Genetic delineation between and within the widespread coccolithophore morpho‐species Emiliania huxleyi and Gephyrocapsa oceanica (Haptophyta)

Emiliania huxleyi and Gephyrocapsa oceanica are abundant coccolithophore morpho‐species that play key roles in ocean carbon cycling due to their importance as both primary producers and cal‐cifiers. Global change processes such as ocean acidification impact these key calcifying species. The physiology of E. huxleyi, a developing model species, has been widely studied, but its genetic delineation from G. oceanica remains unclear due to a lack of resolution in classical genetic markers. Using nuclear (18S rDNA and 28S rDNA), mitochondrial (cox1, cox2, cox3, rpl16, and dam), and plastidial (16S rDNA, rbcL, tufA, and petA) DNA markers from 99 E. huxleyi and 44 G. oceanica strains, we conducted a multigene/multistrain survey to compare the suitability of different markers for resolving phylogenetic patterns within and between these two morpho‐species. The nuclear genes tested did not provide sufficient resolution to discriminate between the two morpho‐species that diverged only 291Kya. Typical patterns of incomplete lineage sorting were generated in phylogenetic analyses using plastidial genes. In contrast, full morpho‐species delineation was achieved with mitochondrial markers and common intra‐morpho‐species phylogenetic patterns were observed despite differing rates of DNA substitution. Mitochondrial genes are thus promising barcodes for distinguishing these coccolithophore morpho‐species, in particular in the context of environmental monitoring.     

Bacterial cellulose as carrier for immobilization of laccase: Optimization and characterization

Bacterial cellulose (BC) has attracted attention as a new functional material due to its excellent mechanical strength, tridimensional nanostructure, high purity, and increased water absorption, compared to plant cellulose. In this work, commercial laccase was immobilized on BC and the influence of enzyme concentration, contact time, and pH was optimized toward the recovery activity of immobilized laccase. This optimization was carried out using a 3³ experimental design and response surface methodology. Enzyme concentration played a critical role in laccase immobilization. Under optimized conditions (0.15 μL L^?1 of enzyme concentration, 4.8 h of contact time, pH 5.4), the predicted and experimental response were equal to 47.88 and 49.30%, respectively. The thermal stability of the immobilized laccase was found to increase notably at 60 and 70°C presenting stabilization factor equal to 1.79 and 2.11, respectively. The immobilized laccase showed high operational stability, since it retained 86% of its initial activity after seven consecutive biocatalytic cycles of reaction with 2,2′‐azinobis‐(3‐ethylbenzothiazoline‐6‐sulfonic acid). Kinetic studies showed that the values of Michaelis–Menten constant and maximum reaction rate decreased upon immobilization (9.9‐ and 1.6‐fold, respectively). Globally, the use of immobilized laccase on BC offers an interesting tool for industrial biocatalytic applications.     

Ion current activity and molecules modulating maturation and growth stages of ascidian (Ciona intestinalis) oocytes

Electrophysiological techniques were used to study ion currents in the ascidian Ciona intestinalis oocyte plasma membranes during different stages of growth and meiosis. Three stages (A, B, C) of immature oocytes were discriminated in the ovary, with the germinal vesicle (GV) showing specific different features of growth and maturation. Stage A (pre‐vitellogenic) oocytes exhibited the highest L‐type Ca²⁺current activity, and were incompetent for meiosis resumption. Stage B (vitellogenic) oocytes showed Na⁺ currents that remained high during the maturation, up to the post‐vitellogenic stage C oocytes. The latter had acquired meiotic competence, undergoing spontaneous maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation plays a specific role in embryo development. Spontaneous maturation was inhibited at low pH whereas trypsin was able to trigger germinal vesicle breakdown (GVBD) regardless of pH; in addition spontaneous maturation was not affected by removal of follicle cells or by inhibiting junctional communication between oocyte and follicle cells. Taken together these results imply: (i) Ca²⁺ and Na⁺ currents are involved in meiotic progression, growth, and acquisition of meiotic competence; (ii) trypsin‐like molecules may have a role as candidates for providing the physiological stimulus to resume meiosis. Finally, we provide evidence that follicle cells in Ciona are not involved in triggering GVBD as it occurs in other ascidians. Mol. Reprod. Dev. 76: 1084–1093, 2009. © 2009 Wiley‐Liss, Inc.     

