Association of glucocorticoid receptor expression with key members of the insulin signaling pathway and heat shock proteins in the bovine ovary.

Glucocorticoids (GCs) act through their receptor (GR) as regulators in different biological processes such as reproduction. In the absence of GCs, the GR remains inactive in the cytoplasm by associating with heat shock proteins (HSPs), which act as molecular chaperones, among which the most relevant are HSP90 and HSP70. Cytoplasmic GC-activated GR mediates non-genomic effects, interacting with members of signaling pathways such as PI3K/Akt, which participates in several metabolic processes, including the insulin signaling pathway. The aim of the present study was to evaluate possible associations between the cytoplasmic GR and the main intermediates of the insulin signaling pathway and HSP90 and HSP70 in ovaries of dairy cows. To this end, the protein expression of cytoplasmic GR, key members of the insulin signaling pathway, and HSPs was evaluated in ovarian preovulatory follicles of non-lactating Holstein cows in proestrus. Positive associations were observed between protein expression of GR and HSP90, IRS1, pIRS1, PI3K and pAkt (p < 0.05; ß > 0) in granulosa cells of dominant follicles of dairy cows. Instead, in theca cells, no associations were observed between protein expression of GR and members of the insulin signaling pathway or HSPs. These data provide evidence of the possible association between the non-genomic mechanisms of action of the GR and the insulin signaling pathway in the bovine ovary.

Extrapyramidal plasticity predicts recovery after spinal cord injury.

Spinal cord injury (SCI) leads to wide-spread neurodegeneration across the neuroaxis. We explored trajectories of surface morphology, demyelination and iron concentration within the basal ganglia-thalamic circuit over 2 years post-SCI. This allowed us to explore the predictive value of neuroimaging biomarkers and determine their suitability as surrogate markers for interventional trials. Changes in markers of surface morphology, myelin and iron concentration of the basal ganglia and thalamus were estimated from 182 MRI datasets acquired in 17 SCI patients and 21 healthy controls at baseline (1-month post injury for patients), after 3, 6, 12, and 24 months. Using regression models, we investigated group difference in linear and non-linear trajectories of these markers. Baseline quantitative MRI parameters were used to predict 24-month clinical outcome. Surface area contracted in the motor (i.e. lower extremity) and pulvinar thalamus, and striatum; and expanded in the motor thalamus and striatum in patients compared to controls over 2-years. In parallel, myelin-sensitive markers decreased in the thalamus, striatum, and globus pallidus, while iron-sensitive markers decreased within the left caudate. Baseline surface area expansions within the striatum (i.e. motor caudate) predicted better lower extremity motor score at 2-years. Extensive extrapyramidal neurodegenerative and reorganizational changes across the basal ganglia-thalamic circuitry occur early after SCI and progress over time; their magnitude being predictive of functional recovery. These results demonstrate a potential role of extrapyramidal plasticity during functional recovery after SCI.

Fetal programming in dairy cows: Effect of heat stress on progeny fertility and associations with the hypothalamic-pituitary-adrenal axis functions.

Ambient temperatures that result in body temperatures beyond those of the thermo-neutral zone for dairy cattle can lead to reduced reproductive efficiencies that have negative effects on economic and productive efficiencies of dairy farms. In addition, in pregnant cows, ambient temperature-induced heat stress leads to modifications in the epigenome of the developing embryo, which, in turn, could lead to phenotypic variations in the sexually mature animal and its offspring. In the mammalian response to stress, adrenocorticotropic hormone stimulates the synthesis and secretion of glucocorticoids, which may have detrimental effects on the hypothalamic-pituitary-gonadal axis and the female estrous cycle. The aim of this review is to describe the effects of ambient heat stress on the reproductive system of dairy cattle and its potential trans-generational effects. There are many heat stress occurrences in dairy cattle during a large portion of the year in many countries and there is an increase in incidence with the onset of global warming. These heat stress conditions make it possible that the embryo/fetus of cows may be affected when heat stress conditions prevail in ways that there is impaired fertility of the sexually mature cows that develop from these embryos/fetuses. This is the outcome because of molecular changes in ovarian glucocorticoid response caused by epigenetic modifications established during fetal development.

Protein and gene expression of relevant enzymes and nuclear receptor of hepatic lipid metabolism in grazing dairy cattle during the transition period.

