The role of the orexin (hypocretin) system in controlling energy homeostasis, endocrine system, and reproduction in pigs and other living organisms.

Orexins A (OXA) and B (OXB) (hypocretin 1 and 2) are neuropeptides produced in the brain and peripheral tissues. Biological activities of orexins are mediated through activation of two G-protein coupled receptors termed as orexin 1 receptor (OX1R) and orexin 2 receptor (OX1R). Orexin system (OXA, OXB, OX1R, OX2R) was implicated in controlling sleep, energy expenditure, appetite, reproduction as well as metabolism and energy homeostasis. In this review, we summarize the current knowledge regarding the role of the orexin system in controlling porcine physiology. Particularly, we review and discuss evidence indicating that in pig and other living organisms, orexins and their receptors modulate the energy homeostasis, reproduction as well as functions of peripheral tissues including the pancreas, adrenal glands, gastro-intestinal tract and adipose tissue.

Adiponectin affects uterine steroidogenesis during early pregnancy and the oestrous cycle: An in vitro study.

Reproduction in females is an energetically demanding process. We assumed that adiponectin (ADPN), known for its role in energy balance maintenance, is also engaged in the regulation of uterine steroidogenesis in the pig. We determined the impact of ADPN alone or in combination with insulin (INS) on testosterone (T), estrone (E1) and estradiol (E2) secretion by porcine endometrium and myometrium, uterine expression of CYP17A1 and CYP19A3 genes, and endometrial abundance of P450C17 and P450AROM proteins during the peri-implantation period and the oestrous cycle, using radioimmunoassay, qPCR, and Western Blot, respectively. During pregnancy, in the endometrial explants from days 10-11, ADPN decreased CYP17A1 gene expression, P450C17 protein abundance and T secretion, whereas increased E1 secretion. On days 12-13 of pregnancy, ADPN decreased CYP17A1 and CYP19A3 expression, P450C17 and P450AROM protein abundance and E1 secretion, but stimulated T secretion. On days 15-16 of pregnancy, ADPN decreased P450C17 protein accumulation but enhanced CYP19A3 expression and E1 secretion. On days 27-28 of pregnancy, ADPN increased CYP17A1 and CYP19A3 mRNA content and T secretion in this tissue and decreased P450C17 content. ADPN effect on myometrial explants was dependent on stage of gestation or oestrous cycle. Moreover, INS treatment modulated basal and ADPN-affected steroidogenic enzymes gene and protein expression and steroids secretion. The results obtained indicate that ADPN may affect processes required for successful implantation such as steroidogenesis. ADPN and INS were also shown to modulate each other action, which indicates that the proper course of uterine steroidogenesis may be dependent on both hormones' interaction.

The expression of chemerin and its receptors (CMKLR1, GPR1, CCRL2) in the porcine uterus during the oestrous cycle and early pregnancy and in trophoblasts and conceptuses.

Recent research has demonstrated that chemerin may take part in the regulation of reproduction. The aim of this study was to determine the expression of chemerin system - chemerin and its receptors, chemokine-like receptor 1 (CMKLR1), G protein-coupled receptor 1 (GPR1) and C-C chemokine receptor-like 2 (CCRL2) - in the porcine uterus during the oestrous cycle and early pregnancy, and in trophoblasts and conceptuses by real-time PCR and western blotting. Chemerin concentrations in uterine luminal flushings (ULF) were determined using ELISA test. In the endometrium, the highest expression of chemerin and GPR1 proteins was observed during the mid-luteal phase; CMKLR1, during the late luteal phase; and CCRL2, during the follicular phase of the cycle. In the myometrium, chemerin protein expression was enhanced during the early luteal phase, and chemerin receptor proteins were highly expressed during the follicular phase. In the endometrium of pregnant pigs, the highest expression of chemerin and CCRL2 protein was observed during implantation; CMKLR1, during placentation; and GPR1, during embryo migration. In the myometrium, chemerin and CCRL2 protein expression increased at the end of implantation, and the expression of CMKLR1 and GPR1 protein was enhanced during implantation. In the conceptuses and trophoblasts, the highest expression of chemerin system proteins was observed during placentation, with the exception of GPR1 protein in the trophoblasts. The highest concentrations of the analysed adipokine were observed in ULF during the luteal phase of the cycle and during maternal recognition of pregnancy. This is the first study to demonstrate that the expression of the chemerin system in the porcine uterus, conceptuses and trophoblasts, and chemerin concentrations in ULF are influenced by the hormonal milieu in different stages of the oestrous cycle and in early pregnancy. The present results also suggest that chemerin is implicated in the regulation of reproductive functions in pigs.

