The Role of Genistein in Mammalian Reproduction.

Genistein is a natural compound belonging to flavonoids, having antioxidant, anti-inflammatory, and anti-neoplastic properties. Genistein is considered a phytoestrogen. As such, genistein can bind estrogen receptors (ERα and ERß), although with a lower affinity than that of estradiol. Despite considerable work, the effects of genistein are not well established yet. This review aims to clarify the role of genistein on female and male reproductive functions in mammals. In females, at a high dose, genistein diminishes the ovarian activity regulating several pathway molecules, such as topoisomerase isoform I and II, protein tyrosine kinases (v-src, Mek-4, ABL, PKC, Syk, EGFR, FGFR), ABC, CFTR, Glut1, Glut4, 5α-reductase, PPAR-γ, mitogen-activated protein kinase A, protein histidine kinase, and recently circulating RNA-miRNA. The effect of genistein on pregnancy is still controversial. In males, genistein exerts an estrogenic effect by inducing testosterone biosynthesis. The interaction of genistein with both natural and synthetic endocrine disruptors has a negative effect on testis function. The positive effect of genistein on sperm quality is still in debate. In conclusion, genistein has a potentially beneficial effect on the mechanisms regulating the reproduction of females and males. However, this is dependent on the dose, the species, the route, and the time of administration.

Identification of Appropriate Endogenous Controls for Circulating miRNA Quantification in Working Dogs under Physiological Stress Conditions.

Cell-free miRNAs, called circulating miRNAs (cmiRNAs), can act in a paracrine manner by facilitating a diversity of signaling mechanisms between cells. Real-time qPCR is the most accepted method for quantifying miRNA expression levels. The use of stable miRNA endogenous control (EC) for qPCR data normalization allows an accurate cross-sample gene expression comparison. The appropriate selection of EC is a crucial step because qPCR data can change drastically when normalization is performed using an unstable versus a stable EC. To find EC cmiRNA with stable expression in search and rescue (SAR) working dogs, we explored the serum miRNome by Next-Generation Sequencing (NGS) at T0 (resting state) and T1 immediately after SAR performance (state of physiologically recovered stress). The cmiRNAs selected in the NGS circulating miRNome as probable ECs were validated by qPCR, and miRNA stability was evaluated using the Delta Ct, BestKeeper, NormFinder, and GeNorm algorithms. Finally, RefFinder was used to rank the stability orders at both T0 and T1 by establishing miR-320 and miR-191 as the best-circulating ECs. We are confident that this study not only provides a helpful result in itself but also an experimental design for selecting the best endogenous controls to normalize gene expression for genes beyond circulating miRNAs.

Interferon Tau (IFNt) and Interferon-Stimulated Genes (ISGs) Expression in Peripheral Blood Leukocytes and Correlation with Circulating Pregnancy-Associated Glycoproteins (PAGs) during Peri-Implantation and Early Pregnancy in Buffalo Cows.

The objective of this study was to analyze interferon-stimulated genes (ISGs) and interferon tau (IFNt) gene expression in peripheral blood leukocytes during the peri-implantation period and until 40 days of pregnancy in buffalo cows. Relationships were also examined between the expression of ISGs and IFNt and pregnancy-associated glycoproteins (PAGs) peripheral plasma concentration. Buffalo cows were synchronized and artificially inseminated (d 0). Blood samples were collected on days 0, 18, 28 and 40 after artificial insemination (AI) for peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) isolation and PAGs radioimmunoassay analysis. The study was carried out on 21 buffalo cows divided ex post into Pregnant (n = 12) and Non-pregnant (n = 9) groups. Steady state levels of OAS1, MX2, ISG15 and IFNt mRNA were measured by RT-qPCR and their estimated marginal means (p < 0.01 for all) were higher in pregnant than non-pregnant buffaloes, both in PBMCs and PMNs. In PBMCs, pairwise comparisons showed that OAS1 and MX2 expressions differed between pregnant and non-pregnant buffaloes on all the days of observation (p < 0.001), while significant differences in ISG15 and IFNt started from day 28 post-AI (p < 0.05). In PMNs, ISG15 expression differed between groups only at days 18 and 28 (p < 0.001), while comparisons were always significant for IFNt (p < 0.05). The expression of all genes, except ISG15 as determined in PMNs, was positively associated with PAGs plasma concentrations (p < 0.05). This work showed a significant increase in ISGs and IFNt expressions in PBMCs and PMNs in buffalo during the peri-implantation period and early pregnancy, and their correlation with PAGs plasma concentration.