We aimed to study the protein and gene expression of some hepatic enzymes of lipid metabolism along with plasma biomarkers in grazing dairy cattle during the transition period. Blood and liver biopsies from a group of eight multiparous cows were sampled at -28, -14, +4, +14, +28 and +56 days relative to parturition. Peak concentrations of NEFA and beta-hydroxybutyric acid with high triacylglycerol content in the liver were recorded on day 4 postpartum. Consistent with blood biomarkers, the gene expression of carnitine palmitoyltransferase 1A (CPT1A) and acyl-CoA oxidase 1 (ACOX1) increased, whereas that of diacylglycerol O-acyltransferase 1 (DGAT1) decreased. Nevertheless, CPT1A protein expression did not change during all the period evaluated and ACOX1 protein expression increased on day 56 postpartum. In addition, the protein expression of peroxisome proliferator-activated receptor alpha (PPAR-alpha) increased on day 28 postpartum. On the other hand, DGAT1 protein expression decreased on day 14 postpartum. As expected, the expression of genes associated with fatty acid oxidation increased on the first days postpartum but, notably, protein expression was highest after transition. Since most infectious diseases and metabolic disorders in dairy cattle occur particularly on the first days postpartum, it is not so clear whether an increase in the oxidation capacity of the liver at that time could help to prevent disease and improve dairy production. The valuable results about protein expression of enzymes involved in liver lipid metabolism could help to better characterize the metabolism of dairy cattle during the transition period.

Alterations in key metabolic sensors involved in bovine cystic ovarian disease.

High-producing dairy cows frequently suffer metabolic alterations that cause different diseases, which could decrease the reproductive efficiency of the herd. Among these reproductive disorders, cystic ovarian disease (COD) has been related to alterations in metabolites and hormonal factors such as insulin, adiponectin and leptin. The aim of this study was to determine the protein expression of adiponectin and some of its downstream targets in ovarian follicles of control cows and cows with clinical diagnosis of COD. We also analyzed some key metabolic sensors in plasma and follicular fluid from both groups. In follicular cysts, we detected higher protein expression of adiponectin receptor 2 (AdipoR2), 5' adenosine monophosphate-activated protein kinase (AMPK), carnitine palmitoyl transferase 1 (CPT1) and acyl-coenzyme A oxidase 1 (ACOX1) relative to control antral follicles (p < 0.05). This was related to higher plasma adiponectin concentration in cows with COD than in control cows (p < 0.05). On the other hand, insulin concentrations showed an opposite pattern (p < 0.05). Furthermore, we found alterations in local and systemic concentrations of several metabolites. In this regard, in follicular fluid of cystic cows, the concentrations of non-esterified fatty acids and beta-hydroxybutyrate were higher (p < 0.05), whereas the concentrations of glucose and triacylglycerol were lower than in follicular fluid from control cows (p < 0.05). Besides, in both follicular fluid and plasma of cows with COD, the concentration of cholesterol was higher than in control animals (p < 0.05). These results evidence a local altered scenario of some metabolic sensors in cystic follicles, which could generate an adverse microenvironment for the resumption of ovarian activity, possibly causing the persistence of follicles and the recurrence of COD.

Impaired insulin signaling pathways affect ovarian steroidogenesis in cows with COD.

Cystic ovarian disease (COD) represents an important cause of infertility in dairy cattle and is associated with multiple physiological disorders. Steroidogenesis, which is necessary to ensure normal ovarian functions, involves multiple enzymatic pathways coordinated by insulin and other proteins. We have previously shown that cows with COD have an altered insulin response. Therefore, in the present study, we evaluated further alterations in intermediates downstream of the PI3K pathway and pathways mediated by ERK as critical signals for the expression of steroidogenic enzymes in the ovaries of control cows and cows with spontaneous COD. To this end, we evaluated the gene and protein expression of pan-AKT, mTOR, ERK1/2, and steroidogenic enzymes by real-time PCR and immunohistochemistry. Steroid hormone concentrations were assessed at systemic and intrafollicular level. Results showed altered expression of intermediate molecules of the insulin signaling pathway, whose action might modify the synthetic pathway of steroidogenic hormones. Similarly, the expression of steroidogenic enzymes and the concentration of progesterone in serum and follicular fluid were altered. These alterations support the hypothesis that systemic factors contribute to the development and/or maintenance of COD, and that metabolic hormones within follicles such as insulin exert determinant effects on ovarian functionality in cows with COD.

Role of Components of the Insulin-like Growth Factor System in the Early Stages of Ovarian Follicular Persistence in Cattle.