Strategies for improving fertility in the modern dairy cow

The high producing dairy cow of the 21st century is subfertile during lactation. Our objectives are to characterize physiological periods limiting reproductive performance and to describe integrated management strategies to improve pregnancy rates. Ovarian recrudescence with normal re-occurring estrous cycles and restoration of fertility to first service are associated with a reduced occurrence of periparturient metabolic and reproductive disorders. Marked negative changes in energy balance and reduced immunocompetence influence gonadotropic and metabolic hormones. Induced ovarian inactivity was associated with enhanced uterine involution. Post-partum health and reproductive performance were improved when by-pass lipids enriched in polyunsaturated fatty acids were fed in the pre- and post-partum periods. Pharmaceutical control of follicle, CL, and uterine function with PGF, GnRH and intravaginal progesterone releasing inserts, has permitted development of more optimal timed-insemination programs for first service. Likewise, resynchronization of nonpregnant cows coupled with the use of ultrasound for early pregnancy diagnosis provides the opportunity for a second timed-insemination within 3 days of a nonpregnant diagnosis. Bovine somatotropin (bST) increases embryo development and embryo survival when coupled with a timed-insemination program or cows detected in estrus. Presence of a conceptus alters endometrial expression of genes and proteins in response to bST and nutraceuticals (i.e., unsaturated fatty acids such as eicosapentaenoic and docosahexaenoic acid in by-pass lipids) to improve pregnancy rates. Postovulatory increases in progesterone may enhance pregnancy rates in targeted populations of lactating dairy cows, but timing and magnitude of the progesterone increases are pharmaceutically dependent.     

The use of a deslorelin implant (GnRH agonist) during the late embryonic period to reduce pregnancy loss

Embryonic and fetal mortality reduce reproductive performance of lactating dairy cows. The objectives of this study were to reduce pregnancy loss by administering a deslorelin implant (GnRH agonist) during the late embryonic period, to reduce follicular growth, induce accessory corpora lutea, and increase plasma progesterone concentrations. Lactating dairy cows received an implant containing 2.1 mg of deslorelin (Deslorelin group; n = 89) or no treatment (Control group; n = 92) on Day 27 of pregnancy. Pregnancy, ovarian structures and plasma progesterone concentrations were determined on Days 27 and 45, and pregnancy was re-confirmed on Day 90. On Day 45, mean +/- S.E.M. numbers of class 2 (6-9 mm; 0.72+/-0.19) and class 3 (> or = 10 mm; 0.86 +/- 0.12) follicles for cows in the Deslorelin group were lower (P < 0.01) than the numbers of class 2 (1.90 +/- 0.18) and class 3 (1.92 +/- 0.12) follicles for cows in the Control group. On Day 45, the number of accessory corpora lutea for cows in the Deslorelin group (1.80 +/- 0.07) were greater (P < 0.01) than for cows in the Control group (1.31 +/- 0.07). On Day 45, plasma progesterone concentration was increased (P < 0.01) for cows in the Deslorelin group (8.03 +/- 0.33 ng/mL) compared to cows in the Control group (6.40 +/- 0.31 ng/mL). Pregnancy losses did not differ between Days 27 and 45 and Days 45 and 90 for cows in the Control (15.2 and 11.0%, respectively) and Deslorelin groups (20.2 and 10.5%, respectively). However, in the Deslorelin group, pregnancy loss between Days 45 and 90 was lower (P < 0.05) for cows that formed an accessory CL (0%) compared to cows that did not form an accessory CL (16.1%).     

