First results about ProAKAP4 concentration in stallion semen after cryopreservation in two different freezing media.

The quality of fresh or thawed sperm in stallions has been generally determined by the viability and total and progressive motility of the sperm. Today, the expression of ProAKAP4, a protein present in the flagellum of spermatozoa, appears to be an innovative and relevant functional marker to assess semen quality and male fertility. This study aims to compare the concentration of ProAKAP4 in the semen from 5 stallions frozen with two different extenders immediately after thawing (T0) and 4 h post-thawing (T4). Viability, total and progressive motility were measured in parallel. Significant differences for sperm viability and total motility were observed between the two extenders, as was the concentration of ProAKAP4 both at T0 and T4. At T4, all quality parameters and ProAKAP4 content significantly decreased compared to T0, but with a considerably slower decrease in one extender than the other. These preliminary results suggest that measuring the concentration of ProAKAP4 is a promising tool for the comparison of different extenders and the selection of the optimal freezing medium for each stallion ejaculate.

Expression, localization, and concentration of A-kinase anchor protein 4 (AKAP4) and its precursor (proAKAP4) in equine semen: Promising marker correlated to the total and progressive motility in thawed spermatozoa.

A-kinase anchor protein 4 (AKAP4) is playing a central role in flagellar structure, chemotaxis, capacitation and sperm motility. In mammals, AKAP4 is expressed during spermatogenesis. AKAP4 is synthesized as a precursor, proAKAP4, which is cleaved into mature AKAP4 during fibrous sheath assembly. The proAKAP4 is a good indicator of sperm quality in humans and boars. The aims of this work were to study the expression, the localization and the concentration of proAKAP4 and AKAP4 in equine semen, and to evaluate the possible correlation between the total and progressive motility and the concentration of proAKAP4 measured by ELISA in post-thawed semen. Frozen sperm from 13 different stallions were used. Semen samples (n = 17) were prepared using the INRA Freeze medium to reach a concentration of 150 million spermatozoa/mL, packaged and frozen in 0.5 mL straws. The precursor proAKAP4 and the mature protein AKAP4 both localize to the fibrous sheath of the principle piece of equine sperm flagellum. The concentrations of proAKAP4 were determined in the post-thawed semen using ELISA method (Horse 4MID® kits, 4BioDx, France). The mean concentration of proAKAP4 was then of 7.372 ±â€¯0.79 ng/µL and was significantly correlated with the post-thawed total motility (Pearson coefficient r = 0.66, p = 0.002) and progressive motility (Pearson coefficient r = 0.76, p = 0.0002) and the amount of proAKAP4 represent the amount of spermatozoa that expressed proAKAP4. Taken together, these preliminary results confirm the interest to use proAKAP4 concentrations as a promising marker of stallion sperm quality as close correlation was observed between the proAKAP4 concentration and sperm motility parameters.

Combining role-play with interactive simulation to motivate informed climate action: Evidence from the World Climate simulation.

Climate change communication efforts grounded in the information deficit model have largely failed to close the gap between scientific and public understanding of the risks posed by climate change. In response, simulations have been proposed to enable people to learn for themselves about this complex and politically charged topic. Here we assess the impact of a widely-used simulation, World Climate, which combines a socially and emotionally engaging role-play with interactive exploration of climate change science through the C-ROADS climate simulation model. Participants take on the roles of delegates to the UN climate negotiations and are challenged to create an agreement that meets international climate goals. Their decisions are entered into C-ROADS, which provides immediate feedback about expected global climate impacts, enabling them to learn about climate change while experiencing the social dynamics of negotiations. We assess the impact of World Climate by analyzing pre- and post-survey results from >2,000 participants in 39 sessions in eight nations. We find statistically significant gains in three areas: (i) knowledge of climate change causes, dynamics and impacts; (ii) affective engagement including greater feelings of urgency and hope; and (iii) a desire to learn and do more about climate change. Contrary to the deficit model, gains in urgency were associated with gains in participants' desire to learn more and intent to act, while gains in climate knowledge were not. Gains were just as strong among American participants who oppose government regulation of free markets-a political ideology that has been linked to climate change denial in the US-suggesting the simulation's potential to reach across political divides. The results indicate that World Climate offers a climate change communication tool that enables people to learn and feel for themselves, which together have the potential to motivate action informed by science.

