Selection of references for quantitative real-time PCR analysis of microRNAs in Nile tilapia (Oreochromis niloticus) under osmotic stress.

MicroRNAs play crucial regulatory roles in various aspects of development and physiology, including environmental adaptation and stress responses in teleosts. RT-qPCR is the most commonly used method for studying microRNA expression, with the accuracy and reliability of results depending on the use of an appropriate reference gene for normalization. This study aimed to evaluate seven miRNAs (U6, Let-7a, miR-23a, miR-25-3, miR-103, miR-99-5, and miR-455) expression stability in different tissues of Nile tilapia subjected to osmotic stress. Fish were divided into two groups: a control and an experimental group, raised in 0 and 12 ppt salinity water respectively. After 21 days, brain, gills, liver, and posterior intestine were collected for analysis. Different mathematical algorithms (geNorm, NormFinder, BestKeeper, and the comparative ΔCt method) were employed to identify the most suitable reference miRNAs. The results indicate that the miR-455/miR-23a combination is a robust reference for normalizing miRNA expression levels in studies of osmotic stress responses in Nile tilapia. The stability of miRNA expression can vary depending on specific stress conditions and biological processes, underscoring the necessity of selecting appropriate normalizing miRNAs for each experimental context. This study identifies reliable reference genes for future RT-qPCR analyses of miRNA expression, thereby enhancing our understanding of molecular responses in fish to environmental challenges. These insights are fundamental to the development of new technologies for the improved management and sustainability of aquaculture practices.

A Pilot Study of Gene Expression Modulation from Antioxidant System of Killifish Austrolebias charrua After Exposure to Roundup Transorb®.

Roundup Transorb® (RDT) is the most popular glyphosate-based herbicide (GHB) used in agriculture, and its impact extends to non-target organisms. The annual killifish Austrolebias charrua is an endangered species endemic to southern South America and inhabits temporary ponds. This study evaluates the effects of RDT concentrations (0.065 and 5 mg/L GAE) on A. charrua exposed for 96 h. Gene expression of cat, sod2, gstα, gclc, and ucp1 was evaluated on the liver and gills. Highlighting that even at low concentrations permitted by Brazilian legislation, the RDT can have adverse effects on A. charrua.

Patentometric analysis of the technological development of biotechnology for health in higher education institutions in Rio Grande do Sul.

Biotechnology offers solutions and opportunities to meet various societal demands, thereby contributing to significant scientific advancements. This study aimed to characterize the technological development of biotechnology in the healthcare sector in the state of Rio Grande do Sul, Brazil, from 2016 to 2022 by analyzing patents filed by and granted to public and private Higher Education institutions. For data collection, a quantitative exploratory approach was employed using statistical methods and a patent analysis of institutions in the patent database of the Brazilian National Institute of Industrial Property (INPI), focusing on patents related to the healthcare field. Data were collected in October, November, and December. A total of 580 patent records were collected from the INPI, belonging to Sections A and C of the International Patent Classification (IPC) related to educational institutions. Furthermore, this study highlighted that higher education institutions have a higher number of patents in the healthcare field. These results provide an understanding of the strategic areas for technological development in biotechnology in Rio Grande do Sul, Brazil.

MicroRNA qPCR normalization in Nile tilapia (Oreochromis niloticus): Effects of acute cold stress on potential reference targets.

The Nile tilapia (Oreochromis niloticus) is one of the most important cultured fish worldwide, but tilapia culture is largely affected by low temperatures. Recent studies suggest that microRNAs (miRNAs) regulate cold tolerance traits in fish. In general, qPCR-based methods are the simplest and most accurate forms of miRNA quantification. However, qPCR data heavily depends on appropriate normalization. Therefore, the aim of the present study is to determine whether the expression of previously tested, stably expressed miRNAs are affected by acute cold stress in Nile tilapia. For this purpose, one small nuclear RNA (U6) and six candidate reference miRNAs (miR-23a, miR-25-3, Let-7a, miR-103, miR-99-5, and miR-455) were evaluated in four tissues (blood, brain, liver, and gills) under two experimental conditions (acute cold stress and control) in O. niloticus. The stability of the expression of each candidate reference miRNA was analyzed by four independent methods (the delta Ct method, geNorm, NormFinder, and BestKeeper). Further, consensual comprehensive ranking of stability was built with RefFinder. Overall, miR-103 was the most stable reference miRNA in this study, and miR-103 and Let-7a were the best combination of reference targets. Equally important, Let-7a, miR-23a, and miR-25-3 remained consistently stable across different tissues and experimental groups. Considering all variables, U6, miR-99-5, and miR-455 were the least stable candidates under acute cold stress. Most important, suitable reference miRNAs were validated in O. niloticus, facilitating further accurate miRNA quantification in this species.