Vitrification of pre-pubertal ovine cumulus-oocyte complexes: effect of cytochalasin B pre-treatment

The aim of this study was to evaluate the effect of cytochalasin B (CCB) pre-treatment before vitrification on ability of immature oocytes from lamb ovaries to progress until metaphase II (MII) stage after vitrification/warming procedure. Cumulus-oocyte complexes (COCs) were obtained from ovaries of lambs, from 80 to 90 days old, collected from a local slaughterhouse. Before vitrification, COCs were randomly distributed in two experimental groups corresponding to the incubation with or without 7.5 microg/ml CCB for 30 min. In order to study cryoprotectant and CCB pre-treatment toxicity (toxicity test), oocytes were exposed to cryoprotectants, with or without CCB pre-treatment, but without plunging into N2 liquid. Vitrification solution was composed by 4.48 M EG plus 3.50 M DMSO supplemented with 0.25 M sucrose. Two-step addition was performed. After vitrification or toxicity test, COCs were matured in bicarbonate-buffered TCM 199 containing 10% foetal calf serum and 10 ng/ml epidermal growth factor. A sample of COCs was directly in vitro matured (control group). Rates of MII oocytes of toxicity groups both, with or without CCB pre-treatment were lower than control group (41.1-50.0 versus 79.9, respectively; P<0.05). After vitrification, a lower number of oocytes progressed to MII stage in comparison with non-vitrification groups (P<0.05). In vitrified groups both with or without CCB pre-treatment 8.0 and 12.7%, respectively, of immature oocytes reached MII stage by the end of in vitro maturation culture. No effect of CCB was observed, either in the toxicity or vitrified groups. In conclusion, no effect of CCB pre-treatment before vitrification was detected in this study with immature oocytes of pre-pubertal sheep. More studies are needed in order to increase ovine oocyte survival after vitrification.     

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) regulates endothelial nitric oxide synthase (eNOS) activity and its localization within the human vein endothelial cells (HUVEC) in culture

We have recently demonstrated that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increases endothelial nitric oxide synthase (eNOS) phosphorylation, NOS activity, and nitric oxide (NO) synthesis in cultured human umbilical vein endothelial cells (HUVEC), without inducing apoptotic cell death. Although an important factor that regulates eNOS activity is its localization within the cells, little is known about the role of TRAIL in the regulation of eNOS trafficking among cellular compartments and the cytoskeleton involvement in this machinery. Then, we did both quantitative and semi-quantitative evaluations with biochemical assays and immune fluorescence microscopy in the presence of specific inhibitors of NOS activity as well as of cytoskeletal microtubule structures. In our cellular model, TRAIL treatment not only increased NO levels but also caused a time-dependent NO migration of fluorescent spots from the plasma membrane to the inner part of the cells. In unstimulated cells, most of the eNOS was localized at the cell membranes. However, within 10 min following addition of TRAIL, nearly all the cells showed an increased cytoplasm localization of eNOS which appeared co-localized with the Golgi apparatus at a higher extent than in unstimulated cells. These effects were associated to an increased formation of trans-cytoplasm stress fibers with no significant changes of the microtubule network. Conversely, microtubule disruption and Golgi scattering induced with Nocodazole treatment inhibited TRAIL-increased NOS activity, indicating that, on cultured HUVEC, TRAIL ability to affect NO production by regulating eNOS sub-cellular distribution is mediated by cytoskeleton and Golgi complex modifications.     

Ca2+ and Na+ current patterns during oocyte maturation, fertilization, and early developmental stages of Ciona intestinalis

