Validation of qPCR reference genes in the endangered annual killifish Austrolebias charrua considering different tissues, gender and environmental conditions.

The annual killifish Austrolebias charrua is an endangered species, endemic to the southern region of South America, which inhabits temporary ponds that emerges in the rainy season. The main anthropogenic threat driving the extinction of A. charrua stems from extensive agriculture, primarily due to the widrespread use of glyphosate-based herbicides near their habitats. Annual killifishes have been used as models for ecotoxicological studies but, up to now, there are no studies about reference genes in any Austrolebias species. This represents an obstacle to the use of qPCR-based technologies, the standard method for gene expression quantification. The present study aimed to select and validate potential reference genes for qPCR normalization in the annual killifish Austrolebias charrua considering different tissues, gender and environmental conditions. The candidate reference genes 18 s, actb, gapdh, ef1a, shox, eif3g, and the control gene atp1a1 were evaluated in male and female individuals in three different tissues (brain, liver, and gills) under two experimental conditions (control and acute exposition to Roundup Transorb®). The collected tissues were submitted to RNA extraction, followed by cDNA synthesis, cloning, sequencing, and qPCR. Overall, 18 s was the most stable reference gene, and 18 s and ef1a were the most stable combination. Otherwise, considering all variables, gapdh and shox were the least stable candidate genes. Foremost, suitable reference genes were validated in A. charrua, facilitating accurate mRNA quantification in this species, which might be useful for developing molecular tools of ecotoxicological assessment based on gene expression analysis for environmental monitoring of annual killifish.

Chemical activation of mammalian oocytes and its application in camelid reproductive biotechnologies: A review.

Mammalian oocyte activation is a critical process occurring post-gamete fusion, marked by a sequence of cellular events initiated by an upsurge in intracellular Ca2+. This surge in calcium orchestrates the activation/deactivation of specific kinases, leading to the subsequent inactivation of MPF and MAPK activities, alongside PKC activation. Despite various attempts to induce artificial activation using distinct chemical compounds as Ca2+ inducers and/or Ca2+-independent agents, the outcomes have proven suboptimal. Notably, incomplete suppression of MPF and MAPK activities persists, necessitating a combination of different agents for enhanced efficiency. Moreover, the inherent specificity of activation methods for each species precludes straightforward extrapolation between them. Consequently, optimization of protocols for each species and for each technique, such as PA, ICSI, and SCNT, is required. Despite recent strides in camelid biotechnologies, the field has seen little advancement in chemical activation methods. Only a limited number of chemical agents have been explored, and the effects of many remain unknown. In ICSI, despite obtaining blastocysts with different chemical compounds that induce Ca2+ and calcium-independent increases, viable offspring have not been obtained. However, SCNT has exhibited varying outcomes, successfully yielding viable offspring with a reduced number of chemical activators. This article comprehensively reviews the current understanding of the physiological activation of oocytes and the molecular mechanisms underlying chemical activation in mammals. The aim is to transfer and apply this knowledge to camelid reproductive biotechnologies, with emphasis on chemical activation in PA, ICSI, and SCNT.

Treatment with Blackberry Extract and Metformin in Sporadic Alzheimer's Disease Model: Impact on Memory, Inflammation, Redox Status, Phosphorylated Tau Protein and Insulin Signaling.

Natural products offer promising potential for the development of new therapies for Alzheimer's disease (AD). Blackberry fruits are rich in phytochemical compounds capable of modulating pathways involved in neuroprotection. Additionally, drug repurposing and repositioning could also accelerate the development of news treatments for AD. In light of the reduced brain glucose metabolism in AD, an alternative approach has been the use of the drug metformin. Thus, the aim of this study was to evaluate the effect of treatment with blackberry extract in a model of AD induced by streptozotocin (STZ) and compare it with metformin treatment. Male rats were divided into groups: I - Control; II - STZ; III - STZ + blackberry extract (100 mg/kg); IV - STZ + blackberry extract (200 mg/kg) and V - STZ + metformin (150 mg/kg). The animals received intracerebroventricular injection of STZ or buffer. Seven days after the surgical procedure, the animals were treated orally with blackberry extract or metformin for 21 days. Blackberry extract and metformin prevented the memory impairment induced by STZ. In animals of group II, an increase in acetylcholinesterase activity, phosphorylated tau protein, IL-6, oxidative damage, and gene expression of GSK-3ß and Nrf2 was observed in the hippocampus. STZ induced a decrease in IL-10 levels and down-regulated the gene expression of Akt1, IRS-1 and FOXO3a. Blackberry extract and metformin prevented the alterations in acetylcholinesterase activity, IL-6, GSK3ß, Nrf2, and oxidative damage. In conclusion, blackberry extract exhibits multi-target actions in a model of AD, suggesting new therapeutic potentials for this neurodegenerative disease.