Ex vivo nail infection as an effective preclinical method for screening of new topical antifungals.

Onychomycosis are fungal nail infections comprising of about 50% of onychopathies and are commonly caused by dermatophytes. The treatment of this dermatomycosis requires a long period of time and is associated with high rates of recurrence. In view of the need to evaluate the antifungal performance of promising preclinical compounds, we developed, in this study, a practical and accessibleex vivo model for establishing a Trichophyton rubrum onychomycosis framework using porcine hooves. This model has as its main advantage the similar structural and three-dimensional characteristics that the porcine hooves have with the human nail. The proposed model allowed to evaluate the antifungal activity of a new antifungal compound and a reference drug (terbinafine), both already incorporated into a nail lacquer for topical use. Treatments with compound 3-selenocyanate-indole (Se4a) and with terbinafine incorporated into this nail lacquer completely inhibited fungal growth, corresponding to the profile of in vitro activity observed against T. rubrum. This study concludes that the ex vivo porcine hoof model is an effective alternative method for preclinical screening of drugs or new topical compounds developed to combat onychomycosis. Further studies are needed to compare the permeability of porcine hooves with human nails permeability.

The effect of estrone and estradiol on the expression of the orexin/hypocretin system in the porcine uterus during early pregnancy.

Orexin A and B (OXA, OXB) are hypothalamic neuropeptides acting via two receptors, type 1 (OX1R) and 2 (OX2R). Orexins, also known as hypocretins, take part in a common endocrine system regulating metabolism and reproductive functions. Changes in the orexin system expression during the estrous cycle and pregnancy suggest dependence on the local hormonal milieu. Estrogens are the key hormones controlling reproductive functions, including maternal recognition of pregnancy and implantation. We hypothesize that estrogens may affect orexin system expression in the early pregnant uterus. The aim of this study was to investigate the influence of estrogens on prepro-orexin (PPO), OX1R, and OX2R gene expression, OX1R and OX2R protein content in the porcine uterine tissue, as well as OXA and OXB secretion on days 10-11, 12-13, 15-16, and 27-28 of pregnancy and on days 10-12 of the estrous cycle (n = 5 per group). The expression of PPO, OX1R, and OX2R genes was examined using qPCR, OX1R and OX2R protein content was evaluated using western blotting, and orexins secretion was determined with ELISA. This is the first study to describe the influence of estrogens on orexin system expression in the porcine uterus. Obtained results revealed that estrogens significantly affect the expression of orexin system and orexins secretion. The influence of estrogens varied between different stages of early pregnancy and the estrous cycle. The steroids showed a tissue-specific and dose-dependent effect. Our findings suggest that orexins could act as a "molecular switch" for estrogen activation in the processes of endometrial decidualization and rapid uterine enlargement during early pregnancy.

Impact of Factors that Affect Reading Skill Level on King-Devick Baseline Performance Time.