Using the whole-cell voltage clamp technique, the electrical changes in oocyte and embryo plasma membrane were followed during different meiotic and developmental stages in Ciona intestinalis. We show, for the first time, an electrophysiological characterization of the plasma membrane in oocytes at the germinal vesicle (GV) stage with high L-type calcium (Ca2+) current activity that decreased through meiosis. Moreover, the absence of Ca2+ reduced germinal vesicle breakdown (GVBD), which is consistent with a role of Ca2+ currents in the prophase/metaphase transition. In mature oocytes at the metaphase I (MI) stage, Ca2+ currents decreased and then disappeared and sodium (Na+) currents first appeared remaining high up to the zygote stage. Intracellular Ca2+ release was higher in MI than in GV, indicating that Ca2+ currents in GV may contribute to fill the stores which are essential for oocyte contraction at fertilization. The fertilization current generated in Na+ free sea water was significantly lower than the control; furthermore, oocytes fertilized in the absence of Na+ showed high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, suggesting that signaling pathways that mediate first cleavage do not rely on ion current activities. At the 8-cell stage embryo, a resumption of Na+ current activity and conductance occurred, without a correlation with specific blastomeres. Taken together, these results imply: (i) an involvement of L-type Ca2+ currents in meiotic progression from the GV to MI stage; (ii) a role of Na+ currents during electrical events at fertilization and subsequent development; (iii) a major role of plasma membrane permeability and a minor function of specific currents during initial cell line segregation events.     

Body composition and physical performance during a National Collegiate Athletic Association Division I men's soccer season

The purpose of this study was to examine changes in body composition (BC) and physical performance tests (PT) resulting from a competitive season in soccer. Twenty-five male collegiate players (age = 19.9 +/- 1.3 years; height = 177.6 +/- 6.4 cm; body mass = 77.6 +/- 8.6 kg, and percentage body fat = 12.8 +/- 5.2%) were tested before (PRE) and after (POST) the 2003-2004 National Collegiate Athletic Association season. The following tests were performed: BC (anthropometric and dual energy x-ray absorptiometry measurements), vertical jump (VJ), 9.1-m (9 m) and 36.5-m (36 m) sprint, lower-body power (LP), total body power (TP), and cardiorespiratory endurance (VO(2)max). Training was divided into soccer-specific training: field warm-up drills, practices, games, and additional conditioning sessions. A daily, unplanned, nonlinear periodization model was used to assign session volume and intensity for strength sessions (total repetitions < or =96 and workload was > or =80% of 1 repetition maximum). For the entire team, body mass significantly increased by 1.5 +/- 0.4 kg from PRE to POST due to a significant increase in total lean tissue (0.9 +/- 0.2 kg). Regionally, lean tissue mass significantly increased in the legs (0.4 +/- 0.0 kg) and trunk (0.3 +/- 0.1 kg). Physical performance variables were very similar for the entire team at PRE and POST; VJ (cm) = 61.9 +/- 7.1 PRE vs. 63.3 +/- 8.0 POST, 9.1-m (s) = 1.7 +/- 0.1 PRE and POST, 36.5-m (s) = 5.0 +/- 0.2 PRE and POST, predicted VO(2)max (ml.kg.min(-1))= 59.8 +/- 3.3 PRE vs. 60.9 +/- 3.4 POST. The only significant improvements across the season were for TP (17.3%) and for LP (10.7%). In conclusion, soccer athletes who begin a season with a high level of fitness can maintain, and in some cases improve, body composition and physical performance from before to after a competitive season. A correct combination of soccer-specific practices and strength and conditioning programs can maintain and develop physical performance, allowing a soccer athlete to perform optimally throughout pre-, in-, and postseason play.     

Robust, easy, and dose-sensitive methylation test for the diagnosis of Prader-Willi and Angelman syndromes

We present a new method for differential diagnosis of Prader-Willi (PWS) and Angelman syndromes (AS) that requires only a small amount of DNA including that obtained from amniocentesis specimens. This method not only proved to be robust and rapid, but, most importantly, it can be dosage sensitive, supplying additional information useful for genetic counselling. After methylation-dependent digestion of DNA with HpaII or McrBC, exon 1 of the SNRPN gene is amplified together with a sequence in the CpG island of the H19 gene. Given the similarities in sequence composition and methylation status between the amplified sequences, their co-amplification under semiquantitative conditions allows an easy discrimination between single dosage (present in deletions or chromosomal translocations) and a double-dosage state (uniparental disomy or imprinting error), when the appropriate controls are included. The method we have developed in combination with standard cytogenetic studies and segregation analysis of microsatellite markers offers a rapid and easy procedure to resolve most suspected cases of PWS and AS, and consequently to provide accurate genetic counselling.