Cardiac troponin T N-domain variant destabilizes the actin interface resulting in disturbed myofilament function.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca2+ activation. Additionally, in the low Ca2+-relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca2+, and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.

GSK-3ß Localizes to the Cardiac Z-Disc to Maintain Length Dependent Activation.

BACKGROUND: Altered kinase localization is gaining appreciation as a mechanism of cardiovascular disease. Previous work suggests GSK-3ß (glycogen synthase kinase 3ß) localizes to and regulates contractile function of the myofilament. We aimed to discover GSK-3ß's in vivo role in regulating myofilament function, the mechanisms involved, and the translational relevance. METHODS: Inducible cardiomyocyte-specific GSK-3ß knockout mice and left ventricular myocardium from nonfailing and failing human hearts were studied. RESULTS: Skinned cardiomyocytes from knockout mice failed to exhibit calcium sensitization with stretch indicating a loss of length-dependent activation (LDA), the mechanism underlying the Frank-Starling Law. Titin acts as a length sensor for LDA, and knockout mice had decreased titin stiffness compared with control mice, explaining the lack of LDA. Knockout mice exhibited no changes in titin isoforms, titin phosphorylation, or other thin filament phosphorylation sites known to affect passive tension or LDA. Mass spectrometry identified several z-disc proteins as myofilament phospho-substrates of GSK-3ß. Agreeing with the localization of its targets, GSK-3ß that is phosphorylated at Y216 binds to the z-disc. We showed pY216 was necessary and sufficient for z-disc binding using adenoviruses for wild-type, Y216F, and Y216E GSK-3ß in neonatal rat ventricular cardiomyocytes. One of GSK-3ß's z-disc targets, abLIM-1 (actin-binding LIM protein 1), binds to the z-disc domains of titin that are important for maintaining passive tension. Genetic knockdown of abLIM-1 via siRNA in human engineered heart tissues resulted in enhancement of LDA, indicating abLIM-1 may act as a negative regulator that is modulated by GSK-3ß. Last, GSK-3ß myofilament localization was reduced in left ventricular myocardium from failing human hearts, which correlated with depressed LDA. CONCLUSIONS: We identified a novel mechanism by which GSK-3ß localizes to the myofilament to modulate LDA. Importantly, z-disc GSK-3ß levels were reduced in patients with heart failure, indicating z-disc localized GSK-3ß is a possible therapeutic target to restore the Frank-Starling mechanism in patients with heart failure.

Timing of puberty in F1-generation hatchery-produced greater amberjack (Seriola dumerili).

We evaluated the onset of puberty of first-generation (F1) hatchery-produced greater amberjack (Seriola dumerili) reared in sea cages for 5 years. Fish were sampled every year in June, at the expected peak of the spawning period in the Mediterranean Sea. No sexual dimorphism in body weight was observed in the study. The ovaries of 1 and 2-year-old (yo) females consisted of primary oocytes only, while at the age of 3-yo early vitellogenic (Vg) oocytes were also identified, but with extensive follicular atresia. At the age of 4-yo, late Vg oocytes were observed, but again extensive follicular atresia characterized the ovaries of 50 % of females. At the age of 5-yo, follicular atresia of Vg oocytes was very limited. In males, gametogenesis was evident already in 1- and 2-yo fish, and 100 % of sampled 3-yo males produced collectable viable sperm. Plasma testosterone (T), 17ß-estradiol (E2), and 17,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P) remained similar in 3 - 5-yo females, with T and E2 levels being highest in females in advanced vitellogenesis or with significant follicular atresia, compared to immature females. In males, plasma T declined over the years, while 11-ketotestosterone (11-KT) and 17,20ß-P were highest in 4 and 5-yo males, with spermatozoa motility characteristics being improved from the 4th year onwards. The administration of GnRHa implants to 5-yo fish induced only two spawns, albeit no fertilized eggs were obtained. The results indicate that hatchery-produced greater amberjack males mature well and within the same age observed in the wild, however with smaller gonad size. On the contrary, females mature later than in the wild, also with a smaller gonad size. Spawning in response to GnRHa treatment was not effective, suggesting that Mediterranean hatchery-produced broodstocks may be dysfunctional, and further research is needed to document any improvement as the fish get older, or to determine if the results may be related to the specific stock of fish.