Morphine, a potential inhibitor of myeloperoxidase activity.

Morphine is an opioid alkaloid commonly used in clinical practice for its analgesic properties. The phenolic hydroxyl group of that phenanthrene derivative is pivotal for binding to opioid receptors but it may also be responsible for the antioxidant behavior of morphine reported in several in vitro experiments. In this study, we assessed the effect of morphine on myeloperoxidase (MPO), a hemic enzyme from azurophilic granules of polymorphonuclear neutrophils involved in the production of cytotoxic and microbicidal reactive oxidants during inflammatory response. Specific immunological extraction followed by enzyme detection (SIEFED) and molecular modeling (docking) were performed to study the potential anti-catalytic action of morphine on MPO in comparison with the inhibitory effects of reference antioxidant molecules quercetin, gallic acid and ascorbic acid. The reducing action of morphine on the MPO peroxidase cycle has been investigated using electron paramagnetic resonance (EPR) and UV-visible absorption spectroscopy. Morphine acted as a reducing substrate in the peroxidase cycle of MPO and therefore protected the enzyme against the suicide action of its natural substrate, hydrogen peroxide. The SIEFED experiments associated with the docking study, further demonstrated a lack of strong and sustained anti-catalytic activity of morphine. In summary, from the results of this study, it appears that morphine acts as a weak and reversible inhibitor of MPO that may nonetheless contribute to immunomodulatory and antioxidant effects of this opioid analgesic in vivo.

JAK2/IDH-mutant-driven myeloproliferative neoplasm is sensitive to combined targeted inhibition.

Patients with myeloproliferative neoplasms (MPNs) frequently progress to bone marrow failure or acute myeloid leukemia (AML), and mutations in epigenetic regulators such as the metabolic enzyme isocitrate dehydrogenase (IDH) are associated with poor outcomes. Here, we showed that combined expression of Jak2V617F and mutant IDH1R132H or Idh2R140Q induces MPN progression, alters stem/progenitor cell function, and impairs differentiation in mice. Jak2V617F Idh2R140Q-mutant MPNs were sensitive to small-molecule inhibition of IDH. Combined inhibition of JAK2 and IDH2 normalized the stem and progenitor cell compartments in the murine model and reduced disease burden to a greater extent than was seen with JAK inhibition alone. In addition, combined JAK2 and IDH2 inhibitor treatment also reversed aberrant gene expression in MPN stem cells and reversed the metabolite perturbations induced by concurrent JAK2 and IDH2 mutations. Combined JAK2 and IDH2 inhibitor therapy also showed cooperative efficacy in cells from MPN patients with both JAK2mut and IDH2mut mutations. Taken together, these data suggest that combined JAK and IDH inhibition may offer a therapeutic advantage in this high-risk MPN subtype.

Jak1 Integrates Cytokine Sensing to Regulate Hematopoietic Stem Cell Function and Stress Hematopoiesis.

JAK1 is a critical effector of pro-inflammatory cytokine signaling and plays important roles in immune function, while abnormal JAK1 activity has been linked to immunological and neoplastic diseases. Specific functions of JAK1 in the context of hematopoiesis, and specifically within hematopoietic stem cells (HSCs), have not clearly been delineated. Here, we show that conditional Jak1 loss in HSCs reduces their self-renewal and markedly alters lymphoid/myeloid differentiation in vivo. Jak1-deficient HSCs exhibit decreased competitiveness in vivo and are unable to rescue hematopoiesis in the setting of myelosuppression. They exhibit increased quiescence, an inability to enter the cell cycle in response to hematopoietic stress, and a marked reduction in cytokine sensing, including in response to type I interferons and IL-3. Moreover, Jak1 loss is not fully rescued by expression of a constitutively active Jak2 allele. Together, these data highlight an essential role for Jak1 in HSC homeostasis and stress responses.

Polyphenols from Silybum marianum inhibit in vitro the oxidant response of equine neutrophils and myeloperoxidase activity.