The Complex Interaction between P53 and miRNAs Joins New Awareness in Physiological Stress Responses.

This review emphasizes the important role of cross-talk between P53 and microRNAs in physiological stress signaling. P53 responds to stress in a variety of ways ranging from activating survival-promotion pathways to triggering programmed cell death to eliminate damaged cells. In physiological stress generated by any external or internal condition that challenges cell homeostasis, P53 exerts its function as a transcription factor for target genes or by regulating the expression and maturation of a class of small non-coding RNA molecules (miRNAs). The miRNAs control the level of P53 through direct control of P53 or through indirect control of P53 by targeting its regulators (such as MDMs). In turn, P53 controls the expression level of miRNAs targeted by P53 through the regulation of their transcription or biogenesis. This elaborate regulatory scheme emphasizes the relevance of miRNAs in the P53 network and vice versa.

Extracellular circulating miRNAs as stress-related signature to search and rescue dogs.

Our research explores serum extracellular circulating miRNAs (ecmiRNAs) involved in dog stress response immediately after the search and rescue (SAR) of missing people. The experimental plan considers four arduous SAR simulations. The SAR dogs are trained by the Alpine School of the Military Force of Guardia di Finanza (Passo Rolle, Italy). The First SAR Trial analyzed dog serum samples at rest time (T0), and immediately after SAR performance (T1) using the miRNome-wide screening next-generation sequencing (NGS). T1 versus T0 NGS results revealed a different expression level of let-7a and let-7f. Subsequently, in a large sample size including: 1st (n = 6), 2nd (n = 6), 3rd (n = 6), and 4th (n = 4) trials, let-7a and let-7f were validated by qPCR. Bioinformatics analysis with TarBase (v.8) and the Diana-mirPath (v.3) revealed a functional role of let-7a and let-7f in the p53 pathway to restore cellular homeostasis. Let-7a and let-7f, highly expressed at T1, could stop MDMs-p53 inhibition inducing the p53 increase in level. In addition, let-7a and let-7f, via p53 post-transcriptional regulation, buffers p53 transcription spikes. During SAR stress, the possibility of p53 preconditioning could explain the phenomenon of "stress hardening" where the tolerance of particular stress increases after preconditioning.

How Do Avalanche Dogs (and Their Handlers) Cope with Physical Exercise? Heart Rate Changes during Endurance in a Snowy Environment.

This study aimed to assess the heart rate (HR) responses of avalanche SAR dogs and handlers under working field conditions. Thirteen SAR units (dogs and handlers) performed an exercise (Endurance) consisting of approximately 5.5 km of rough tracks through deep snow, at an altitude of 1991-2250 m.a.s.l. The exercise was repeated twice for each of the two different tracks. Both handlers and dogs were equipped with a global positioning satellite/heart rate (GPS/HR) system (Polar®). Multivariable models were used to evaluate the effects of environmental (i.e., gradient, altitude, track, and time) and intrinsic (i.e., speed, repetition, and breed) factors on changes from baseline HR (Δ%HR). The dog's Δ%HR was greater in the flat and uphill compared with downhill, and increased progressively as the speed increased (p < 0.001). Moreover, it rose at altitudes above 2100 m.a.s.l. and peaked after 30 min of the Endurance activity (p < 0.01). These findings indicated that HR monitors could be a valuable tool to contribute to the evaluation of avalanche dogs' fitness in their real working environment. In contrast, the lack of correlation between the dogs' and handlers' HR changes suggests that handlers might not perceive the physical conditions of their dog in real-time. Thus, implementing protocols to monitor avalanche SAR dogs' fitness using a GPS/HR monitoring system could help handlers to tailor the training and workload and to detect the risk factors for physical distress of working dogs.