The King-Devick (K-D) test is often used as part of a multimodal assessment to screen for sport-related concussion. However, the test involves reading numbers, and little is known about variation in baseline performance on the K-D by reading skill level. We conducted a cross-sectional study analyzing data from the Concussion Assessment, Research and Education (CARE) Consortium to assess differences in baseline performance on the K-D associated with factors that impact reading skill level (learning disorder [LD] and primary home language other than English [PHLOTE]), while controlling for covariates (gender, type of sport, attentional issues, history of concussion and modality of administration). We had a sample of 2311 student-athletes (47% female), and multivariate regression indicated an average K-D performance time of 40.4 s. Presence of LD was associated with a 3.3 s slower K-D time (95% CI 1.9-4.7, p < 0.001), and PHLOTE was associated with a 2.6 s slower K-D time (95% CI 1.2-4.0, p < 0.001), after controlling for other covariates. These results suggest caution in the use of normative data with the K-D. Future studies should explore the impact of factors associated with reading skill level on sensitivity of the K-D in detecting concussion.

Health-Related Quality of Life Following Concussion in Collegiate Student-Athletes With and Without Concussion History.

The purpose of this study was to compare global and specific health-related quality of life (HRQOL) throughout concussion recovery between those with and without concussion history. Student-athletes diagnosed with concussion completed global (Short Form-12v2; SF-12) and specific (Hospital Anxiety and Depression Scale: HADS) HRQOL assessments at baseline, 24-48 h, asymptomatic, return-to-play, and 6-months post-injury. Baseline scores were compared to post-injury time points for SF-12 subscores (physical and mental; PCS-12, MCS-12) and HADS subscores (depression and anxiety; HADS-D, HADS-A). We conducted a 2 × 5 mixed model ANOVA for group (with and without concussion history) and time (four post-injury assessments compared to baseline). We did not observe interaction or main effects for group, except those with concussion history had worse HADS-D subscores than those without concussion history. PCS-12 subscores were worse at 24-48 h, asymptomatic, and return-to-play compared to baseline, but returned to baseline 6-months post-injury. MCS-12 subscores did not differ at any time points. HADS-D subscores worsened 24-48 h post-injury, but improved for additional assessments compared to baseline. HADS-A improved post-injury compared to baseline at asymptomatic, return-to-play, and 6-month assessments, but was similar to baseline 24-48 h post-injury. HRQOL physical aspects slightly worsened post-injury and restored to baseline after returning to play.

New insight into organic anion transporters from the perspective of potentially important interactions and drugs toxicity.

The family of organic anion transporters (OATs) includes a group of over 10 transmembrane transporting proteins belonging to the solute carrier 22 subfamilies of the major facilitator superfamily. Their function is related to the transport of a great variety of organic anions against the electrical and chemical gradient. OATs are present in most types of human tissues, including the kidneys, liver, placenta, olfactory epithelium, retina, and choroid plexus tissues. The OATs family plays an important role in the cellular uptake, distribution, excretion, and detoxification of many water-soluble drugs, endogenous compounds, nutrition ingredients, environmental contaminants and toxins, and significantly impacts their efficacy, pharmacokinetics and toxicity, both in a preferable and unfavorable way. OATs demonstrated great potential to participate in many potentially relevant interactions, which may lead to unexpected, but not always detrimental, effects. Wider knowledge about their specific functions in the body, role in disease states, pharmacokinetics interactions, and intraindividual response to therapeutic treatment will allow to predict and prevent OAT-related adverse effects or use favorable interactions in pharmacotherapy, as well as to rationally design therapeutics targeted at individual transporter drugs with improved bioavailability, prolonged half-life or reduced toxicity, and improve safety guidelines concerning drug dosage. This review gathers recent reports regarding OAT-related essential interactions involving components of popular therapeutic herbal products, dietary supplements, and clinically important drugs, their significance and potential suitability in modulating the severity of drug-related side effects and toxicity mechanisms.

Effect of orexin B on CYP17A1 and CYP19A3 expression and oestradiol, oestrone and testosterone secretion in the porcine uterus during early pregnancy and the oestrous cycle.