Modulation of miR-429 during osmotic stress in the silverside Odontesthes humensis.

Silverside fish inhabit marine coastal waters, coastal lagoons, and estuarine regions in southern South America. Although silversides are not fully adapted to freshwater, they can tolerate a wide range of salinity variations. MicroRNAs (miRNAs) are a class of ∼22 nucleotide noncoding RNAs, which are crucial regulators of gene expression at post-transcriptional level. Current data indicate that miRNAs biogenesis is altered by situations of environmental stress, thereby altering the expression of target mRNAs. Foremost, the silversides were acutely exposed to 30 g.L-1 of salt to reveal in which tissue miR-429 could be differentially expressed. Thus, fish were acclimated to freshwater (0 g.L-1) and to brackish water (10 g.L-1), and then exposed to opposite salinity treatment. Here, we reveal that miR-429, a gill-enriched miRNA, emerges as a prime osmoregulator in silversides. Taken together, our findings suggest that miR-429 is an endogenous regulator of osmotic stress, which may be developed as a biomarker to assist silverside aquaculture.

Exposure to salinity induces oxidative damage and changes in the expression of genes related to appetite regulation in Nile tilapia (Oreochromis niloticus).

Variations in water salinity and other extrinsic factors have been shown to induce changes in feeding rhythms and growth in fish. However, it is unknown whether appetite-related hormones mediate these changes in Nile tilapia (Oreochromis niloticus), an important species for aquaculture in several countries. This study aimed to evaluate the expression of genes responsible for appetite regulation and genes related to metabolic and physiological changes in tilapia exposed to different salinities. Moreover, the study proposed to sequence and to characterize the cart, cck, and pyy genes, and to quantify their expression in the brain and intestine of the fish by quantitative polymerase chain reaction (qPCR). The animals were exposed to three salinities: 0, 6, and 12 parts per thousand (ppt) of salt for 21 days. Furthermore, lipid peroxidation, reactive oxygen species, DNA damage, and membrane fluidity in blood cells were quantified by flow cytometry. The results indicated an increased expression of cart, pyy, and cck and a decreased expression of npy in the brain, and the same with cck and npy in the intestine of fish treated with 12 ppt. This modulation and other adaptive responses may have contributed to the decrease in weight gain, specific growth rate, and final weight. In addition, we showed oxidative damage in blood cells resulting from increasing salinity. These results provide essential data on O. niloticus when exposed to high salinities that have never been described before and generate knowledge necessary for developing biotechnologies that may help improve the production of economically important farmed fish.

Chronic cold exposure modulates genes related to feeding and immune system in Nile tilapia (Oreochromis niloticus).

Nile tilapia is the fourth most produced species in the global aquiculture panorama. This species requires water temperatures higher than 16 °C to grow and survive, and so, little is known about the effects of low temperatures on genes related to food intake and inflammatory responses. This study brought insights about the modulation of genes in different tissues of Nile tilapia chronically exposed to low temperatures. Thus, sixty animals were divided in two experimental groups: a control group in which the animals remained at the optimum temperature of 24 °C; and an exposed to cold group, in which a decrease in the water temperature was applied until reaching 15 °C. These conditions were maintained for 28 days. Blood samples were collected for flow cytometry analysis, while brain, spleen, liver, and kidney tissues were collected for total RNA extraction, followed by quantitative PCR (RT-qPCR). For genes related to feeding process pathway, it was observed an upregulation in pyy and a downregulation of npy and cart gene expression. Also, pro-inflammatory cytokine genes were modulated in the spleen, kidney and liver with a higher expression of il-1b and tnfα and a reduction in the il-8 and nf-κß gene expressions in the group exposed to 15 °C. The fish exposed to cold presented higher serum cortisol levels than the ones from control group. The blood cell analysis showed a lower level of membrane fluidity and a higher DNA fragmentation and cell disruption in the group exposed to cold. These findings suggest an important effect of a stressful situation in the tilapia organism due to cold exposure. This study brings insights on tilapia wellbeing under low temperature stress. It can be a first step to understanding the appropriate way to cope with cold impacts on aquaculture.