MicroRNA Dysregulation in Canine Meningioma: RT-qPCR Analysis of Formalin-Fixed Paraffin-Embedded Samples.

MicroRNAs (miRNAs) are small non-coding RNAs that play key roles in tumorigenesis as modulators of cell signaling pathways. miRNA expression has been found to be dysregulated in several human and canine tumors, but data are not yet available on canine meningioma. In this study, we analyzed the expression of 12 miRNAs (i.e. miR-335, miR-200a, miR-98, miR-96, miR-190a, miR-29c, miR-219-5p, miR-155, miR-146a, miR-145, miR-136, miR-451) by RT-qPCR in a series of 41 formalin-fixed, paraffin-embedded canine meningiomas, and normal arachnoid samples. We identified 8 dysregulated miRNAs that might be involved in canine meningioma pathogenesis. Five miRNAs (i.e. miR-96, miR-145, miR-335, miR-200a, miR-29c), were downregulated in tumor samples and 3 (i.e. miR-136, miR-155, miR-146a) were upregulated. Moreover, miR-200a was overexpressed in grade III compared to grade I and grade II meningiomas, suggesting that it might have a dual role in tumor initiation and progression. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses suggest that dysregulated miRNAs might influence cellular processes and pathways mainly involved in tumor cell migration, extracellular matrix interactions, cell proliferation, and inflammatory responses. The characterization of miRNA functions in canine meningiomas is needed to assess their potential clinical utility, also in view of the relevance of the dog as a potential spontaneous animal model of human disease.

The Prophylactic Use of Bovine Colostrum in a Murine Model of TNBS-Induced Colitis.

This study investigated the effects of a short-term administration of bovine colostrum (BC) in a TNBS model of induced colitis. Colitis was induced by TNBS treatment after seven days of BC (BC group, n = 12) or saline (control group, n = 12) administration in mice. Clinical signs, histopathological characteristics, expression levels of Toll-like receptor 4 (TLR4), pro- and anti-inflammatory cytokines, and microbial composition were assessed. BC was well tolerated and did not induce any histological damage or clinical symptoms. After TNBS treatment, the BC group showed a reduction in body weight (BW) loss compared to Control (p < 0.05). Moreover, expression levels of TLR4 (p < 0.01), Interleukin-1ß (IL-1ß; p < 0.001), Interleukin-8 (IL-8; p < 0.001), and Interleukin-10 (IL-10; p < 0.001) were lower in mice administered with BC. Finally, Escherichia coli were higher (p < 0.05), while Enterococci (p < 0.001), Lactobacillus spp. (p < 0.001), and Bifidobacterium spp. (p < 0.05) were lower in Control than BC group. This study confirms that pre-treatment with BC modulates the expression of genes and the count of microbes involved in the etiopathogenesis of colitis.

Beta-lactam resistance in Campylobacter coli and Campylobacter jejuni chicken isolates and the association between blaOXA-61 gene expression and the action of ß-lactamase inhibitors.

The objectives of this work were to evaluate ß-lactamase-mediated ß-lactam resistance in Campylobacter coli and Campylobacter jejuni isolates obtained from broiler chickens, expression of the blaOXA-61 gene in relation to ß-lactamase production, and the possible association between blaOXA-61 gene expression and the action of inhibitors when combined with ß-lactams. All strains were tested by disk diffusion and nitrocefin methods to assess antibiotic susceptibility and ß-lactamase production, respectively. PCR and qPCR amplification were performed to evaluate qualitative and quantitative blaOXA-61 expression. Campylobacter spp. showed a high level of resistance to the most of antimicrobials tested. C. coli strains were ampicillin resistant and blaOXA-61 positive, and 59 out of 60 isolates were positive in the nitrocefin test. Twenty C. jejuni isolates were positive for blaOXA-61 and the nitrocefin test, although two isolates were ampicillin sensitive. Amoxicillin/clavulanic acid and ticarcillin/clavulanic acid do not seem to be active against C. coli, as 73.3 %, and 88.3 % of isolates were resistant to amoxicillin/clavulanic acid and ticarcillin/clavulanic acid, respectively. C. jejuni was not susceptible to amoxicillin/clavulanic acid, with 90 % of the strains showing resistance, whereas ticarcillin associated with clavulanic acid was significantly more efficient than ticarcillin alone (P < 0.01), with 90 % of the strains found to be susceptible. An association between blaOXA-61 expression and amoxicillin/clavulanic acid and ticarcillin/clavulanic acid resistance (P = 0.0001) was seen in C. coli, as well as in C. jejuni for ampicillin/sulbactam (P = 0.0001). Our results suggest that the clavulanic acid only shows an inhibitory effect on C. jejuni when combined with ticarcillin and that the inhibitors action is lower if the blaOXA-61 gene is highly expressed.