Orexin A (OXA) and B (OXB) are hypothalamic neuropeptides identified as regulators of food intake, energy homoeostasis, sleep-wake cycle and arousal. They also create an integrative link between energy homoeostasis and reproduction. Although their functions in the ovaries and testes have been partially explored, to date, less attention has been focused on the role of the peptides in the uterus. The aim of this study was to investigate the effect of one of orexins - orexin B on oestradiol (E2), oestrone (E1) and testosterone (T) secretion by porcine endometrial and myometrial slices as well as the gene expression of key steroidogenic enzymes responsible for steroid production (CYP17A1, CYP19A3) during the luteal phase of the oestrous cycle (days 10 to 11) and early pregnancy (days 10 to 11, 12 to 13, 15 to 16, 27 to 28). Orexin B suppressed E2 secretion by endometrial slices on days 10 to 11 and 15 to 16 of pregnancy, and days 10 to 11 of the cycle. In the myometrium, OXB inhibited E2 production on days 10 to 11 of pregnancy, whereas on days 12 to 13 it enhanced steroid output. Endometrial E1 release was potentiated by the peptide during all studied periods of the cycle and pregnancy, with the exception of days 12 to 13, when an inhibitory effect was observed. Myometrial secretion of E1 was increased, except on days 27 to 28. Testosterone secretion by endometrial slices was increased on days 12 to 13 and 27 to 28 of pregnancy. On days 10 to 11 of the cycle, T release was stimulated in response to the lowest and decreased under the influence of the highest dose of OXB. In the myometrium, T production was inhibited by OXB on days 10 to 11 of pregnancy and during the corresponding period of the cycle. On days 27 to 28 of pregnancy, T release was potentiated by the lowest dose of OXB. Expression of both genes was modified by OXB depending on the period of pregnancy and the type of examined uterine tissues. Our findings suggest that OXB, through modulation of uterine steroidogenesis, may have a regulatory role in the uterus.

Controlled droplet microfluidic systems for multistep chemical and biological assays.

Droplet microfluidics is a relatively new and rapidly evolving field of science focused on studying the hydrodynamics and properties of biphasic flows at the microscale, and on the development of systems for practical applications in chemistry, biology and materials science. Microdroplets present several unique characteristics of interest to a broader research community. The main distinguishing features include (i) large numbers of isolated compartments of tiny volumes that are ideal for single cell or single molecule assays, (ii) rapid mixing and negligible thermal inertia that all provide excellent control over reaction conditions, and (iii) the presence of two immiscible liquids and the interface between them that enables new or exotic processes (the synthesis of new functional materials and structures that are otherwise difficult to obtain, studies of the functions and properties of lipid and polymer membranes and execution of reactions at liquid-liquid interfaces). The most frequent application of droplet microfluidics relies on the generation of large numbers of compartments either for ultrahigh throughput screens or for the synthesis of functional materials composed of millions of droplets or particles. Droplet microfluidics has already evolved into a complex field. In this review we focus on 'controlled droplet microfluidics' - a portfolio of techniques that provide convenient platforms for multistep complex reaction protocols and that take advantage of automated and passive methods of fluid handling on a chip. 'Controlled droplet microfluidics' can be regarded as a group of methods capable of addressing and manipulating droplets in series. The functionality and complexity of controlled droplet microfluidic systems can be positioned between digital microfluidics (DMF) addressing each droplet individually using 2D arrays of electrodes and ultrahigh throughput droplet microfluidics focused on the generation of hundreds of thousands or even millions of picoliter droplets that cannot be individually addressed by their location on a chip.

A precise and accurate microfluidic droplet dilutor.