7-Chloro-4-(Phenylselanyl) Quinoline Is a Novel Multitarget Therapy to Combat Peripheral Neuropathy and Comorbidities Induced by Paclitaxel in Mice.

Paclitaxel-induced peripheral neuropathy (PIPN) is a very common and complex painful condition related to paclitaxel (PTX) exposure, severely impacting patients' quality of life, and contributing to the emergence of clinical signs of anxiety and cognitive loss. At present, no sufficient treatment options are available for PIPN and its exact pathophysiology remains unclear. Based on the therapeutic potential of the 7-chloro-4-(phenylselanyl) quinoline (4-PSQ), we assessed its ability to reverse PIPN and its comorbities induced by PTX. The effect of 4-PSQ was evaluated on pathophysiological processes involved in PIPN, such as oxidative stress (oxidative damage and antioxidant enzymes), neuroinflammation (mRNA expression levels of nuclear factor-kappa B, interleukin-1beta, tumor necrosis factor-alpha, and inducible nitric oxide synthase), and calcium homeostasis (Ca2+ATPase activity) in the spinal cord, cerebral cortex, and hippocampus of mice. Male Swiss mice received PTX (2 mg/kg) or vehicle by intraperitoneal route (days 1, 2, and 3). Oral administration of 4-PSQ (1 mg/kg) or vehicle was performed on days 3 to 14. It was observed that 4-PSQ reduced the mechanical and thermal hypersensitivities induced by PTX. Likewise, 4-PSQ reduced both anxious behavior and cognitive impairment in mice with PIPN. We believe that effects of 4-PSQ may be associated, at least in part, with the modulation of oxidative stress, reduction of neuroinflammation, and normalizing Ca2+ATPase activity in the spinal cord, cerebral cortex, and hippocampus of mice with PIPN. Taken together, the 4-PSQ might be a good prototype for the development of a more effective drug for the treatment of PIPN and its comorbities.

DNA microarray for forensic intelligence purposes: High-density SNP profiles obtained directly from casework-like samples with and without a DNA purification step.

SNP analyses from a forensic intelligence perspective have proven to be an important tool to restrict the number of suspected offenders and find missing persons. DNA microarray assays have been demonstrated as a potential feature in forensic analysis, like such as forensic genetic genealogy. The objective of this study was to describe the results from DNA microarray assay from saliva samples deposited on a glass surface collected from by a double swab technique, commonly applied in crime scenes. Eighteen samples from the same person were subjected to Infinium® Global Screening Array-24 v1.0 (~642.824 SNP markers) in two different protocols - with or without the DNA purification procedure. The measured genotype was compared with a Consensus Genotype, obtained from standard control samples, and the parameters such as Call Rate and GenCall Scores were evaluated. Results showed that the Call Rate parameter is enough to estimate the probability of obtaining a correct genotype in the SNP assay. Reliable genotypes with a confidence level of more than 90% (at least 90.15%) were observed in Call Rates above 69.41%, regardless of the experimental condition. Our data demonstrate that DNA Microarray from samples collected under conditions such as those found at crime scenes can generate high-density SNP genetic profiles with a confidence level greater than 90%. Enzymatic adjustments and protocol changes may enable DNA microarray assays for crime analysis and investigation purposes eliminating the purification step in the future. Our data suggest that DNA microarray can support criminal investigation teams from a forensic intelligence perspective.

Acute exposition to Roundup Transorb® induces systemic oxidative stress and alterations in the expression of newly sequenced genes in silverside fish (Odontesthes humensis).