Gonadotropin expression, pituitary and plasma levels in the reproductive cycle of wild and captive-reared greater amberjack (Seriola dumerili).

We compared the endocrine status of the pituitary-gonad axis of wild and captive-reared greater amberjack (Seriola dumerili) during the reproductive cycle (April - July), reporting on the expression and release of the two gonadotropins for the first time in the Mediterranean Sea. Ovaries from wild females were characterized histologically as DEVELOPING in early May and SPAWNING capable in late May-July, the latter having a 3 to 4-fold higher gonadosomatic index (GSI). SPAWNING capable wild females exhibited an increase in pituitary follicle stimulating hormone (Fsh) content, plasma testosterone (T) and 17,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P), while almost a 10-fold increase was observed in pituitary luteinizing hormone (Lh) content. An increasing trend of plasma 17ß-estradiol (E2) was also recorded between the two reproductive stages in wild females. Captive-reared females sampled during the reproductive cycle exhibited two additional reproductive categories, with REGRESSED females having extensive follicular atresia and fish in the REGENERATING stage having only primary oocytes in their ovaries. Pituitary content of Fsh and Lh, fshb and lhb expression and plasma levels of Fsh and Lh remained unchanged among the four reproductive stages in captive females, in contrast with plasma E2 and T that decreased in the REGENERATING stage, and 17,20ß-P which increased after the DEVELOPING stage. In general, no significant hormonal differences were recorded between captive-reared and wild DEVELOPING females, in contrast to SPAWNING capable females, where pituitary Lh content, plasma Fsh and T were found to be lower in females in captivity. Overall, the captive females lagged behind in reproductive development compared to the wild ones and this was perhaps related to the multiple handling of the sea cages where all the sampled fish were maintained. Between wild males in the DEVELOPING and SPAWNING capable stages, pituitary Lh content, plasma T and 17,20ß-P, and GSI exhibited 3 to 4-fold increases, while an increasing trend of pituitary Fsh content, lhb expression levels and plasma 11-ketotestosterone (11-KT) was also observed, and an opposite trend was observed in plasma Lh. Captive males were allocated to one more category, with REGRESSED individuals having no spermatogenic capacity. During the SPAWNING capable phase, almost all measured parameters were lower in captive males compared to wild ones. More importantly, captive males showed significant differences from their wild counterparts throughout the reproductive season, starting already from the DEVELOPING stage. Therefore, it appears that captivity already exerted negative effects in males prior to the onset of the study and the multiple handling of the cage where sampled fish were reared. Overall, the present study demonstrated that female greater amberjack do undergo full vitellogenesis in captivity, albeit with some dysfunctions that may be related to the husbandry of the experiment, while males, on the other hand, may be more seriously affected by captivity even before the onset of the study.

Extraction of High Value Products from Zingiber officinale Roscoe (Ginger) and Utilization of Residual Biomass.

Zingiber officinale Roscoe (ginger) is a plant from the Zingiberaceae family, and its extracts have been found to contain several compounds with beneficial bioactivities. Nowadays, the use of environmentally friendly and sustainable extraction methods has attracted considerable interest. The main objective of this study was to evaluate subcritical propane (scPropane), supercritical CO2 (scCO2), and supercritical CO2 with ethanol (scCO2 + EtOH) as co-solvent methods for the extraction of high value products from ginger. In addition, the reuse/recycling of the secondary biomass in a second extraction as a part of the circular economy was evaluated. Both the primary and the secondary biomass led to high yield percentages, ranging from 1.23% to 6.42%. The highest yield was observed in the scCO2 + EtOH, with biomass prior used to scCO2 extraction. All extracts presented with high similarities as far as their total phenolic contents, antioxidant capacity, and chemical composition. The most abundant compounds, identified by the two different gas chromatography-mass spectrometry (GC-MS) systems present, were a-zingiberene, ß- sesquiphellandrene, a-farnesene, ß-bisabolene, zingerone, gingerol, a-curcumene, and γ-muurolene. Interestingly, the reuse/recycling of the secondary biomass was found to be promising, as the extracts showed high antioxidant capacity and consisted of significant amounts of compounds with beneficial properties.