We demonstrate a microfluidic system for the precise (coefficient of variance between repetitions below 4%) and highly accurate (average difference from two-fold dilution below 1%) serial dilution of solutions inside droplets with a volume of ca. 1 µl. The two-fold dilution series can be prepared with the correlation coefficient as high as R2 = 0.999. The technique that we here describe uses hydrodynamic traps to precisely meter every droplet used in subsequent dilutions. We use only one metering trap to meter each and every droplet involved in the process of preparation of the dilution series. This eliminates the error of metering that would arise from the finite fidelity of fabrication of multiple metering traps. Metering every droplet at the same trap provides for high reproducibility of the volumes of the droplets, and thus high reproducibility of dilutions. We also present a device and method to precisely and accurately dilute one substance and simultaneously maintain the concentration of another substance throughout the dilution series without mixing their stock solutions. We compare the here-described precise and accurate dilution systems with a simple microdroplet dilutor that comprises several traps - each trap for a subsequent dilution. We describe the effect of producing more reproducible dilutions in a simple microdroplet dilutor thanks to the application of an alternating electric field.

Optimized droplet digital CFU assay (ddCFU) provides precise quantification of bacteria over a dynamic range of 6 logs and beyond.

Standard digital assays need a large number of compartments for precise quantification of a sample over a broad dynamic range. We address this issue with an optimized droplet digital approach that uses a drastically reduced number of compartments for quantification. We generate serial logarithmic dilutions of an initial bacterial sample as an array of microliter-sized droplet plugs. In a subsequent step, these droplets are split into libraries of nanoliter droplets and pooled together for incubation and analysis. We show that our technology is at par with traditional dilution plate count for quantification of bacteria, but has the advantage of simplifying the experimental setup and reducing the manual workload. The method also has the potential to reduce the assay time significantly.

A passive microfluidic system based on step emulsification allows the generation of libraries of nanoliter-sized droplets from microliter droplets of varying and known concentrations of a sample.

We present a novel geometry of microfluidic channels that allows us to passively generate monodisperse emulsions of hundreds of droplets smaller than 1 nL from collections of larger (ca. 0.4 µL) mother droplets. We introduce a new microfluidic module for the generation of droplets via passive break-up at a step. The module alleviates a common problem in step emulsification with efficient removal of the droplets from the vicinity of the step. In our solution, the droplets are pushed away from the step by a continuous liquid that bypasses the mother droplets via specially engineered bypasses that lead to the step around the main channel. We show that the bypasses tighten the distribution of volume of daughter droplets and eliminate subpopulations of daughter droplets. Clearing away the just produced droplets from the vicinity of the step provides for similar conditions of break-up for every subsequent droplet and, consequently, leads to superior monodispersity of the generated emulsions. Importantly, this function is realized autonomously (passively) in a protocol in which only a sequence of large mother droplets is forced through the module. Our system features the advantage of step emulsification systems in that the volumes of the generated droplets depend very weakly on the rate of flow through the module - an increase in the flow rate by 300% causes only a slight increase of the average diameter of generated droplets by less than 5%. We combined our geometry with a simple T-junction and a simple trap-based microdroplet dilutor to produce a collection of libraries of droplets of gradually changing and known concentrations of a sample. The microfluidic system can be operated with only two syringe pumps set at constant rates of flow during the experiment.

Effect of progesterone on adiponectin system in the porcine uterus during early pregnancy.