Roundup Transorb® (RDT) is a glyphosate-based herbicide commonly used in agricultural practices worldwide. This herbicide exerts negative effects on the aquatic ecosystem and affects bioenergetic and detoxification pathways, oxidative stress, and cell damage in marine organisms. These effects might also occur at the transcriptional level; however, the expression of genes associated with oxidative stress has not been studied well. Odontesthes humensis is a native Brazilian aquatic species naturally distributed in the habitats affected by pesticides, including Roundup Transorb® (RDT). This study evaluated the toxic effects of short-term exposure to RDT on O. humensis. Moreover, the genes related to oxidative stress were sequenced and characterized, and their expressions in the gills, hepatopancreas, kidneys, and brain of the fish were quantified by quantitative reverse transcription-polymerase chain reaction. The animals were exposed to two environmentally relevant concentrations of RDT (2.07 and 3.68 mg L-1) for 24 h. Lipid peroxidation, reactive oxygen species (ROS), DNA damage, and apoptosis in erythrocytes were quantified by flow cytometry. The expression of the target genes was modulated in most tissues in the presence of the highest tested concentration of RDT. In erythrocytes, the levels of lipid peroxidation, ROS, and DNA damage were increased in the presence of both the concentrations of RDT, whereas cell apoptosis was increased in the group exposed to 3.68 mg L-1 RDT. In conclusion, acute exposure to RDT caused oxidative stress in the fish, induced negative effects on cells, and modulated the expression of genes related to the enzymatic antioxidant system in O. humensis.

Evaluation of qPCR reference genes in GH-overexpressing transgenic zebrafish (Danio rerio).

Reference genes (RGs) must have a stable expression in tissues in all experimental conditions to normalize real-time quantitative reverse transcription PCR (qRT-PCR) data. F0104 is a highly studied lineage of zebrafish developed to overexpress the growth hormone (GH). It is assumed that the transgenic process may influence the expression levels of commonly used RGs. The objective of the present study was to make a comprehensive analysis of stability of canditade RGs actb1, actb2, b2m, eif2s2, eef1a1, gapdh, rplp2, rpl7, rpl13α, tuba1, and rps18, in gh-transgenic and non-transgenic zebrafish. Liver, brain, intestine and muscle samples from both groups had qRT-PCR results analyzed by dCt, geNorm, NormFinder, BestKeeper, and RefFinder softwares. Consensus analyses among software concluded that rpl13α, rpl7, and eef1a1 are the most stable genes for zebrafish, considering the studied groups and tissues. Gapdh, rps18, and tuba1 suffered variations in stability among different tissues of both groups, and so, they were listed as the genes with lowest stability. Results from an average pairwise variations test indicated that the use of two RGs would generate reliable results for gene expression analysis in the studied tissues. We conclude that genes that are commonly used in mammals for qRT-PCR assays have low stability in both non-transgenic and gh-transgenic zebrafish reinforcing the importance of using species-specific RGs.

The anxiolytic effect of a promising quinoline containing selenium with the contribution of the serotonergic and GABAergic pathways: Modulation of parameters associated with anxiety in mice.

Recently, we demonstrated the promising anxiolytic action of 7-chloro-4-(phenylselanyl) quinoline (4-PSQ) in mice. For this reason, the objective of this study was to expand our previous findings by investigating the contribution of serotoninergic and GABAergic systems to the anxiolytic action of this compound. Pretreatment with different serotoninergic antagonists (pindolol, WAY100635 and ketanserin) blocked the anxiolytic effect caused by 4-PSQ (50 mg/kg, per oral) in the elevated plus maze (EPM) test. The contribution of the GABAergic system was investigated by pretreatment with pentylenetetrazole (a GABAA receptor antagonist) (PTZ). 4-PSQ diminished the PTZ-induced anxiety, and did not modify the locomotor, exploratory and motor activities of mice. Later, this group of animals was euthanized and the blood was removed to determine the levels of corticosterone, and cerebral cortex and hippocampus to determine the mRNA expression levels of cAMP response element binding protein (CREB), brain derived neurotrophic factor (BDNF) and nuclear factor kappa B (NF-κB), as well as the Na+, K+ ATPase activity and reactive species (RS) levels. 4-PSQ was able to significantly reverse the increase in RS and corticosterone levels, as well as the decrease of CREB and BDNF expression in the cerebral structures and increase of NF-κB expression in the hippocampus. Finally, 4-PSQ restored the Na+, K+ ATPase activity in the cerebral structures evaluated. Here, we showed that the modulation of serotonergic and GABAergic systems, factors related to neurogenesis, oxidative status and Na+, K+ ATPase activity contributes to the anxiolytic effect of 4-PSQ and reinforces the therapeutical potential of this compound for the treatment of anxiety.