Myofibril orientation as a metric for characterizing heart disease.

Myocyte disarray is a hallmark of many cardiac disorders. However, the relationship between alterations in the orientation of individual myofibrils and myofilaments to disease progression has been largely underexplored. This oversight has predominantly been because of a paucity of methods for objective and quantitative analysis. Here, we introduce a novel, less-biased approach to quantify myofibrillar and myofilament orientation in cardiac muscle under near-physiological conditions and demonstrate its superiority as compared with conventional histological assessments. Using small-angle x-ray diffraction, we first investigated changes in myofibrillar orientation at increasing sarcomere lengths in permeabilized, relaxed, wild-type mouse myocardium from the left ventricle by assessing the angular spread of the 1,0 equatorial reflection (angle σ). At a sarcomere length of 1.9 µm, the angle σ was 0.23 ± 0.01 rad, decreased to 0.19 ± 0.01 rad at a sarcomere length of 2.1 µm, and further decreased to 0.15 ± 0.01 rad at a sarcomere length of 2.3 µm (p < 0.0001). Angle σ was significantly larger in R403Q, a MYH7 hypertrophic cardiomyopathy model, porcine myocardium (0.24 ± 0.01 rad) compared with wild-type myocardium (0.14 ± 0.005 rad; p < 0.0001), as well as in human heart failure tissue (0.19 ± 0.006 rad) when compared with nonfailing samples (0.17 ± 0.007 rad; p = 0.01). These data indicate that diseased myocardium suffers from greater myofibrillar disorientation compared with healthy controls. Finally, we showed that conventional, histology-based analysis of disarray can be subject to user bias and/or sampling error and lead to false positives. Our method for directly assessing myofibrillar orientation avoids the artifacts introduced by conventional histological approaches that assess myocyte orientation and only indirectly evaluate myofibrillar orientation, and provides a precise and objective metric for phenotypically characterizing myocardium. The ability to obtain excellent x-ray diffraction patterns from frozen human myocardium provides a new tool for investigating structural anomalies associated with cardiac diseases.

Myofilament glycation in diabetes reduces contractility by inhibiting tropomyosin movement, is rescued by cMyBPC domains.

Diabetes doubles the risk of developing heart failure (HF). As the prevalence of diabetes grows, so will HF unless the mechanisms connecting these diseases can be identified. Methylglyoxal (MG) is a glycolysis by-product that forms irreversible modifications on lysine and arginine, called glycation. We previously found that myofilament MG glycation causes sarcomere contractile dysfunction and is increased in patients with diabetes and HF. The aim of this study was to discover the molecular mechanisms by which MG glycation of myofilament proteins cause sarcomere dysfunction and to identify therapeutic avenues to compensate. In humans with type 2 diabetes without HF, we found increased glycation of sarcomeric actin compared to non-diabetics and it correlated with decreased calcium sensitivity. Depressed calcium sensitivity is pathogenic for HF, therefore myofilament glycation represents a promising therapeutic target to inhibit the development of HF in diabetics. To identify possible therapeutic targets, we further defined the molecular actions of myofilament glycation. Skinned myocytes exposed to 100 µM MG exhibited decreased calcium sensitivity, maximal calcium-activated force, and crossbridge kinetics. Replicating MG's functional affects using a computer simulation of sarcomere function predicted simultaneous decreases in tropomyosin's blocked-to-closed rate transition and crossbridge duty cycle were consistent with all experimental findings. Stopped-flow experiments and ATPase activity confirmed MG decreased the blocked-to-closed transition rate. Currently, no therapeutics target tropomyosin, so as proof-of-principal, we used a n-terminal peptide of myosin-binding protein C, previously shown to alter tropomyosin's position on actin. C0C2 completely rescued MG-induced calcium desensitization, suggesting a possible treatment for diabetic HF.

Development of an in vitro homeostasis model between airway epithelial cells, bacteria and bacteriophages: a time-lapsed observation of cell viability and inflammatory response.