Omics Insights into Animal Resilience and Stress Factors.

Resilience is conceived as a dynamic developmental process involving the achievement of positive adaptation within the context of significant adversity. Resilience is not a unique ability but rather a set of capacities of a system put in place to absorb a disturbance and to reorganize while trying to retain the same function, structure, and identity. This review describes the characteristics and the molecular mechanisms of resilience to understand the core elements of resilience and its indicators. The objectives of this review are: (1) to define some of the leading environmental stressors and clarify the mechanism of vulnerability or resilience outcomes; (2) to clarify some of the prominent epigenetic modulations mediating resilience or vulnerability as a stress response; (3) to highlight the neural mechanisms related to stress resilience since the central nervous system is a highly dynamic structure characterized by an everlasting plasticity feature, which therefore has the opportunity to modify resilience. The review aims to introduce the reader to the concept of resilience seen as an ability acquired in life and not only inherited from birth.

Benefits of dietary supplements on the physical fitness of German Shepherd dogs during a drug detection training course.

A high standard of physical fitness is an essential characteristic of drug detection dogs because it affects not only their ability to sustain high activity levels but also their attention and olfaction efficiency. Nutritional supplements could improve physical fitness by modulating energy metabolism, oxidative processes, and perceived fatigue. The aim of this study was to investigate the physiological and biochemical changes induced by submaximal exercise on drug detection dogs (German Shepherd breed) and to assess whether a dietary supplement improves their physical fitness. During a drug detection dog training course, seven dogs were fed with a basal diet (Control Group) for three-month period, while a further seven dogs were fed with a basal diet as well as a daily nutritional supplement containing branched-chain and limiting amino acids, carnitine, vitamins, and octacosanol (Treatment Group). At the end of this period, individual physical fitness was assessed by making each subject take a graded treadmill exercise test. A human heart rate monitor system was used to record the dog's heart rate (HR) during the treadmill exercise and the subsequent recovery period. The parameters related to HR were analysed using nonparametric statistics. Blood samples were collected before starting the nutritional supplement treatment, before and after the treadmill exercise and following recovery. Linear mixed models were used. The dietary supplements accelerated HR recovery, as demonstrated by the lower HR after recovery (P<0.05) and Time constants of HR decay (P<0.05), and by the higher Absolute HR Recovered (P<0.05) recorded in the Treatment group compared with the Control dogs. The supplemented dogs showed the lowest concentrations of creatine kinase (CK; P<0.001), aspartate aminotransferase (AST, P<0.05) and non-esterified fatty acids (NEFA; P<0.01) suggesting a reduction in muscle damage and improvement of energy metabolism. These data suggest that this combined supplement can significantly enhance the physical fitness of drug detection dogs.

Investigation of PAG2 mRNA Expression in Water Buffalo Peripheral Blood Mononuclear Cells and Polymorphonuclear Leukocytes from Maternal Blood at the Peri-Implantation Period.

: The main objective of this study was to assess PAG2 mRNA expression in maternal blood cells at the peri-implantation period in water buffalo; moreover, we wanted to evaluate the earliest time in which PAG-2 could be detected in maternal blood. Thirty-two lactating buffaloes artificially inseminated (AI) were utilized. Blood was collected at Days 0, 14, 18, 28, 40 after AI (AI = day 0). Pregnancy was diagnosed by ultrasound at Days 28 and 40 post AI. Out of 32 buffaloes, 14 were pregnant (P group) and 18 were not pregnant (NP group). The plasma PAG-2 threshold of 1.0 ng/mL in the P group was reached at day 40 post AI. PAG2 mRNA expression differed between the P and NP groups, and was either evaluated in Peripheral Blood Mononuclear Cells (PBMC) or Polymorphonuclear Leukocytes (PMN), starting from day 14. However, both the estimated marginal means and multiple comparisons showed that PAG2 mRNA expression was higher in PMN than PBMC. In the present study, PAG-2 appeared in the blood (40 Days post AI), and an early expression of PAG2 mRNA at Day 14 post AI was also observed. Although further research is undoubtedly required, PAG2 mRNA in peripheral blood leukocytes could be using to better understand the role that PAGs play during pregnancy in buffalo.

