cell adhesion to extracellular matrix molecules, and migration in transwell chambers. Results: Although GH-treated cells from old mice exhibited decreased ROS production, we did not observe the effects of GH on macrophage morphology or macrophage phagocytic activity in young and old mice-derived cell cultures. Macrophages from old mice had increased adhesion to laminin and fibronectin substrates, as did cells obtained from young mice treated with GH, but no change was observed in the expression of integrin receptors. Furthermore, cells from old mice exhibited increased migration compared to young mice and a significant increase in macrophage migration was observed under GH stimulation. Conclusion: Our results showed that GH can interfere with the motility of macrophages from old mice, advancing our understanding of the interactions between the immune and neuroendocrine systems during aging.

Various beneficial biological activities have been reported for passion fruit (Passiflora edulis), grown in tropical and subtropical regions. However, no study has yet demonstrated its action against the Zika virus (ZIKV) infection. The present study aimed to investigate the anti-ZIKV of the ethanolic passion fruit seed extract (PFSE), from which, liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis allowed to putatively annotate the occurrence of piceatannol, astringin, scirpusin A, scirpusin B, isookanin-7-O-glucoside and naringenin-7-O-glucoside, two quadranguloside derivatives, tyrosine, and phenylalanine. PFSE is well tolerated by cell lines (Vero E6 and HTR-8/SVneo) and tissue explants from the human placenta. With 1- and 24-h treatments, PFSE consistently reduced the viral load and NS1 protein expression of the two strains of ZIKV (MR766 and PE243) in placental explants and cell lines. Thus, PFSE has a promising potential for developing anti-ZIKV treatments, protecting the placenta against infection caused by ZIKV, along with relevant antioxidant potential, represented by ferric reducing antioxidant power (FRAP) of 390.5 Trolox eq per g dry extract and half maximal inhibitory concentration (IC50) for 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) of 12.1 μg mL–1, and more than 90% protection from lipid peroxidation. Although further studies need to be conducted, PFSE treatment was safe and effective in trophoblast cells and placental explants, thus representing a promising class of all-accessible products based on the reuse of industrial by-products.

Adaptive immune responses are acknowledged to evolve from innate immunity. However, limited information exists regarding whether encounters between innate cells direct the generation of specialized T-cell subsets. We aim to understand how natural killer (NK) cells modulate cell-mediated immunity in humans. We found that human CD14+CD16− monocytes that differentiate into inflammatory dendritic cells (DCs) are shaped at the early stages of differentiation by cell-to-cell interactions with NK cells. Although a fraction of monocytes is eliminated by NK-cell–mediated cytotoxicity, the polarization of interferon-γ (IFN-γ) at the NKp30-stabilized synapses triggers a stable IFN-γ signature in surviving monocytes that persists after their differentiation into DCs. Notably, NK-cell–instructed DCs drive the priming of type 17 CD8+ T cells (Tc17) with the capacity to produce IFN-γ and interleukin-17A. Compared with healthy donors, this cellular network is impaired in patients with classical NK-cell deficiency driven by mutations in the GATA2 gene. Our findings reveal a previously unrecognized connection by which Tc17-mediated immunity might be regulated by NK-cell–mediated tuning of antigen-presenting cells.

Breast cancer is a highly frequent and lethal malignancy which metastasis and relapse frequently associates with the existence of breast cancer stem cells (CSCs). CSCs are undifferentiated, aggressive and highly resistant to therapy, with traits modulated by microenvironmental cells and the extracellular matrix (ECM), a biologically complex and dynamic structure composed mainly by type I collagen (Col-I). Col-I enrichment in the tumor-associated ECM leads to microenvironment stiffness and higher tumor aggressiveness and metastatic potential. While Col-I is also known to induce tumor stemness, it is unknown if such effect is dependent of Col-I density. To answer this question, we evaluated the stemness phenotype of MDA-MB-231 and MCF-7 human breast cancer cells cultured within gels of varying Col-I densities. High Col-I density increased CD44⁺CD24⁻ breast cancer stem cell (BCSC) immunophenotype but failed to potentiate Col-I fiber alignment, cell self-renewal and clonogenicity in MDA-MB-231 cells. In MCF-7 cells, high Col-I density decreased total levels of variant CD44 (CD44v). Common to both cell types, high Col-I density induced neither markers related to CSC nor those related with mechanically-induced cell response. We conclude that high Col-I density per se is not sufficient to fully develop the BCSC phenotype.

Versican is a proteoglycan known to interact with cells to influence their ability to proliferate, differentiate, migrate, invade and assemble extracellular matrix, with all of these cell functions present during placentation. In the placenta, cytotrophoblast cells have the ability to differentiate into the syncytiotrophoblast, a mechanism that is greatly increased in gestational trophoblastic diseases (GTD). Nevertheless, the molecular signaling underlying the increased syncytiotrophoblast differentiation are still being unveiled and may result in novel therapeutic targets for GTD. Versican expression was investigated to establish its differential expression among GTD (partial moles, complete moles, invasive moles and choriocarcinoma) and the possible functional outcomes from versican gene silencing. Tissue samples had their versican expression evaluated using immunohistochemistry and RT-PCR. BeWo cells were employed for versican silencing with siRNA and the efficiency was confirmed by RT-PCR, immunofluorescence and flow cytometry. Cell death and forskolin-induced syncytialization were analyzed by a morphological analysis and human chorionic gonadotropin (hCG) production using immunofluorescence. Versican V0 and V1 isoforms were mainly expressed in the syncytiotrophoblast and they were the most expressed in benign rather than in malignant tumors. BeWo cells also expressed V0 and V1 isoforms, but only in cells undergoing syncytial fusion. After versican silencing, cell death was greatly increased, whereas spontaneous and forskolin-induced syncytialization decreased as well as hCG production. Versican is differentially expressed in GTD and is important for hydatidiform moles pathophysiology, protecting trophoblast cells from death and playing a role in their differentiation and functionality.

Growth hormone (GH) plays a physiological role in the immune system. In macrophages, GH enhances the production of hydrogen peroxide, superoxide anions, nitric oxide, cytokines, and chemokines, including interferon-γ and macrophage inflammatory protein-1α. However, some of the effects of GH stimulation on the biological functions of macrophages remain to be elucidated. Herein we showed that in vivo GH treatment resulted in decreased expression of VLA-5 and VLA-6 integrins on the macrophage surface, accompanied by a reduction in macrophage adhesion to extracellular matrix (ECM) ligands, fibronectin and laminin. Additionally, a decrease in macrophage adhesion to laminin was observed when the cells were treated in vitro with GH. In transwell migration assays, GH-treated macrophages showed increased migration after 6 hours. Although in vitro GH treatment did not influence the phagocytic activity of macrophages, when the treatment was performed in vivo, peritoneal macrophages from GH-treated mice showed a higher percentage of phagocytosis and higher phagocytic capacity than cells from control animals. These results led us to analyse the role of insulin-like growth factor-1 (IGF-1), a GH stimulated factor, on macrophage phagocytosis. We observed an increase in phagocytic activity when J774 murine macrophages were treated with IGF-1 for 24 hours. Our results revealed an important role for GH in resident macrophage migration and phagocytic activity. Specifically, we demonstrate that IGF-1 may be the GH stimulated factor that induces macrophage phagocytosis in vivo.