Bacteriophages represent the most extensive group of viruses within the human virome and have a significant impact on general health and well-being by regulating bacterial population dynamics. Staphylococcus aureus, found in the anterior nostrils, throat and skin, is an opportunistic pathobiont that can cause a wide range of diseases, from chronic inflammation to severe and acute infections. In this study, we developed a human cell-based homeostasis model between a clinically isolated strain of S. aureus 141 and active phages for this strain (PYOSa141) isolated from the commercial Pyophage cocktail (PYO). The cocktail is produced by Eliava BioPreparations Ltd. (Tbilisi, Georgia) and is used as an add-on therapy for bacterial infections, mainly in Georgia. The triptych interaction model was evaluated by time-dependent analysis of cell death and inflammatory response of the nasal and bronchial epithelial cells. Inflammatory mediators (IL-8, CCL5/RANTES, IL-6 and IL-1ß) in the culture supernatants were measured by enzyme-linked immunosorbent assay and cell viability was determined by crystal violet staining. By measuring trans-epithelial electrical resistance, we assessed the epithelial integrity of nasal cells that had differentiated under air-liquid interface conditions. PYOSa141 was found to have a prophylactic effect on airway epithelial cells exposed to S. aureus 141 by effectively down-regulating bacterial-induced inflammation, cell death and epithelial barrier disruption in a time-dependent manner. Overall, the proposed model represents an advance in the way multi-component biological systems can be simulated in vitro.

Temperature-Biased miRNA Expression Patterns during European Sea Bass (Dicentrarchus labrax) Development.

Environmental effects and, particularly, temperature changes have been demonstrated to influence the activity, function, and well-being of teleosts. Temperature may change seasonally in the wild, and in captivity under aquaculture operations. Moreover, climate change is expected to shift temperature profiles worldwide. MicroRNAs (miRNA) are important temperature-sensitive gene-expression regulators acting at the post-transcriptional level. They are known to be key regulators in development, reproduction, and immune responses. Therefore, early larval development of the European sea bass (Dicentrarchus labrax), one of the most extensively cultured species in Mediterranean aquaculture, was investigated at early rearing temperatures, i.e., 15, 17.5, and 20 °C, in regard to the impact of temperatures on miRNAs through sncRNA high-throughput sequencing but also at the phenotypic level in terms of growth, sex, vision, and skeletal deformities. Expression profiling revealed stage- and temperature-specific miRNA expression targeting genes with roles in reproduction and immune response mainly at the flexion and all-fins stages. Similar stage- and temperature-specific results were also observed concerning the number of rod cells and lower jaw elongation. The present work presents for the first time highly promising results on the influence of early rearing temperature at the post-transcriptional level during European sea bass development, with a putative impact on reproduction and immune response, as well as regarding teleost vision and larval development.

Assessment of the Efficacy and Clinical Utility of Different Circulating Tumor Cell (CTC) Detection Assays in Patients with Chemotherapy-Naïve Advanced or Metastatic Non-Small Cell Lung Cancer (NSCLC).

We herein investigated the detection frequency and clinical relevance of circulating tumor cells (CTCs) in chemotherapy-naïve stage IIIB/IV non-small cell lung cancer (NSCLC), by using the CellSearch and real-time CEACAM5mRNA assays. Blood samples from 43 patients were obtained at different time points during first-line chemotherapy. CellSearch revealed the detection of ≥1 CTCs in 41.9%, 40.9%, and 16.7% of patients at baseline, post-1st, and post-2nd treatment cycle, respectively, and of ≥5 CTCs in 11.6%, 9.1%, and 5.6%, respectively. CEACAM5mRNA+ CTCs were detected in 29.3% and 16% of patients pre- and post-treatment, respectively. The positivity concordance between the two assays was 2.2%. CTC-detection by CellSearch (≥5 CTCs: p = 0.004), CEACAM5mRNA (p = 0.010), or by any assay (p = 0.000) was associated with disease progression. Reduced survival was demonstrated for patients harboring ≥5 CTCs (progression-free survival; PFS: p = 0.000; overall survival; OS: p = 0.009), CEACAM5mRNA+ CTCs (PFS: p = 0.043; OS: p = 0.039), and CTCs by any assay (PFS: p = 0.005; OS: p = 0.006, respectively). CTC-detection by any assay independently predicted for increased risk of relapse (hazard ratio; HR: 3.496; p = 0.001) and death (HR: 2.866; p = 0.008). CellSearch-positivity either pre-, post-1st, or post-2nd cycle, was predictive for shorter PFS (p = 0.036) compared to negativity in all time points. Persistent CEACAM5mRNA-positivity pre- and post-treatment was associated with reduced PFS (p = 0.036) and OS (p = 0.026). In conclusion, CTC detection and monitoring using the CellSearch and CEACAM5mRNA assays provides valuable and complementary clinical information for chemo-naïve advanced or metastatic NSCLC.