A recent study showed that silymarin, a standardized extract of S. marianum might be used in the prevention of equine laminitis. We investigated the effects of quercetin and some compounds found in silymarin (silybin, taxifolin and dehydrosilybin) on reactive oxygen species (ROS) production and myeloperoxidase (MPO) release by stimulated equine neutrophils (PMNs) and on MPO activity. All compounds (tested between 100 nm and 100 µm) inhibited superoxide anion production by stimulated PMNs in a dose-dependent manner. Dehydrosilybin and quercetin inhibited superoxide production and MPO release from 10 µm. Classical MPO assay showed quercetin as the most potent inhibitor, followed by taxifolin, dehydrosilybin and silybin. SIEFED MPO assay highlighting the binding of tested compounds to MPO showed that only quercetin and taxifolin maintained an efficient inhibition above 90% at 10 µm. Altogether, our results showed a strong inhibition of PMN activation by planar compounds such as quercetin and dehydrosilybin and a strong inhibition of MPO activity by the smallest molecules, quercetin and taxifolin. In conclusion, the compounds from silymarin may be useful for modulating the oxidative response of PMNs, involved in the pathogenesis of laminitis, but further in vivo studies are needed.

An immunological method to combine the measurement of active and total myeloperoxidase on the same biological fluid, and its application in finding inhibitors which interact directly with the enzyme.

Myeloperoxidase (MPO) is a pro-oxidant enzyme involved in inflammation, and the measurement of its activity in biological samples has emerged essential for laboratory and clinical investigations. We will describe a new method which combines the SIEFED (specific immunological extraction followed by enzymatic detection) and ELISA (ELISAcb) techniques to measure the active and total amounts of MPO on the same human sample and with the same calibration curve, as well as to define an accurate ratio between both the active and total forms of the enzyme. The SIEFED/ELISAcb method consists of the MPO extraction from aqueous or biological samples by immobilized anti-MPO antibodies coated onto microplate wells. After a washing step to eliminate unbound material, the activity of MPO is measured in situ by adding a reaction solution (SIEFED). Following aspiration of the reaction solution, a secondary anti-MPO antibody is added into the wells and the ELISAcb test is carried out in order to measure the total MPO content. To validate the combined method, a comparison was made with SIEFED and ELISA experiments performed separately on plasma samples isolated from human whole blood, after a neutrophil stimulation. The SIEFED/ELISAcb provides a suitable tool for the measurement of specific MPO activity in biological fluids and for the estimation of the inhibitory potential of a fluid. The method can also be used as a pharmacological tool to make the distinction between a catalytic inhibitor, which binds to MPO and inhibits its activity, and a steric inhibitor, which hinders the enzyme and prevents its immunodetection.

Somatic mutations in leukocytes infiltrating primary breast cancers.

BACKGROUND: Malignant transformation requires the interaction of cancer cells with their microenvironment, including infiltrating leukocytes. However, somatic mutational studies have focused on alterations in cancer cells, assuming that the microenvironment is genetically normal. Because we hypothesized that this might not be a valid assumption, we performed exome sequencing and targeted sequencing to investigate for the presence of pathogenic mutations in tumor-associated leukocytes in breast cancers. METHODS: We used targeted sequencing and exome sequencing to evaluate the presence of mutations in sorted tumor-infiltrating CD45-positive cells from primary untreated breast cancers. We used high-depth sequencing to determine the presence/absence of the mutations we identified in breast cancer-infiltrating leukocytes in purified tumor cells and in circulating blood cells. RESULTS: Capture-based sequencing of 15 paired tumor-infiltrating leukocytes and matched germline DNA identified variants in known cancer genes in all 15 primary breast cancer patients in our cohort. We validated the presence of mutations identified by targeted sequencing in infiltrating leukocytes through orthogonal exome sequencing. Ten patients harbored alterations previously reported as somatically acquired variants, including in known leukemia genes (DNTM3A, TET2, and BCOR). One of the mutations observed in the tumor-infiltrating leukocytes was also detected in the circulating leukocytes of the same patients at a lower allele frequency than observed in the tumor-infiltrating cells. CONCLUSIONS: Here we show that somatic mutations, including mutations in known cancer genes, are present in the leukocytes infiltrating a subset of primary breast cancers. This observation allows for the possibility that the cancer cells interact with mutant infiltrating leukocytes, which has many potential clinical implications.

Concentration, activity and biochemical characterization of myeloperoxidase in fresh and post-thaw equine semen and their implication on freezability.