MicroRNA Transcriptomes Reveal Prevalence of Rare and Species-Specific Arm Switching Events During Zebrafish Ontogenesis.

In metazoans, microRNAs (miRNAs) are essential regulators of gene expression, affecting critical cellular processes from differentiation and proliferation, to homeostasis. During miRNA biogenesis, the miRNA strand that loads onto the RNA-induced Silencing Complex (RISC) can vary, leading to changes in gene targeting and modulation of biological pathways. To investigate the impact of these "arm switching" events on gene regulation, we analyzed a diverse range of tissues and developmental stages in zebrafish by comparing 5p and 3p arms accumulation dynamics between embryonic developmental stages, adult tissues, and sexes. We also compared variable arm usage patterns observed in zebrafish to other vertebrates including arm switching data from fish, birds, and mammals. Our comprehensive analysis revealed that variable arm usage events predominantly take place during embryonic development. It is also noteworthy that isomiR occurrence correlates to changes in arm selection evidencing an important role of microRNA distinct isoforms in reinforcing and modifying gene regulation by promoting dynamics switches on miRNA 5p and 3p arms accumulation. Our results shed new light on the emergence and coordination of gene expression regulation and pave the way for future investigations in this field.

Whole-genome sequencing of Leptospira interrogans from southern Brazil: genetic features of a highly virulent strain

BACKGROUND Leptospirosis is the most widespread zoonotic disease. It is caused by infection with pathogenic Leptospira species, of which over 300 serovars have been described. The accurate identification of the causative Leptospira spp. is required to ascertain the pathogenic status of the local isolates. OBJECTIVES This study aimed to obtain the complete genome sequence of a virulent Leptospira interrogans strain isolated from southern Brazil and to describe its genetic features. METHODS The whole genome was sequenced by next-generation sequencing (Ion Torrent). The genome was assembled, scaffolded, annotated, and manually reviewed. Mutations were identified based on a variant calling analysis using the genome of L. interrogans strain Fiocruz L1-130 as a reference. FINDINGS The entire genome had an average GC content of 35%. The variant calling analysis identified 119 single nucleotide polymorphisms (SNPs), from which 30 led to a missense mutation. The structural analyses identified potential evidence of genomic inversions, translocations, and deletions in both the chromosomes. MAIN CONCLUSIONS The genome properties provide comprehensive information about the local isolates of Leptospira spp., and thereby, could facilitate the identification of new targets for the development of diagnostic kits and vaccines.

Effect of porcine somatotropin on metabolism and testicular characteristics of prepubertal pigs / Efeito da somatotrofina suína sobre o metabolismo e características testiculares de suínos pré-púberes

The effect of pST on the testicular characteristics and metabolic parameters of prepubertal pigs was evaluated. Experiment 1 aimed to determine the interval between applications of pST based on the concentrations of circulating IGF-I. Experiment 2 aimed to evaluate the effect of pST on metabolic parameters, testicular characteristics, and expression of GHR, IGF-I and PCNA. In Experiment 1 twelve piglets with 30 days of age were used. The pST Group (n = 6) was submitted to one i.m. injection of pST and the Control Group (n = 6) to one placebo injection. Blood collections were performed until day 7 post pST application to determine IGF-I concentration and metabolic profile. In Experiment 2 twelve piglets with 22 days of age were used. The pST Group was submitted to pST injections every three days, and the Control Group received placebo doses during 30 days. Blood collections were performed every 3 days. Samples of liver and testicular tissue were collected to determine gene expression and testicular characteristics. In Experiment 1 IGF-I concentration was higher for the pST Group (P = 0.02). In Experiment 2 the pST Group had higher body and testicular weight (P=0.06) and increased gene expression of PCNA in testes (P < 0.05). However, a reduction in the number of seminiferous tubules, and Sertoli cells, and in GHR expression (P < 0.05) was observed. Thus, pST administration increased body and testis development in prepubertal pigs, however it reduced the density of seminiferous tubules and Sertoli cells.