Transfection of exogenous DNA complexed to cationic dendrimer induces alterations of bovine sperm microRNAome.

MicroRNAs have been hypothesized to be involved in the regulation of male fertility potential. The primary aim of our study was to demonstrate the effects of transfection with dendrimer nanostructure on the parameters of bovine sperm quality and to investigate whether the microRNA profile could be disturbed after cationic dendrimer-mediated exogenous DNA transfection of bovine spermatozoa. The binding of exogenous DNA was significantly increased when dendrimer-based transfection was implemented. However, cationic dendrimer transfection induced detrimental changes in the kinetics and sperm quality parameters, such as membrane integrity, acrosome reaction, and mitochondrial membrane potential, when compared to the control group. Sperm microRNA sequencing revealed 218 known and 106 novel microRNAs in the sperm samples, among which nine were dysregulated after transfection (one was upregulated and eight were downregulated), in comparison to the non-transfected sperm. All the dysregulated microRNAs were related to sperm quality and embryonic development. These results suggest that the transfection process using the dendrimer nanostructure has an impact on the quality and microRNA profile of bovine sperm.

Efficiency and cell viability implications using tip type electroporation in zebrafish sperm cells.

Sperm-mediated gene transfer (SMGT) has a potential use for zebrafish transgenesis. However, transfection into fish sperm cells still needs to be improved. The objective was to demonstrate the feasibility of tip type electroporation in zebrafish sperm, showing a protocol that provide high transfection efficiency, with minimal side-effects. Sperm was transfected with a Cy3-labelled DNA using tip type electroporation with voltages ranging from 500 to 1500 V. Sperm kinetics parameters were assessed using Computer Assisted Semen Analysis (CASA) and cell integrity, reactive oxygen species (ROS), mitochondrial functionality and transfection rate were evaluated by flow cytometry. The transfection rates were positively affected by tip type electroporation, reaching 64.9% ± 3.6 in the lowest voltage used (500 V) and 86.6% ± 1.9 in the highest (1500 V). The percentage of overall motile sperm in the electrotransfected samples was found to decrease with increasing field strength (P < 0.05). Increase in the sperm damaged plasma membrane was observed with increasing field strength (P < 0.05). ROS and sperm mitochondrial functionality did not present a negative response after the electroporation (P > 0.05). Overall results indicate that tip type electroporation enhances the internalization of exogenous DNA into zebrafish sperm cells with minimal harmful effects to sperm cells.

Voltages up to 600V did not affect cryopreserved bovine spermatozoa on capillary-type electroporation.

Physical methods such as electroporation have been used to improve the DNA uptake efficiency of sperm cells. This study aims to develop an efficient capillary-type electroporation method for incorporation of exogenous DNA into bovine cryopreserved sperm cells with minimal detrimental effects for later use in SMGT. Electroporation of the samples was performed in 2 different groups (with 1 µg of DNA and without DNA transfection) and under five different voltages: 500 V, 600 V, 700 V, 800 V and 900 V. Non-electroporated sperm cells (with and without DNA) were used as control. Kinetics parameters were determined using computer assisted semen analyses, whereas membrane integrity, fluidity, mitochondrial function and DNA uptake were evaluated by flow cytometry. Results revealed that all tested voltages reduced electroporated sperm motility (P < 0.05) when compared to the control (non-electroporated cells). Mitochondrial function results showed no statistical difference among groups. Similarly, groups electroporated with lower (500 V, 600 V and 700 V) voltages showed no difference in cell membrane integrity and fluidity. Groups electroporated at higher voltages (800 V and 900 V) demonstrated negative effects in cells membrane integrity when compared to other groups and control. Also, all electroporated groups demonstrated significant higher percentages of transfected sperm cells when compared to the control group (P < 0.05). Under the recommendation of using voltages up to 600 V, this method represents a safe and efficient alternative for electroporation of bovine spermatozoa.