Lack of negative effects of fasting of gilthead seabream (Sparus aurata) breeders during the spawning period on maternal and egg nutrient composition, fertilization success, and early embryo/larval development.

The effect of fasting on spawning performance, maternal, and egg nutrient composition, and on embryo/larval development was monitored in gilthead seabream (Sparus aurata). Two broodstocks were fasted during two consecutive years, for a period of 43 and 54 days within the spawning season, in a preliminary (year 1, 5-year-old breeders) and the main study (year 2, 6-year-old breeders), respectively. Mean daily fecundity showed a declining trend during fasting in the main study only, while fertilization success was high in both years and it was not affected by fasting, as was hatching and 5-day larval survival. There was a loss of 23.5% of maternal body mass due to fasting, and a reduction in gonadosomatic and hepatosomatic indexes, as well as crude protein in maternal muscle and gonads, but not in the liver. After fasting, muscle Σω-6 PUFA and C18:3ω-3 were reduced while C20:4ω-6, 20:5ω-3/20:4ω-6, and C22:6ω-3/20:4ω-6 increased; in the liver, significant reductions were observed in C16:0, C18:3 ω-3, 20:5ω-3/C22:6ω-3 and increases in C18:0, C20:5ω-3, Σω-6 PUFA, and 20:5ω-3/20:4ω-6; in gonads, C15:0, ΣMUFA, 20:5ω-3/C22:6ω-3, 20:5ω-3/20:4ω-6 were increased, while C18:1ω-9 and C20:5ω-3 decreased. Contrary to maternal tissues, the energy density and proximate composition of the eggs did not change due to fasting. The study suggests that fasting of gilthead seabream breeders for 6-8 weeks during the spawning period does not affect spawning performance, egg proximate composition, or embryo and early larval development since maternal nutrient reserves are mobilized to maintain optimal egg nutrient composition.

Histological evaluation of sex differentiation and early sex identification in hatchery-produced greater amberjack (Seriola dumerili) reared in sea cages.

The histological process of gonadal differentiation, together with the endocrine changes of sex steroid hormones and some of their precursors, was studied in hatchery-produced greater amberjack Seriola dumerili from 101 until 408 days post-hatching (dph), with samplings conducted every 50 days. Histological processing showed that sex differentiation began at 101 dph with the formation of the ovarian cavity in females, while the presumptive males did not yet contain any germ cells in their gonad. At 150 dph, we observed the first germ cells in the developing testes. Sex differentiation in almost all sampled individuals was complete at 408 dph. No size dimorphism was observed between the sexes, and the sex ratio was 1:1, suggesting that there was no influence of early rearing in captivity on sex differentiation. Plasma concentrations of adrenosterone (Ad), androstenedione (Δ4), 11-ketotestosterone (11ΚΤ), testosterone (Τ), estradiol (Ε2), progesterone (P4) and 17,20ß-dihydroxy-4-pregnen-3-one (17,20ßP) were measured in males and females with the use of liquid chromatography tandem mass spectrometry (LC-MS/MS) to examine their role in the sex differentiation process. From the seven hormones, the only one that exhibited differences between the sexes was 11-KT and the plasma 11-KT concentration was found to be a useful indication of greater amberjack sex. Variations were observed in the mean values of Ad, Δ4, 11-KT, T, P4 and 17,20ßP over time in one or both sexes, indicating their involvement in the sex differentiation process.