Foi investigado o efeito da pST sobre características testiculares e metabolismo de suínos pré-púberes. O Experimento 1 determinou o intervalo entre aplicações de pST, baseado nas concentrações de IGF-I. O Experimento 2 avaliou o efeito da pST sobre o metabolismo, características testiculares e expressão gênica de GHR, IGF-I e PCNA. No Experimento 1, foram usados 12 leitões com 30 dias de idade. O grupo pST (n = 6) foi submetido a uma injeção IM de pST e o grupo Controle (n = 6) a uma injeção de placebo. Coletas de sangue foram realizadas até o dia sete após a aplicação de pST para determinação dos níveis de IGF-I e parâmetros metabólicos. No Experimento 2, foram usados 12 leitões com 22 dias de idade. O grupo pST foi submetido às aplicações de pST a cada 3 dias, e o grupo Controle, às doses de placebo, durante 30 dias. Coletas de sangue foram realizadas a cada três dias. Amostras de fígado e testículo foram coletadas para determinar a expressão gênica e características testiculares. No Experimento 1, a concentração de IGF-I foi maior no grupo pST (P = 0,02). No Experimento 2, o grupo pST teve maior peso corporal e testicular (P = 0,06) e aumento na expressão de PCNA no testículo (P < 0,05). Contudo, foi observada uma redução no número de túbulos seminíferos, células de Sertoli e GHR (P < 0,05). Assim, a administração de pST aumentou o desenvolvimento testicular e corporal de suínos pré-púberes, porém reduziu a densidade de túbulos seminíferos e células de Sertoli.

MicroRNAs e seu papel no desenvolvimento embrionário / MicroRNAs and its role in embryonic development

MicroRNAs (miRNAs) são pequenas moléculas de RNA com aproximadamente 22 nucleotídeos incapazes de codificar proteínas e que apresentam função na regulação pós-transcricional da expressão gênica. Vários estudos vêm demonstrando o importante papel dos miRNAs na regulação do desenvolvimento embrionário de diferentes espécies, desde o controle da expressão de RNAs mensageiros durante o desenvolvimento inicial embrionário até a determinação de linhagens celulares durante a organogênese. Esta revisão irá abordar os principais miRNAs e seu papel na biologia reprodutiva, com ênfase no desenvolvimento embrionário de mamíferos.

MicroRNAs (miRNAs) are small RNA molecules with around 22 nucleotides that are unable to encode proteins and play a key role on post-transcription regulation process. Several studies have demonstrated the relevant role of miRNAs on the regulation of embryonic development on different species, from the control of gene expression during the early embryo development to determination of cellular lineages over the organogenesis. This review will present the miRNAs and its role on reproductive biology focusing on the mammalian embryo development.

NPY and sbGnRH gene expression in juvenile and adult male Brazilian flounder Paralichthys orbignyanus / Expressão gênica do NPY e do sbGnRH em machos juvenis e adultos de linguado Paralichthys orbignyanus

The objective of this study was to evaluate neuropeptide Y (NPY) and sea bream gonadotropin-release hormone (sbGnRH) gene expression in juvenile and adult males of Brazilian flounder. Hypothalamuses from fish were sampled for total RNA extraction. After cDNA synthesis, real-time PCR was used to measure gene expression. NPY showed approximately 2-fold increases in their mRNA levels while sbGnRH showed 3-fold increases in adult fish. These results suggest that these peptides could be involved on hypothalamic regulation of Brazilian flounder sexual maturation.

O objetivo deste estudo foi avaliar a expressão gênica do neuropeptídeo Y (NPY) e da variante sea bream do hormônio liberador de gonadotrofinas (sbGnRH) em linguados machos juvenis e adultos. O hipotálamo foi isolado para a extração de RNA total. Após a síntese de cDNA, a PCR em tempo real foi usada para avaliar a expressão gênica. Foi observado um aumento de aproximadamente duas vezes nos níveis de NPY e de aproximadamente três vezes nos níveis de sbGnRH nos peixes adultos. Esses resultados demonstram que estes peptídeos podem estar envolvidos na regulação, via hipotálamo, da maturação sexual no linguado.