Preventive effects of bovine colostrum supplementation in TNBS-induced colitis in mice.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder for which the current medical therapy is not completely effective. Bovine colostrum (BC) is a biological fluid rich in bioactive molecules that may have beneficial effects on several gastrointestinal disorders. The objectives of this study were to assess the preventive effects of BC supplementation in a mouse model of 2,4,6 trinitrobenzene sulfonic acid (TNBS)-induced colitis using a multidisciplinary approach. Specifically, the following parameters were evaluated: (i) disease activity index (DAI), (ii) histological score, (iii) expression levels of TLR4, anti- and pro-inflammatory cytokines, and (iv) count of some bacterial species of the intestinal microbiota. Mice received a daily suspension of BC (BC group, n = 12) or saline solution (control, CN group, n = 12) for 21 days before the intrarectal inoculation with 1% of TNBS solution. BC was well tolerated and did not induce any histological damage or clinical symptoms. After TNBS treatment, BC group showed a reduction of body weight (BW) loss (P<0.01) and histological score (P<0.05) compared to CN. Moreover, the expression levels of TLR4 (P<0.05), IL-1ß (P<0.001), IL-8 (P<0.001), and IL-10 (P<0.001) were lower in mice administered with BC, while the concentrations of TNF-α did not show any differences between groups. Finally, the supplementation with BC resulted in a differential response to TNBS treatment in the bacterial count. In CN group, E. coli and Enterococci increased (P<0.001), while Anaerobes (P<0.01), Lactobacilli, and Bifidobacteria (P<0.001) reduced. Conversely, no significant changes in bacterial load were found after the inoculation of TNBS in BC pre-treated mice. This study confirms that TNBS-induced colitis model in mice is useful for studying the mechanisms involved in IBD pathogenesis and shows that pre-treatment with BC reduces the intestinal damages and clinical signs of the colitis. Molecular mechanisms and intestinal microflora could be involved in the protective effect of colostrum.

My Dog Is Not My Cat: Owner Perception of the Personalities of Dogs and Cats Living in the Same Household.

This study aims to define the personality traits perceived by the owners of multiple pets and to evaluate how they are modulated by experiential-environmental factors. A questionnaire was administered to 1270 owners of multiple pets (dogs and cats) to collect data on the demographics, management, and personality of their pets. Data were analysed by principal component analysis, bivariate, and multivariable models. Five personality traits emerged in dogs and cats: sociability, reactivity, protectiveness, neuroticism, and fearfulness. The owners perceived differences in the personality of their pet: dogs scored higher in sociability, protectiveness, and reactivity, while lower in the neuroticism dimension compared with cats (p < 0.001). Age similarly affected sociability (p < 0.01), and reactivity (p < 0.001) in both dogs and cats, while species-specific gender differences were found as to fearfulness (p < 0.05) and neuroticism (p < 0.001). The age of acquisition modulated several traits in dog personality, while living with conspecifics especially influenced cats. Physiological, behavioural, and evolutionary characteristics could explain species differences. Moreover, intrinsic and extrinsic factors modulated the five dimensions of dogs and cats in a diversified fashion, suggesting complex interactions between species and the environment. However, owners could have had different attitudes with their animals which could have influenced personality perception.

Next Generation Sequencing of urine exfoliated cells: an approach of prostate cancer microRNAs research.