Myeloperoxidase (MPO) is a pro-oxidant enzyme associated with decreased motility in thawed equine semen. This study aimed to describe MPO concentration, activity and subunits in raw and thawed semen and to correlate these data with motilities in raw and thawed semen. Semen samples from five stallions were collected four times. Motilities were assessed in raw and thawed semen. MPO assays were performed in raw seminal plasma, raw sperm-rich pellet and thawed semen. Total and active MPO concentrations were, respectively, assayed by enzyme-linked immunosorbent assay and specific immunological extraction followed by enzymatic detection. MPO subunits present in semen were characterized by Western blot. Purified active MPO was added in saline solution and freezing extender to control its activity during freezing procedure. Differences between medians were determined using Kruskal-Wallis test, and correlations were determined using Spearman's test for nonparametric data. Active MPO concentration was low in seminal plasma and thawed semen, but high in pellet (p = 0.0058), as the opposite relation was observed for total MPO concentration (p < 0.0001). In seminal plasma and post-thaw semen, inactive 86-kDa MPO precursor was mainly observed. Purified MPO activity was decreased in the extender (p = 0.0286). MPO activity in pellet was highly correlated with thawed progressive motility (r = -0.5576, p = 0.0086). Inactive MPO precursor and unknown low molecular weight inactive MPO precursor subunits explain low MPO activity in semen. Major MPO activity was observed in pellet, and post-thaw loss of activity is partially explained by MPO inactivation in extender. Thawed semen motility was negatively correlated with MPO activity in pellet, becoming a potential freezability predictor.

Myeloperoxidase and its products in synovial fluid of patients with treated or untreated rheumatoid arthritis.

OBJECTIVE: Plasma and synovial myeloperoxidase (MPO) and its products were strongly associated with osteoarthritis (OA) and rheumatoid arthritis (RA). In addition, it is well known that there is a link between oxidative stress and cytokines. The present study aims at investigating the link between synovial MPO (and its products), interleukin (IL)-18, which is involved in the degradation of articular cartilage in RA, and IL-8, which is involved in recruitment and activation of neutrophils during inflammation. Effects of the treatment of RA on the biological parameters were also investigated. METHODS: Patients (n = 105) were studied including 39 patients with OA, 33 with RA and 33 with RA receiving a specific treatment. Disease activity score (DAS-28) was calculated whereas MPO antigen/activity, neutrophils, chloro-tyrosine (Cl-Tyr), homocitrulline (Hcit), IL-8, and IL-18 were measured in synovial fluid (SF) and CRP was measured in serum. RESULTS: DAS-28 and CRP levels were not significantly different between groups. MPO activity, and MPO, Cl-Tyr, and Hcit levels were significantly higher in SF of RA patients than OA patients. MPO specific activity (MPO activity/antigen ratio) was significantly lower in treated than in untreated RA patients as was IL-8. MPO activity and concentration were correlated with IL-8 and IL-18 in untreated but not in treated RA patients. CONCLUSIONS: MPO level is related to IL-8 and IL-18 levels in untreated RA patients. A link has been shown between treatment and decrease of IL-8, MPO specific activity and Hcit in SF. The causal role of MPO in SF inflammation and how treatment can affect MPO specific activity need further investigations.

Equine myeloperoxidase: a novel biomarker in synovial fluid for the diagnosis of infection.