The aim of this study was to investigate the influence of progesterone (P4) on adiponectin system genes and protein expression in the endometrium and myometrium during early gestation. Twenty-five gilts were assigned to 1 of 5 groups ( = 5): d 10 to 11 (embryo migration), 12 to 13 (maternal recognition of pregnancy), 15 to 16 (implantation), and 27 to 28 (end of implantation) of pregnancy and d 10 to 11 of the cycle (fully active corpora lutea, corresponding to the corpora lutea activity during gestation). The endometrial and myometrial tissues were cut into 100 mg slices, treated with P4 (10, 100, 1000 nM) and incubated for 24 h. Gene expression was analyzed by the real-time PCR method. Adiponectin secretion was determined by ELISA. Receptor protein content was defined using Western Blot analysis. In the endometrium, on d 10 to 11 of pregnancy, P4 stimulated adiponectin protein secretion. On those days, P4 enhanced adiponectin receptor type 1 () and type 2 () gene expression but inhibited both receptors' protein content. On d 12 to 13 of pregnancy, P4 inhibited adiponectin gene expression. During those period, P4 enhanced gene expression but suppressed both receptors' protein content. On d 15 to 16 of gestation, P4 increased adiponectin gene expression but inhibited the protein secretion. During those days, P4 suppressed gene expression and enhanced AdipoR2 protein content. On d 27 to 28 of gestation, P4 enhanced gene and AdipoR1 protein expression ( < 0.05). In the myometrium, on d 10 to 11 of gestation, P4 increased both receptors' gene expression but suppressed their protein content. On d 12 to 13 of pregnancy, P4 increased adiponectin and genes and AdipoR1 protein expression but decreased AdipoR2 protein content. On d 15 to 16 of gestation, P4 inhibited adiponectin gene expression. On those days, P4 enhanced gene and protein expression. On d 27 to 28 of gestation, P4 decreased adiponectin gene expression. On those days, P4 increased the myometrial AdipoR2 protein concentration and decreased gene protein expression ( < 0.05). Overall, the influence of P4 was found to be tissue specific and dose dependent. Results presented in this study indicate the modulatory effect of P4 on adiponectin system in the porcine uterus during early pregnancy, which may suggest the involvement of this adipokine in the early pregnancy establishment.

Validation and calibration of HeadCount, a self-report measure for quantifying heading exposure in soccer players.

The long-term effects of repetitive head impacts due to heading are an area of increasing concern, and exposure must be accurately measured; however, the validity of self-report of cumulative soccer heading is not known. In order to validate HeadCount, a 2-week recall questionnaire, the number of player-reported headers was compared to the number of headers observed by trained raters for a men's and a women's collegiate soccer teams during an entire season of competitive play using Spearman's correlations and intraclass correlation coefficients (ICCs), and calibrated using a generalized estimating equation. The average Spearman's rho was 0.85 for men and 0.79 for women. The average ICC was 0.75 in men and 0.38 in women. The calibration analysis demonstrated that men tend to report heading accurately while women tend to overestimate. HeadCount is a valid instrument for tracking heading behaviour, but may have to be calibrated in women.

Effect of adiponectin on the steroidogenic acute regulatory protein, P450 side chain cleavage enzyme and 3ß-hydroxysteroid dehydrogenase gene expression, progesterone and androstenedione production by the porcine uterus during early pregnancy.

Adiponectin and its receptors are expressed in the human and porcine uterus and this endocrine system has important role in the regulation of reproductive processes. The expression of steroidogenic acute regulatory protein (StAR) and 3ß-hydroxysteroid dehydrogenase (HSD3B1) were observed in the human and porcine uterus during the oestrous cycle and pregnancy. The de novo synthesis of steroids in the uterus might be a crucial factor for effective implantation and maintenance of pregnancy. We hypothesized that adiponectin modulates the expression of key enzymes in the synthesis of the steroids: StAR, P450 side chain cleavage enzyme (CYP11A1) and HSD3B1, as well as progesterone (P4) and androstenedione (A4) secretion by the porcine uterus. Endometrial and myometrial explants harvested from gilts (n = 5) on days 10 to 11, 12 to 13, 15 to 16 and 27 to 28 of pregnancy and on days 10 to 11 of the oestrous cycle were cultured in vitro in the presence of adiponectin (1, 10 µg/ml), adiponectin with insulin (10 ng/ml) and insulin alone (10 ng/ml). Gene expression was examined by real-time PCR, and the secretion of the steroids was determined by radioimmunoassay. The content of StAR, CYP11A1 and HSD3B1 mRNAs and the secretion of P4 and A4 was modulated by adiponectin in endometrial and myometrial tissue explants during early pregnancy and the oestrous cycle. In this action adiponectin interacted with insulin. Insulin itself also regulated the steroidogenic activity of the porcine uterus. ere we reported, for the first time, the expression of CYP11A1 genes in the porcine endometrium and myometrium. Our novel findings indicate that adiponectin affects basal and insulin-stimulated expression of key steroidogenic genes and production of steroid hormones by the porcine uterus during maternal recognition of pregnancy and implantation.