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been postulated that the insulin-like growth factor (IGF) system may contribute to follicular persistence and development of COD. The initiation of the IGF response is a result of interactions between IGF-binding proteins (IGFBPs) and IGFBP proteases, mainly pregnancy-associated plasma protein A (PAPP-A). IGFBPs bind IGFs with high affinity and consequently regulate their access to IGF receptors (IGFRs). The aim of this research was to determine variations in components of the IGF system in the ovaries of cows with persistent follicles induced by long-term administration of progesterone. Proteins of the IGF system were evaluated at 0 (expected day of ovulation), 5, 10 and 15 days of follicular persistence to determine whether the changes occur early in the development of COD. The concentrations of IGF1 and IGFBP4 in follicular fluid were similar in all groups with follicular persistence and in control antral follicles. IGFR1 and IGFBP4 expression in situ were higher in granulose cells in persistent follicles than in control follicles. No differences were found in PAPP-A concentration within follicular fluid in persistent follicles relative to control antral follicles. These data support the hypothesis that the IGF system is altered in the initial stages of development of follicular persistence and has a determinant role in ovarian function in cattle.

Intertester and intratester reliability of a movement control test battery for patients with knee osteoarthritis and controls.

OBJECTIVES: To develop a test battery of movement control (MC) tests and assess its intertester and intratester reliability. METHODS: 29 subjects with knee OA with mean age of 64.7 (SD 8.7) years and 12 controls without either knee pain or previous diagnosis of OA (mean age 36.6 (SD 16.2) years) were included. Two experienced physiotherapists rated the filmed test performance of six MC tests blinded to the patients and to each other on 3-point scale as correct, incorrect or failed. Weighted kappa coefficient (wK) with 95% confidence interval (95%CI) and the percentage of agreement were calculated for each test. RESULTS: One-leg stance, one-leg squat 30 degrees and step down tests showed moderate to excellent inter- and intratester reliability with wK ranging between 0.43-0.85 for intertester and 0.51-0.80 for intratester reliability. The reliability of the 90 degrees squat test, small squat and step up tests was poor (wK ranging between 0.09-0.50). CONCLUSIONS: One-leg stance test, one-leg squat 30 degrees and step down test are reliable in the subjects with knee OA and controls. Further studies are needed to evaluate the discriminative validity of the reliable tests.

Detection and activity of 11 beta hydroxylase (CYP11B1) in the bovine ovary.

Glucocorticoids (GCs) such as cortisol and corticosterone are important steroid hormones with different functions in intermediate metabolism, development, cell differentiation, immune response and reproduction. In response to physiological and immunological stress, adrenocorticotropic hormone (ACTH) acts on the adrenal gland by stimulating the synthesis and secretion of GCs. However, there is increasing evidence that GCs may also be synthesized by extra-adrenal tissues. Here, we examined the gene and protein expression of the enzyme 11ß-hydroxylase P450c11 (CYP11B1), involved in the conversion of 11-deoxycortisol to cortisol, in the different components of the bovine ovary and determined the functionality of CYP11B1 in vitro CYP11B1 mRNA was expressed in granulosa and theca cells in small, medium and large antral ovarian follicles, and CYP11B1 protein was expressed in medium and large antral follicles. After stimulation by ACTH, we observed an increased secretion of cortisol by the wall of large antral follicles. We also observed a concentration-dependent decrease in the concentration of cortisol in response to metyrapone, an inhibitor of CYP11B1. This decrease was significant at 10-5 µM metyrapone. In conclusion, this study demonstrated for the first time the presence of CYP11B1 in the bovine ovary. This confirms that there could be a local synthesis of GCs in the bovine ovary and therefore a potential endocrine responder to stress through these hormones.

Enhanced MAPK signaling is essential for CSF3R-induced leukemia.

Both membrane-proximal and truncation mutations in CSF3R have recently been reported to drive the onset of chronic neutrophilic leukemia (CNL). Here we show that although truncation mutation alone cannot induce leukemia, both proximal and compound mutations (proximal and truncation mutations on same allele) are leukemogenic with a disease latency of 90 and 23 days, respectively. Comparative whole-genome expression profiling and biochemical experiments revealed that induced expression of Mapk adaptor protein Ksr1 and enhanced Mapk signaling are crucial to leukemogenesis by CSF3R proximal and compound mutants. Moreover, inhibition of Mek1/2 by trametinib alone is sufficient to suppress leukemia induced by both CSF3R proximal and ruxolitinib-resistant compound mutations. Together, these findings elucidate a Mapk-dependent mechanism of CSF3R-induced pathogenesis, and they establish the rationale for clinical evaluation of MEK1/2 inhibition in CNL.

