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.

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.

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.

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.

GH Overexpression Alters Spermatic Cells MicroRNAome Profile in Transgenic Zebrafish.

Overexpression of growth hormone (GH) in gh-transgenic zebrafish of a highly studied lineage F0104 has earlier been reported to cause increased muscle growth. In addition to this, GH affects a broad range of cellular processes in transgenic fish, such as morphology, physiology, and behavior. Reports show changes such as decreased sperm quality and reduced reproductive performance in transgenic males. It is hypothesized that microRNAs are directly involved in the regulation of fertility potential during spermatogenesis. The primary aim of our study was to verify whether gh overexpression disturbs the sperm miRNA profile and influences the sperm quality in transgenic zebrafish. We report a significant increase in body weight of gh-transgenic males along with associated reduced sperm motility and other kinetic parameters in comparison to the non-transgenic group. MicroRNA transcriptome sequencing of gh-transgenic zebrafish sperms revealed expressions of 186 miRNAs, among which six miRNA were up-regulated (miR-146b, miR-200a-5p, miR-146a, miR-726, miR-184, and miR-738) and sixteen were down-regulated (miR-19d-3p, miR-126a-5p, miR-126b-5p, miR-22a-5p, miR-16c-5p, miR-20a-5p, miR-126b-3p, miR-107a-3p, miR-93, miR-2189, miR-202-5p, miR-221-3p, miR-125a, miR-125b-5p, miR-126a-3p, and miR-30c-5p) in comparison to non-transgenic zebrafish. Some of the dysregulated miRNAs were previously reported to be related to abnormalities in sperm quality and reduced reproduction ability in other species. In this study, an average of 134 differentially expressed miRNAs-targeted genes were predicted using the in silico approach. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis demonstrated that the genes of affected pathways were primarily related to spermatogenesis, sperm motility, and cell apoptosis. Our results suggested that excess GH caused a detrimental effect on sperm microRNAome, consequently reducing the sperm quality and reproductive potential of zebrafish males.

Se-[(2,2-Dimethyl-1,3-dioxolan-4-yl)methyl] 4-Chlorobenzoselenolate Attenuates Inflammatory Response, Nociception, and Affective Disorders Related to Rheumatoid Arthritis in Mice.

Despite major advances, not all patients achieve rheumatoid arthritis (RA) remission, thus highlighting a pressing need for new therapeutic treatments. Given this scenario, this study sought to evaluate Se-[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl] 4-chlorobenzoselenolate (Se-DMC) potential on a complete Freund's adjuvant (CFA)-induced unilateral arthritis model. The effects of Se-DMC (5 mg/kg; oral dose) and meloxicam (5 mg/kg; oral dose), both administered to animals daily for 14 days, on paw edema, mechanical sensitivity, neurobehavioral deficits (anxiogenic- and depressive-like behaviors), Na+/K+-ATPase activity, oxidative stress, and inflammation were evaluated in male Swiss mice exposed to CFA (intraplantar injection of 0.1 mL; 10 mg/mL). Se-DMC reduced the paw withdrawal threshold and CFA-induced paw edema. Histopathological results revealed the antiedematogenic potential of the compound, which was evidenced by lower quantities of dilated lymphatic vessels compared with the CFA group. Se-DMC reduced mRNA relative expression levels of tumor necrosis factor-α (TNF-α) and nuclear factor-κB (NF-κB) in the hippocampus and paw of CFA mice. The CFA-induced anxiogenic- and depressive-like behaviors were reversed by Se-DMC to the control levels in the elevated plus-maze and tail suspension tests. Se-DMC reduced the paw reactive species levels and restored the superoxide dismutase (hippocampus and paw) and Na+/K+-ATPase (hippocampus) activities previously increased by CFA. Moreover, CFA administration inhibited serum creatinine kinase activity, albeit the Se-DMC effects did not appear to involve the modulation of this enzyme and were equal to or greater than meloxicam. Se-DMC attenuates CFA-induced inflammatory response, nociception, and neurobehavioral deficits in mice.

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.