REASONS FOR PERFORMING STUDY: Equine joint infection is a life-threatening disorder, and confirmation of the diagnosis can be difficult. Synovial fluid biomarkers may assist the discrimination between infectious and noninfectious joint disease. OBJECTIVES: This study investigates whether the immunological detection of total and enzymatically active myeloperoxidase (MPO) assists the diagnosis of joint infection in horses. METHODS: The following 4 sample groups were included: healthy; osteochondritis dissecans (OCD); traumatic synovitis; and culture-confirmed infected joints. Synovial fluid was analysed for total MPO by a horse-specific sandwich enzyme-linked immunosorbent assay (ELISA) and for active MPO using the specific immunological extraction followed by enzymatic detection (SIEFED) technique. Western blot analysis was performed to confirm the antibody specificity. RESULTS: Synovial fluid from infected joints contained significantly more total and active MPO than samples from healthy joints, joints affected by OCD and joints with traumatic synovitis. Cut-off values were set at 5000 and 350 ng/ml for total and active MPO, respectively, with fair sensitivity, specificity, positive and negative predictive values and likelihood ratios for infection. Correlation coefficients were reported between the total as well as the active MPO levels and the routine synovial fluid parameters, i.e. the white blood cell count, the neutrophil count and the total protein level. No correlation was observed between MPO and either the age of the horse or the joint affected. Western blotting confirmed the antibody specificity for equine MPO. CONCLUSIONS AND POTENTIAL RELEVANCE: Synovial fluid MPO was identified as a very promising biomarker to augment the discrimination of infectious vs. noninfectious joint disease in horses. Both ELISA and SIEFED techniques can be used for its specific and rapid detection. The analysis of synovial fluid MPO can be used as a complementary test to aid in the discrimination between infectious and noninfectious joint disease, especially when the white blood cell counts and the total protein level are inconclusive.

Relationship between arthroscopic joint evaluation and the levels of Coll2-1, Coll2-1NO(2), and myeloperoxidase in the blood and synovial fluid of horses affected with osteochondrosis of the tarsocrural joint.

OBJECTIVE: To evaluate the levels of plasmatic and synovial Coll2-1, Coll2-1NO(2) and myeloperoxidase (MPO) in horses with osteochondral lesions of the tarsocrural joint and to investigate how these levels relate to arthroscopic findings of inflammation and degeneration. MATERIALS AND METHODS: Venous blood and synovial fluid samples were collected from 63 horses presented for arthroscopic removal of osteochondral fragments in the tarsocrural joint. Prior to removal of the osteochondral fragment, an exploration of the joint was performed and an inflammatory and degenerative score was determined. The blood and synovial levels of Coll2-1, Coll2-1NO(2) and MPO were also measured. The effects of the arthroscopic evaluation (inflammatory and degenerative classes) on the blood and synovial markers were evaluated using a linear model (GLM procedure), and correlations between biochemical markers in the blood and synovial fluid and the arthroscopic evaluation (inflammatory and degenerative classes) were established (Pearson's correlations). RESULTS: Significantly higher levels of Coll2-1 were detected in synovial fluid of higher degenerative classes. There was a significant correlation between the degenerative score and the synovial levels of Coll2-1 (r=0.27). According to the logistic regression model, there was a significant effect of the degenerative class on synovial levels of Coll2-1. CONCLUSIONS: Coll2-1 correlates well with the degenerative state of tarsocrural joints as evaluated by arthroscopy. This marker can therefore be classified as a burden-of-disease marker in the assessment of joint disease in horses.

Effect of intensive exercise on plasmatic neutrophil elastase level in eventing and endurance horses.

REASONS FOR PERFORMING STUDY: Intensive exercise induces a systemic inflammatory response characterised by an increase of blood neutrophil count and myeloperoxidase (MPO) release. Neutrophil elastase (NE) could also contribute to tissues lesions by its proteinase activities. OBJECTIVE: To compare plasmatic NE concentrations before and after different forms of intensive exercise. MATERIALS AND METHODS: EDTA blood samples were taken from 51 eventing horses (EvH) and 32 endurance horses (EndH) were sampled before the race (T0). Blood sampling was performed 2 h (T1) after completing either phase D of a one or 2 star eventing competition (n = 51), or a 120 or 160 km endurance race (n = 32). Plasmatic NE and MPO were measured by a specific equine ELISA. Neutrophil counts and creatine kinase (CK) levels were also measured. A Wilcox on test for paired samples was used to compare mean values of neutrophils, CK, MPO and NE at T0 and T1 in EvH and in EndH. Correlations were calculated between all the 4 parameters in EvH and EndH. RESULTS: At T0, mean NE levels were 14.43 ± 3.63 ng/ml for EvH and 11.7 ± 2.11 ng/ml for EndH. The competition induced a significant increase of NE levels in (58.57 ± 24.06 ng/ml) EvH and (95.74 ± 22.70 ng/ml) EndH (P < 0.05). NE was significantly (P < 0.0001) correlated to MPO in EvH (r = 0.293) and EndH (r = 0.594) and to CK (r = 0.297) in EndH (P < 0.0001). Neutrophils, CK and MPO were significantly increased between T0 and T1 in both types of horses. CONCLUSIONS: Significant increase of NE (EndH) was observed after intense exercise with a significant correlation between NE and MPO. The huge variability in MPO and NE indicates that not all horses show the same intensity of systemic inflammatory response.