There is emerging evidence that microRNAs (miRNAs) dysregulation is involved in the genesis and the progression of Prostate Cancer (PCa), thus potentially increasing their use in urological clinical practice. This is the first pilot study which utilizes Illumina Deep Sequencing to examine the entire miRNAs spectrum existent in urine exfoliated prostate cells (UEPCs) of PCa patients. A total of 11 male patients with histological diagnosis of PCa were enrolled in the present study. First-catch urine (30 mL) was collected following a prostate massage. Total RNA was extracted from urine and sequenced using an HiSeq2500 System (Illumina). QPCR assay was used to validate the highest NGS results in PCA patients and in age-matched, caucasian men. Remarkably, PCA let-7 family was down-regulated (P < 0.01), compared to the controls. The results of our study support the notion of a relatively high diagnostic value of miRNA family for PCa detection, especially in the let-7 family. The present research confirmed the potential use of miRNAs as non-invasive biomarkers in the diagnosis of PCa, potentially reducing the invasiveness of actual clinical strategy.

Effects of local lipopolysaccharide administration on the expression of Toll-like receptor 4 and pro-inflammatory cytokines in uterus and oviduct of rabbit does.

Inflammation of the uterus and oviduct is associated with reduced reproductive performance in humans and domestic animals. Toll-like receptors are expressed in various immune and non-immune cells and play a crucial role in innate immunity. Toll-like receptor - 4 (TLR4) can detect lipopolysaccharide (LPS) from Gram-negative bacteria leading to the secretion of pro-inflammatory cytokines, chemokines, antimicrobial peptides and other inflammatory mediators. To investigate the effects of a local inflammation on the expression levels of TLR4 and pro-inflammatory cytokines, 12 female rabbits received an intracervical infusion with either saline solution endotoxin-free (carrier, 2 mL; n = 6) or LPS (500 µg diluted in 2 mL of saline solution; n = 6). Blood samples were performed at 0, 30, 60 and 90 min and 2,4,6 and 24 h after treatment to evaluate interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) plasma concentrations. Animals were sacrificed 24 h post-treatment. The uterus and oviducts were immediately collected. The gene expression and protein levels of TLR4 and pro-inflammatory cytokines were detected by quantitative real-time PCR (qRT-PCR) and immuno-histochemical assay, respectively. Our study showed that the intracervical administration of LPS induced local inflammation given that the animals showed no clinical signs, the histological samples revealed signs of inflammation and plasma levels of pro-inflammatory cytokines were unchanged compared to the control. LPS produced an increase in the TLR4 mRNA expression levels in the uterus with respect to the control (P < 0.05). In LPS-treated rabbits the gene expression of IL-1ß was higher in the uterus and oviducts and TNF-α only in the oviduct (P < 0.05) as compared to the control. The immuno-histochemical assay showed that TLR4, IL-1ß and TNF-α were expressed in the reproductive tissues of the rabbit. Moreover, after the LPS stimulation the stromal cells of the uterus exhibited a higher staining for TLR4 (P < 0.05) and the epithelial cells of the oviduct for TNF-α and IL-1ß (P < 0.05) with respect to the control. These results suggest that (1) TRL4, IL-1ß and TNF-α are expressed in uterus and oviducts of the doe, and (2) LPS up-regulates the gene and protein expression of TLR4 and pro-inflammatory cytokines in uterus and oviducts. Therefore, the rabbit could be a useful animal model for studying the local mechanisms involved in reproductive dysfunctions caused by subclinical infections.

Gold nanoparticles approach to detect chondroitin sulphate and hyaluronic acid urothelial coating.

This study investigated the location of hyaluronic acid (HA)- and chondroitin sulphate (CS)-coated gold nanoparticles in rabbit bladder and evaluated gene expression of CD44, RHAMM and ICAM-1 receptors involved in HA and CS transport into the cell. Gold nanoparticles were synthesised by reduction of gold salts with HA or CS to form HA-AuNPs and CS-AuNPs. Bladder samples were incubated with CS-AuNPs and HA-AuNPs or without glycosaminoglycans. Transmission electron microscopy, optic microscopy and scanning electron microscopy were used to determine the location of the synthesised AuNPs. Real-time PCR was used to analyse expression of urothelial cell receptors CD44, RHAMM, ICAM-1, after ex vivo administration of CS-AuNPs and HA-AuNPs. We showed that HA-AuNPs and CS-AuNPs were located in the cytoplasm and tight junctions of urothelial umbrella cells; this appearance was absent in untreated bladders. There were no significant differences in gene expression levels for CD44, RHAMM and ICAM-1 receptors in treated versus control bladder tissues. In conclusion, we clearly showed the presence of exogenous GAGs in the bladder surface and the tight junctions between umbrella cells, which is important in the regeneration pathway of the urothelium. The GAGs-AuNPs offer a promising approach to understanding the biophysical properties and imaging of urothelial tissue.