Involvement of PAPP-A and IGFR1 in Cystic Ovarian Disease in Cattle.

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra-ovarian factors, such as members of the insulin-like growth factor (IGF) system, may contribute to follicular persistence. The bioavailability of IGF to initiate its response by binding to specific receptors (IGFRs) depends on interactions with related compounds, such as pregnancy-associated plasma protein A (PAPP-A). The aim of this study was to determine IGFR1 and PAPP-A expression both in follicles at different stages of development and in cysts, to evaluate the roles in the etiopathogenesis of COD in cattle. The mRNA expression of PAPP-A was higher in granulosa cells of large tertiary follicles than in cysts, whereas the protein PAPP-A present in the follicular fluid from these follicles showed no differences. Although no PAPP-A mRNA expression was detected in smaller tertiary follicles, in their follicular fluid, this protease was detected in lesser concentration than in cysts. The mRNA expression of IGFR1 was lower in granulosa cells from cystic follicles than in those from tertiary ones. However, the protein expression of this receptor presented the highest levels in cystic structures, probably to increase the possibility of IGF response. The data obtained would indicate that animals with COD have an altered regulation of the IGF system in the ovary, which could be involved in the pathogenesis of this disease in cattle.

Altered expression of transforming growth factor-beta isoforms in bovine cystic ovarian disease.

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra-ovarian factors may contribute to follicular persistence. Transforming growth factor-beta (TGFB) isoforms are important paracrine and autocrine signalling molecules that regulate ovarian follicle growth and physiology. Considering the importance of these factors in the ovarian physiology, in this study, we examined the expression of TGFB isoforms (TGFB1, TGFB2 and TGFB3) in the ovary of healthy cows and animals with spontaneous and adrenocorticotrophic hormone (ACTH)-induced COD. In the oestrous-synchronized control group, the expression of TGFB1 in granulosa and theca cells was higher in spontaneous cysts than in atretic or tertiary follicles. When we compared TGFB2 expression in granulosa cells from atretic or tertiary follicles from the oestrous-synchronized control group with that in ACTH-induced or spontaneous follicular cysts, we found a higher expression in the latter. The expression of the TGFB isoforms studied was also altered during folliculogenesis in both the spontaneous and ACTH-induced COD groups. As it has been previously shown that TGFB influences steroidogenesis, ovarian follicular proliferation and apoptosis, an alteration in its expression may contribute to the pathogenesis of this disease.

Effects of river morphology, hydraulic gradients, and sediment deposition on water exchange and oxygen dynamics in salmonid redds.

Fine sediment decreasing gravel permeability and oxygen supply to incubating salmonid embryos, is often considered the main contributing factor for the observed decline of salmonid populations. However, oxygen supply to salmonid embryos also depends on hydraulic conditions driving water flow through the redd. A more generalized perspective is needed to better understand the constraints on successful salmonid incubation in the many heavily modified fluvial ecosystems of the Northern Hemisphere. The effects of hydraulic gradients, riverbed and redd morphology as well as fine sediment deposition on dissolved oxygen (DO) and water exchange was studied in 18 artificial redds at three sites along a modified river. Fifty percent of the redds in the two downstream sites were lost during high flow events, while redd loss at the upstream site was substantially lower (8%). This pattern was likely related to increasing flood heights from up- to downstream. Specific water infiltration rates (q) and DO were highly dynamic and driven on multiple temporal and spatial scales. Temporally, the high permeability of the redd gravel and the typical pit-tail structure of the new built redds, leading to high DO, disappeared within a month, when fine sediment had infiltrated and the redd structure was leveled. On the scale of hours to days, DO concentrations and q increased during high flows, but decreased during the falling limb of the water level, most likely related to exfiltration of oxygen depleted groundwater or hyporheic water. DO concentrations also decreased under prolonged base flow conditions, when increased infiltration of silt and clay particles clogged the riverbed and reduced q. Spatially, artificial log steps affected fine sediment infiltration, q and interstitial DO in the redds. The results demonstrate that multiple factors have to be considered for successful river management in salmonid streams, including riverbed structure and local and regional hydrogeological conditions.

[Macrophage activation syndrome]. / Makrophagenaktivierungssyndrom.