Endocrine regulation of long-term enhancement of spermiation in meagre (Argyrosomus regius) with GnRHa controlled-delivery systems.

Meagre (Argyrosomus regius) undergo spermatogenesis and spermiation when reared in captivity, but often produce low milt volumes, sometimes with reduced quality and for a limited time period. In the present study we a) compared the efficiency of gonadotropin releasing hormone agonist (GnRHa) implants versus injections on testicular stimulation and spermiation enhancement, b) investigated the effect of GnRHa on the endocrine spermiation regulation (sex steroid hormones), and c) evaluated a commercial induced spawning simulation scenario. Firstly, males (n = 5) were injected with 15.0 ± 0.2 µg GnRHa kg-1 (Injections) or implanted with 51.0 ± 5.1 µg GnRHa kg-1 (Implant) and compared their sperm production response. Secondly, the best hormonal treatment (Implant) was tested treating males (n = 8) with 57.5 ± 7.5 µg GnRHa kg-1 every 3 weeks for a period of 70 days. Milt production was improved by the GnRHa implants with only minor sperm quality alterations (improved sperm motility percentage). Elevated plasma testosterone (T) and 11-ketotestosterone (11-KT) levels were recorded in response to GnRHa implants, while no significant difference for 17,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P) was observed. In the commercial induced spawning simulation, it was shown that meagre females are capable of on-demand induction of spawning at random intervals (5-21 days) using GnRHa injections, over a period of at least 2.5 months. During this period, spermiation enhancement was achieved with GnRHa implants every 3 weeks, producing sperm with stable, in general, quality and motility parameters. Percentage of motile cells, motility duration and density fluctuated significantly, but remained within levels that are considered appropriate for high fertilization success in this species.

Novel insights into SLC25A46-related pathologies in a genetic mouse model.

The mitochondrial protein SLC25A46 has been recently identified as a novel pathogenic cause in a wide spectrum of neurological diseases, including inherited optic atrophy, Charcot-Marie-Tooth type 2, Leigh syndrome, progressive myoclonic ataxia and lethal congenital pontocerebellar hypoplasia. SLC25A46 is an outer membrane protein, member of the Solute Carrier 25 (SLC25) family of nuclear genes encoding mitochondrial carriers, with a role in mitochondrial dynamics and cristae maintenance. Here we identified a loss-of-function mutation in the Slc25a46 gene that causes lethal neuropathology in mice. Mutant mice manifest the main clinical features identified in patients, including ataxia, optic atrophy and cerebellar hypoplasia, which were completely rescued by expression of the human ortholog. Histopathological analysis revealed previously unseen lesions, most notably disrupted cytoarchitecture in the cerebellum and retina and prominent abnormalities in the neuromuscular junction. A distinct lymphoid phenotype was also evident. Our mutant mice provide a valid model for understanding the mechanistic basis of the complex SLC25A46-mediated pathologies, as well as for screening potential therapeutic interventions.

Estrogen but not testosterone preserves myofilament function from doxorubicin-induced cardiotoxicity by reducing oxidative modifications.

Here, we aimed to explore sex differences and the impact of sex hormones on cardiac contractile properties in doxorubicin (DOX)-induced cardiotoxicity. Male and female Sprague-Dawley rats were subjected to sham surgery or gonadectomy and then treated or untreated with DOX (2 mg/kg) every other week for 10 wk. Estrogen preserved maximum active tension (Tmax) with DOX exposure, whereas progesterone and testosterone did not. The effects of sex hormones and DOX correlated with both altered myosin heavy chain isoform expression and myofilament protein oxidation, suggesting both as possible mechanisms. However, acute treatment with oxidative stress (H2O2) or a reducing agent (DTT) indicated that the effects on Tmax were mediated by reversible myofilament oxidative modifications and not only changes in myosin heavy chain isoforms. There were also sex differences in the DOX impact on myofilament Ca2+ sensitivity. DOX increased Ca2+ sensitivity in male rats only in the absence of testosterone and in female rats only in the presence of estrogen. Conversely, DOX decreased Ca2+ sensitivity in female rats in the absence of estrogen. In most instances, this mechanism was through altered phosphorylation of troponin I at Ser23/Ser24. However, there was an additional DOX-induced, estrogen-dependent, irreversible (by DTT) mechanism that altered Ca2+ sensitivity. Our data demonstrate sex differences in cardiac contractile responses to chronic DOX treatment. We conclude that estrogen protects against chronic DOX treatment in the heart, preserving myofilament function. NEW & NOTEWORTHY We identified sex differences in cardiotoxic effects of chronic doxorubicin (DOX) exposure on myofilament function. Estrogen, but not testosterone, decreases DOX-induced oxidative modifications on myofilaments to preserve maximum active tension. In rats, DOX exposure increased Ca2+ sensitivity in the presence of estrogen but decreased Ca2+ sensitivity in the absence of estrogen. In male rats, the DOX-induced shift in Ca2+ sensitivity involved troponin I phosphorylation; in female rats, this was through an estrogen-dependent mechanism.