FOXO1 is a TXN- and p300-dependent sensor and effector of oxidative stress in diffuse large B-cell lymphomas characterized by increased oxidative metabolism.

Molecular profiling has led to identification of subtypes of diffuse large B-cell lymphomas (DLBCLs) differing in terms of oncogenic signaling and metabolic programs. The OxPhos-DLBCL subtype is characterized by enhanced mitochondrial oxidative phosphorylation. As increased oxidative metabolism leads to overproduction of potentially toxic reactive oxygen species (ROS), we sought to identify mechanisms responsible for adaptation of OxPhos cells to these conditions. Herein, we describe a mechanism involving the FOXO1-TXN-p300 redox-dependent circuit protecting OxPhos-DLBCL cells from ROS toxicity. We identify a BCL6-dependent transcriptional mechanism leading to relative TXN overexpression in OxPhos cells. We found that OxPhos cells lacking TXN were uniformly more sensitive to ROS and doxorubicin than control cells. Consistent with this, the overall survival of patients with high TXN mRNA expression, treated with doxorubicin-containing regimens, is significantly shorter than of those with low TXN mRNA expression. TXN overexpression curtails p300-mediated FOXO1 acetylation and its nuclear translocation in response to oxidative stress, thus attenuating FOXO1 transcriptional activity toward genes involved in apoptosis and cell cycle inhibition. We also demonstrate that FOXO1 knockdown in cells with silenced TXN expression markedly reduces ROS-induced apoptosis, indicating that FOXO1 is the major sensor and effector of oxidative stress in OxPhos-DLBCLs. These data highlight dynamic, context-dependent modulation of FOXO1 tumor-suppressor functions via acetylation and reveal potentially targetable vulnerabilities in these DLBCLs.

Role of xanthine oxidoreductase in the anti-thrombotic effects of nitrite in rats in vivo.

The mechanisms underlying nitrite-induced effects on thrombosis and hemostasis in vivo are not clear. The goal of the work described here was to investigate the role of xanthine oxidoreductase (XOR) in the anti-platelet and anti-thrombotic activities of nitrite in rats in vivo. Arterial thrombosis was induced electrically in rats with renovascular hypertension by partial ligation of the left renal artery. Sodium nitrite (NaNO2, 0.17 mmol/kg twice daily for 3 days, p.o) was administered with or without one of the XOR-inhibitors: allopurinol (ALLO) and febuxostat (FEB) (100 and 5 mg/kg, p.o., for 3 days). Nitrite treatment (0.17 mmol/kg), which was associated with a significant increase in NOHb, nitrite/nitrate plasma concentration, resulted in a substantial decrease in thrombus weight (TW) (0.48 ± 0.03 mg vs. vehicle [VEH] 0.88 ± 0.08 mg, p < 0.001) without a significant hypotensive effect. The anti-thrombotic effect of nitrite was partially reversed by FEB (TW = 0.63 ± 0.06 mg, p < 0.05 vs. nitrites), but not by ALLO (TW = 0.43 ± 0.02 mg). In turn, profound anti-platelet effect of nitrite measured ex vivo using collagen-induced whole-blood platelet aggregation (70.5 ± 7.1% vs. VEH 100 ± 4.5%, p < 0.05) and dynamic thromboxaneB2 generation was fully reversed by both XOR-inhibitors. In addition, nitrite decreased plasminogen activator inhibitor-1 concentration (0.47 ± 0.13 ng/ml vs. VEH 0.62 ± 0.04 ng/ml, p < 0.05) and FEB/ALLO reversed this effect. In vitro the anti-platelet effect of nitrite (1 mM) was reversed by FEB (0.1 mM) under hypoxia (0.5%O2) and normoxia (20%O2). Nitrite treatment had no effect on coagulation parameters. In conclusion, the nitrite-induced anti-platelet effect in rats in vivo is mediated by XOR, but XOR does not fully account for the anti-thrombotic effects of nitrite.