Macrophage activation syndrome (MAS) is an infrequent but potentially life-threatening complication of various autoimmune diseases. It is characterized by excessive activation of macrophages induced by a dysfunction of the immune system, although the underlying abnormalities remain to be delineated. Sepsis-like symptoms accompanied by cytopenia, hepatosplenomegaly, coagulopathy and multiple organ dysfunctions are typical disease manifestations. Due to the lack of validated classification criteria and distinct laboratory markers, the diagnosis of MAS is often difficult to establish. However, early diagnosis and therapy is of utmost importance to ensure best possible outcome. Treatment regimens include high doses of glucocorticoids, classical immunosuppressive agents as well as novel biologics.

Oxygen measurements in platelet fluids - a new non-invasive method to detect bacterial contaminations in platelets.

BACKGROUND: The residual risk for bacterial contamination in blood components especially in platelets is one to two orders of magnitude higher than for transfusion relevant viral infections. The majority of all bacterial transmitted fatalities occurred at the end of platelet shelf life. Therefore, the maximum shelf life of platelet concentrates (PC) was reduced to 4 days after blood donation in Germany in 2008. METHODS: A new continuous non-invasive bacterial detection method was developed by O(2) measurements in the platelet fluids and tested with 10 transfusion relevant bacteria species. RESULTS: The bacterial concentration at the time point of a positive signal of PreSense O(2) ranged between 10(2) and 10(5) CFU mL(-1) . Harmful transfusion-transmitted bacterial infection would have probably been prevented by this novel technology. Only strict anaerobic bacteria strains like Clostridium perfringens were not detected within the study period of 72 h. CONCLUSIONS: The described non-invasive bacterial detection method represents a new approach to prevent transmission of bacterial infection in platelets. The method is characterised by the advantage that all investigations can be performed until right up to the time of transfusion, and therefore, reduce the risk for sample errors to a minimum.

Multimodal approach to therapy-related neuropathic pain in breast cancer.

PURPOSE: This study evaluated the effects of the multimodal therapy (gabapentin-non steroidal anti inflammatory drug [NSAID]-morphine) on intensity and relief of treatmentrelated neuropathic pain in patients with breast cancer. METHODS: This study involved 75 breast cancer outpatients who had previously undergone anti-neoplastic therapy (surgery, chemotherapy, radiotherapy). The patients were randomly divided into 3 groups, which were formed depending on the planned analgesic therapy (gabapentin, gabapentin- NSAID, gabapentin-NSAID-morphine). Each group was a control group to itself. The pain intensity difference and scores of daily activities were collated and assessed by the modified Brief Pain Inventory (BPI) questionnaire (VAS/Likert Scale). Monitoring the additional medication and side effects optimized the therapy efficiency evaluation. RESULTS: During this 6-week study, the decrease of pain intensity was significant in all 3 groups (p <0.0001). Although there was intergroup difference, it was statistically not significant (p >0.05). The variant analysis of pain relief showed differences both among and within the groups in the first 3 weeks of the study (F(1)=7.79, p=0.000; F(2)=7.01, p=0.001; F(3) =5.49, p=0.001). The multimodal group needed the least of additional medication and the variant analysis showed a statistically significant difference (p=0.001) from the 4th week of the trial period. The correlation between the increase trend of side effects and the frequency of additional medication was significant (p <0.05). CONCLUSION: The multimodal therapy ensures adequate cancer-related neuropathic pain control with minimal side effects.

The role of prior model calibration on predictions with ensemble Kalman filter.

This paper, based on a real world case study (Limmat aquifer, Switzerland), compares inverse groundwater flow models calibrated with specified numbers of monitoring head locations. These models are updated in real time with the ensemble Kalman filter (EnKF) and the prediction improvement is assessed in relation to the amount of monitoring locations used for calibration and updating. The prediction errors of the models calibrated in transient state are smaller if the amount of monitoring locations used for the calibration is larger. For highly dynamic groundwater flow systems a transient calibration is recommended as a model calibrated in steady state can lead to worse results than a noncalibrated model with a well-chosen uniform conductivity. The model predictions can be improved further with the assimilation of new measurement data from on-line sensors with the EnKF. Within all the studied models the reduction of 1-day hydraulic head prediction error (in terms of mean absolute error [MAE]) with EnKF lies between 31% (assimilation of head data from 5 locations) and 72% (assimilation of head data from 85 locations). The largest prediction improvements are expected for models that were calibrated with only a limited amount of historical information. It is worthwhile to update the model even with few monitoring locations as it seems that the error reduction with EnKF decreases exponentially with the amount of monitoring locations used. These results prove the feasibility of data assimilation with EnKF also for a real world case and show that improved predictions of groundwater levels can be obtained.