Spawning kinetics and egg/larval quality of greater amberjack (Seriola dumerili) in response to multiple GnRHa injections or implants.

The greater amberjack (Seriola dumerili) is one of the most promising finfish species for aquaculture, due to its cosmopolitan distribution and acceptability, high growth rates and large size. However, lack of reproduction control has been one of the main bottlenecks for its commercialization. The study examined two endocrine methods for the induction of oocyte maturation/ovulation and spawning, which are based on the induction of endogenous Luteinizing Hormone (LH) release from the pituitary, through the use of Gonadotropin Releasing Hormone agonist (GnRHa) either in the form of sustained-release delivery systems (implants) or injections (acute release). The stock (n = 28) consisted of wild fish captured in 2011 and each GnRHa administration method was conducted in two replicates, with 6 and 8 fish per tank, respectively, at a 1:1 sex ratio. Fish were given a GnRHa injection once a week (three administrations), or a GnRHa implant every 2 weeks (two administrations). Mean daily relative fecundity was significantly higher (t1.3 = -5.24, P = 0.012) in the implanted fish (15,170 ±â€¯2,738 eggs kg-1 female day-1) compared to the injected fish (6,119 ±â€¯2,790 eggs kg-1 female day-1). Total relative fecundity was also significantly higher (t1.3 = -9.93, P = 0.003) in the implanted fish (102,402 ±â€¯20,337 eggs kg-1 female) compared to the injected ones (26,517 ±â€¯9,938 eggs kg-1 female), but there were no differences in the quality of eggs in terms of fertilization, 24-h embryo survival, hatching and 5-d larval survival. The number of females with fully vitellogenic oocytes eligible for induction of spawning decreased from the initial to the final sampling, from 7 to 6 females for the GnRHa implant treatment and from 7 to 3 females for the GnRHa injection treatment. In addition to apparently promoting the proper endocrine changes leading to multiple cycles of oocyte maturation, ovulation and spawning, and thus producing larger numbers of eggs; the use of GnRHa implants may be more appropriate in greater amberjack than multiple injections because (a) it also stimulates vitellogenesis and (b) involves less handling of the fish.

Effects of diisononyl phthalate (DiNP) on the endocannabinoid and reproductive systems of male gilthead sea bream (Sparus aurata) during the spawning season.