Serum microRNAs in buffalo cows: Potential biomarkers of pregnancy.

MiRNAs (microRNA) constitute a large family of single-stranded, non-coding small RNAs. Although the functions and target genes of most miRNAs are still unknown, it has been well established that they are involved in embryogenesis, organogenesis and neonatal birth. In recent decades, interest in buffalo breeding has largely increased worldwide thus focusing the attention on this species as a dairy purpose animal. Problems related to long calving interval, late puberty and seasonal anestrus hamper reproductive efficiency in this species. Early pregnancy diagnosis is important to shorten the calving interval and increase lifetime production on dairy animals. MicroRNAs have recently emerged as key molecules in fertilization of several species even though in buffalo, few previous studies have investigated miRNAs. The aim of this research was to identify the best miRNA reference in serum among miR-191, miR-25-3p, SNORD44, and SNORD48. Consequently, assess the expression levels of miR-103, miR-200b, miR-301a, miR-423-5p, miR-375, miR-451 and miR-452 involved in buffalo progesterone-maturation oocyte and pregnancy. Interestingly, we found that all the miRNAs analyzed at 40days after artificial insemination were differentially expressed among pregnant and non-pregnant buffalo. Our research may be a first step for gain further insight in the biological function of circulating miRNAs in this species.

Different levels of serum microRNAs in prostate cancer and benign prostatic hyperplasia: evaluation of potential diagnostic and prognostic role.

INTRODUCTION: Diagnosis of prostate cancer (PCa) is based on prostate biopsy that is performed when prostate specific antigen (PSA) is persistently altered over time and/or abnormal digital rectal examination is found. Serum PSA levels increase in both PCa and benign prostatic hyperplasia, leading to an increased number of unnecessary biopsies. There is an urgent need to unravel PCa-specific molecular signatures. PATIENTS AND METHODS: This study aimed at characterizing a panel of circulating micro RNAs (miRNAs) that could distinguish PCa from benign prostatic hyperplasia in a population of age-matched patients with increased PSA levels. Both miRNAs targeting genes involved in PCa onset and miRNAs whose role in PCa has been highlighted in other studies were included. For this purpose, let-7c, let-7e, let-7i, miR-26a-5p, miR-26b-5p, miR-24-3p, miR-23b-3p, miR-27-b-3p, miR-106a-5p, miR-20b-5p, miR-18b-5p, miR-19b-2-5p, miR-363-3p, miR-497, miR-195, miR-25-3p, miR-30c-5p, miR-622, miR-874-3p, miR-346 and miR-940 were assayed through real-time PCR in 64 patients with PCa and compared with 60 patients with benign prostatic hyperplasia. The ability of miRNAs to predict the stage of disease was also analyzed. RESULTS: Let-7c, let-7e, let-7i, miR-26a-5p, miR-26b-5p, miR-18b-5p and miR-25-3p were able to discriminate patients with PCa from those harboring benign prostatic hyperplasia, both presenting altered PSA levels (>3 ng/mL). MiR-25-3p and miR-18b-5p showed the highest sensitivity and specificity to predict PCa, respectively. The combination of these two miRNAs improved the overall sensitivity. A correlation between pathological Gleason score and miRNA expression levels was reported; miR-363-3p, miR-26a-5p, miR-26b-5p, miR-106a-5p, miR-18b-5p, miR-25-3p and let-7i decreased in expression concomitantly with an increase in malignancy. CONCLUSION: This study confirms serum miRNAs to be reliable candidates for the development of minimally invasive biomarkers for the diagnosis and prognosis of PCa, particularly in those cases where PSA acts as a flawed marker.