Alterations in mitochondrial respiratory function in response to endurance training and endurance racing.

REASONS FOR PERFORMING STUDY: Limited information exists about the muscle mitochondrial respiratory function changes that occur in horses during an endurance season. OBJECTIVES: To determine effects of training and racing on muscle oxidative phosphorylation (OXPHOS) and electron transport system (ETS) capacities in horses with high resolution respirometry (HRR). METHODS: Mitochondrial respiration was measured in microbiopsies taken from the triceps brachii (tb) and gluteus medius (gm) muscles in 8 endurance horses (7 purebred Arabians and 1 crossbred Arabian) before training (T0), after two 10 week training periods (T1, T2) and after 2 CEI** endurance races (R1, R2). Muscle OXPHOS capacity was determined using 2 titration protocols without (SUIT 1) or with pyruvate (SUIT 2) as substrate. Electrons enter at the level of Complex I, Complex II or both complexes simultaneously (Complexes I+II). Muscle ETS capacity was obtained by uncoupling Complexes I+II sustained respiration. RESULTS: T1 improved OXPHOS and ETS capacities in the tb as demonstrated by the significant increase of oxygen fluxes vs. T0 (Complex I: +67%; ETS: +37%). Training improved only OXPHOS in the gm (Complex I: +34%). Among horses that completed the race, a significant decrease in OXPHOS (Complex I: ∼ -35%) and ETS (-22%) capacities was found in the tb with SUIT 2 indicating a reduced aerobic glycolysis. Significant correlations between CK activities and changes in OXPHOS were found suggesting a relationship between exercise-induced muscle damage and depression of mitochondrial respiration. CONCLUSIONS: For the first time, OXPHOS and ETS capacities in equine muscle at different steps of an endurance season have been determined by HRR. Significant alterations in mitochondrial respiratory function in response to endurance training and endurance racing have been observed although these changes appeared to be muscle group specific.

Effect of a 120 km endurance race on plasma and muscular neutrophil elastase and myeloperoxidase concentrations in horses.

REASONS FOR PERFORMING STUDY: Intense physical exercise can induce the degranulation of neutrophils leading to an increase in plasma concentration of the neutrophil marker enzymes myeloperoxidase (MPO) and elastase (ELT). These enzymes have pro-oxidative and pro-inflammatory properties and may play a role in the exercised-induced muscular damage. OBJECTIVES: To measure MPO and ELT concentrations in plasma and muscles of endurance horses and to correlate them to the extent of exercise-induced muscular damage. METHODS: Seven endurance horses qualified on 120 km races were tested in this study. Neutrophil count, serum creatine kinase (CK), plasmatic and muscular MPO and ELT concentrations were measured before and 2 h after a 120 km endurance race. RESULTS: The race produced a significant increase of neutrophils, CK, and plasma MPO and ELT levels. A significant correlation was observed between the MPO and ELT values in plasma (r(2) = 0.92, P < 0.01) and in muscles (r(2) = 0.89, P < 0.01) while plasmatic concentrations of MPO and ELT were not significantly correlated to muscular ones. An increase of mean concentrations (± s.e.) of MPO (T0: 9.85 ± 3.9, T1: 228.9 ± 95.9 ng/mg proteins) and ELT (T0: 8.4 ± 2.4, T1: 74.5 ± 39.7 ng/mg proteins) in the muscles were observed after the race. Interestingly, the individual data showed large differences between the horses. Muscular MPO and ELT concentrations were significantly correlated to plasma CK levels. The coefficient of correlation (r(2)) was 0.69 (P < 0.01) for MPO and 0.66 (P < 0.01) for ELT, respectively. CONCLUSIONS: Results underline the possible role of MPO and ELT in exercise-induced muscular damage. POTENTIAL RELEVANCE: Further studies should investigate the effect of exercise type and intensity, as well as the role of the training state on MPO and ELT involvement in muscular damage. The assessment of the intensity of exercise-induced neutrophilic degranulation may have a potential role in the monitoring of the athletic career.