Diisononyl phthalate (DiNP) is a plasticizer used to improve plastic performance in a large variety of items which has been reported as an endocrine-disrupting chemical (EDC) in several organisms. The endocannabinoid system (ECS) is a cellular signaling system, whose functionality is tightly involved with reproductive function. The aim of the present study was the assessment of the effects of DiNP on the gonadal ECS and on the reproductive function of male gilthead sea bream Sparus aurata, an important marine aquacultured species in Europe, during the reproductive season. Fish were fed for 21 days with two diets contaminated with different nominal concentrations of DiNP (DiNP LOW at 15 µg DiNP kg-1 bw day-1 and DiNP HIGH at 1500 µg DiNP kg-1 bw day-1), based on the tolerable daily intake (TDI) ruled by the European Food Safety Authority for humans. The transcription of several genes related to the ECS was affected by the DiNP. Specifically, DiNP reduced the levels of endocannabinoids and endocannabinoid-like mediators, concomitant with the increase of fatty acid amide hydrolase (FAAH) activity. At the histological level, DiNP LOW induced the highest occurrence of individuals with regressed testes. Steroidogenesis was affected significantly, since plasma 11-ketotestosterone (11-KT), the main active androgen in fish, was significantly decreased by the DiNP HIGH treatment, while plasma 17ß-estradiol (E2) levels were raised, associated with an increase of the gonadosomatic index (GSI). Additionally, the level of testosterone (T) was significantly increased in the DiNP LOW group, however, the same DiNP concentration reduced the levels of 17,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P). The production of sperm was in general not affected, since spermiation index, sperm density, survival and the duration of forward motility did not exhibit any changes compared to controls. However, computer-assisted sperm analysis (CASA) showed that DiNP reduced the percentage of motile cells. The results clearly suggest a negative effect of DiNP via the diet on the male endocrine system of gilthead sea bream during the reproductive season.

Mutations in troponin T associated with Hypertrophic Cardiomyopathy increase Ca(2+)-sensitivity and suppress the modulation of Ca(2+)-sensitivity by troponin I phosphorylation.

We investigated the effect of 7 Hypertrophic Cardiomyopathy (HCM)-causing mutations in troponin T (TnT) on troponin function in thin filaments reconstituted with actin and human cardiac tropomyosin. We used the quantitative in vitro motility assay to study Ca(2+)-regulation of unloaded movement and its modulation by troponin I phosphorylation. Troponin from a patient with the K280N TnT mutation showed no difference in Ca(2+)-sensitivity when compared with donor heart troponin and the Ca(2+)-sensitivity was also independent of the troponin I phosphorylation level (uncoupled). The recombinant K280N TnT mutation increased Ca(2+)-sensitivity 1.7-fold and was also uncoupled. The R92Q TnT mutation in troponin from transgenic mouse increased Ca(2+)-sensitivity and was also completely uncoupled. Five TnT mutations (Δ14, Δ28 + 7, ΔE160, S179F and K273E) studied in recombinant troponin increased Ca(2+)-sensitivity and were all fully uncoupled. Thus, for HCM-causing mutations in TnT, Ca(2+)-sensitisation together with uncoupling in vitro is the usual response and both factors may contribute to the HCM phenotype. We also found that Epigallocatechin-3-gallate (EGCG) can restore coupling to all uncoupled HCM-causing TnT mutations. In fact the combination of Ca(2+)-desensitisation and re-coupling due to EGCG completely reverses both the abnormalities found in troponin with a TnT HCM mutation suggesting it may have therapeutic potential.

Troponin structure: its modulation by Ca(2+) and phosphorylation studied by molecular dynamics simulations.

The only available crystal structure of the human cardiac troponin molecule (cTn) in the Ca(2+) activated state does not include crucial segments, including the N-terminus of the cTn inhibitory subunit (cTnI). We have applied all-atom molecular dynamics (MD) simulations to study the structure and dynamics of cTn, both in the unphosphorylated and bis-phosphorylated states at Ser23/Ser24 of cTnI. We performed multiple microsecond MD simulations of wild type (WT) cTn (6, 5 µs) and bisphosphorylated (SP23/SP24) cTn (9 µs) on a 419 amino acid cTn model containing human sequence cTnC (1-161), cTnI (1-171) and cTnT (212-298), including residues not present in the crystal structure. We have compared our results to previous computational studies, and proven that longer simulations and a water box of at least 25 Å are needed to sample the interesting conformational shifts both in the native and bis-phosphorylated states. As a consequence of the introduction into the model of the C-terminus of cTnT that was missing in previous studies, cTnC-cTnI interactions that are responsible for the cTn dynamics are altered. We have also shown that phosphorylation does not increase cTn fluctuations, and its effects on the protein-protein interaction profiles cannot be assessed in a significant way. Finally, we propose that phosphorylation could provoke a loss of Ca(2+) by stabilizing out-of-coordination distances of the cTnC's EF hand II residues, and in particular Ser 69.