Expression of the orexin system in the porcine uterus, conceptus and trophoblast during early pregnancy.

Orexin A and B are hypothalamic peptides derived from the prepro-orexin (PPO) precursor. Orexins stimulate food intake and arousal. Those peptides bind and activate two G protein-coupled receptors: orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). Numerous authors have suggested that orexins play an important role in the regulation of the reproductive functions. The objective of the present study was to analyse the presence of and changes in the gene and protein expression pattern of the orexin system in the porcine uterus, conceptus and trophoblast (chorioallantois) during early pregnancy. In the endometrium, the highest PPO and OX1R gene expression was detected on days 15 to 16 of gestation. The OX2R mRNA content in the endometrium was higher on days 10 to 11 and 15 to 16 than on days 12 to 13 and 27 to 28. In the trophoblasts, PPO gene expression was higher on days 30 to 32 than on days 27 to 28. The highest PPO protein content in the endometrium was noted on days 12 to 13. The highest OX1R protein content in the endometrium was detected on days 10 to 11, whereas OX2R protein on days 15 to 16. In the trophoblasts, PPO and OX1R protein levels were more pronounced on days 27 to 28 than on days 30 to 32, but OX2R expression was higher on days 30 to 32. The expression of PPO, OX1R and OX2R was different in the conceptuses and trophoblasts during early pregnancy. Local orexin production and the presence of the specific orexin receptors suggest that the orexin system may participate in the control of porcine reproductive functions by exerting endocrine and auto/paracrine effects on the uterus, conceptuses and trophoblasts during early pregnancy. This study provides the first evidence for the presence of orexins and their receptors in the uteri, conceptuses and trophoblasts in pigs during early pregnancy. The local orexin system is dependent on the stage of pregnancy.

An acoustic startle alters knee joint stiffness and neuromuscular control.

Growing evidence suggests that the nervous system contributes to non-contact knee ligament injury, but limited evidence has measured the effect of extrinsic events on joint stability. Following unanticipated events, the startle reflex leads to universal stiffening of the limbs, but no studies have investigated how an acoustic startle influences knee stiffness and muscle activation during a dynamic knee perturbation. Thirty-six individuals were tested for knee stiffness and muscle activation of the quadriceps and hamstrings. Subjects were seated and instructed to resist a 40-degree knee flexion perturbation from a relaxed state. During some trials, an acoustic startle (50 ms, 1000 Hz, 100 dB) was applied 100 ms prior to the perturbation. Knee stiffness, muscle amplitude, and timing were quantified across time, muscle, and startle conditions. The acoustic startle increased short-range (no startle: 0.044 ± 0.011 N·m/deg/kg; average startle: 0.047 ± 0.01 N·m/deg/kg) and total knee stiffness (no startle: 0.036 ± 0.01 N·m/deg/kg; first startle 0.027 ± 0.02 N·m/deg/kg). Additionally, the startle contributed to decreased [vastus medialis (VM): 13.76 ± 33.6%; vastus lateralis (VL): 6.72 ± 37.4%] but earlier (VM: 0.133 ± 0.17 s; VL: 0.124 ± 0.17 s) activation of the quadriceps muscles. The results of this study indicate that the startle response can significantly disrupt knee stiffness regulation required to maintain joint stability. Further studies should explore the role of unanticipated events on unintentional injury.