Incidence and severity of ovarian hyperstimulation syndrome (OHSS) in high responders after gonadotropin-releasing hormone (GnRH) agonist trigger in "freeze-all" approach.

OBJECTIVE: To determine the incidence and severity of ovarian hyperstimulation syndrome (OHSS) in high responders (25-35 follicles with a diameter of ≥12 mm on day of triggering) who received a gonadotropin-releasing hormone (GnRH) agonist to trigger final follicular maturation. METHODS: We used individual data from women who participated in four different clinical trials and were high responders to ovarian stimulation in a GnRH antagonist protocol in this retrospective combined analysis. All women were evaluated for signs and symptoms of OHSS using identical criteria based on Golan's system (1989). RESULTS: High responders (n = 77) were of different ethnicities. There were no differences in baseline characteristics between women with or without signs and symptoms of OHSS. Mean ± standard deviation baseline data were: age, 32.3 ± 3.5 years; anti-Müllerian hormone, 42.4 ± 20.7 pmol/L; antral follicle count, 21.5 ± 9.2. Before triggering, duration of stimulation was 9.5 ± 1.6 days and the mean number of follicles with a diameter of ≥12 mm and ≥17 mm was 26.5 ± 4.4 and 8.8 ± 4.7, respectively. Mean serum estradiol (17,159 pmol/l) and progesterone (5.1 nmol/l) levels were high at 36 h after triggering. Overall, 17/77 high responders (22%) developed signs and symptoms of mild OHSS which lasted 6-21 days. The most frequently prescribed medication was cabergoline to prevent worsening of OHSS. No severe OHSS occurred and no OHSS cases were reported as serious adverse events. CONCLUSIONS: High responders receiving GnRH agonist for triggering should be informed that they may experience signs and symptoms of mild OHSS.

Is coracohumeral distance associated with pain-function, and shoulder range of movement, in chronic anterior shoulder pain?

BACKGROUND: The aim of this study was twofold: (i) to assess the intrarater reliability of coracohumeral distance; (ii) to investigate the level of association between coracohumeral distance measured by ultrasonography, and pain-disability and shoulder range of movement, in patients suffering from chronic anterior shoulder pain. METHODS: An observational, cross sectional study was carried out. A convenience sample comprised of 87 patients with chronic anterior shoulder pain was assessed from 3 primary care centres. Main outcomes as pain and function were measured through the shoulder pain and disability index. Furthermore, shoulder range of movement-free of pain in shoulder elevation, as well as coracohumeral distance at both 0 and 60 degrees, were collected. RESULTS: Absence of any correlation was found between coracohumeral distance and shoulder pain and disability index at both 0 and 60 degrees of shoulder elevation. Furthermore, absence of any correlation was found between coracohumeral distance measurements and active shoulder range of movement -free of pain. CONCLUSIONS: There was poor association between coracohumeral distance and shoulder pain and function, as well as with shoulder range of movement, in patients with chronic anterior shoulder pain. Hence, clinicians should consider, not only increasing this space, but also other possibilities in their therapies, when patients with anterior shoulder pain are treated. TRIAL REGISTRATION: ACTRN12614000144617 . Registered: 1st March 2014.

Insulin and Leptin Signaling in Placenta from Gestational Diabetic Subjects.

Insulin and leptin receptors are known to share signaling pathways, such as JAK2/STAT-3 (Janus kinase2/signal transduction and activator of transcription3), MAPK (Mitogen activated protein kinase), and PI3K (phosphoinositide 3-kinase). Both positive and negative cross-talk have been previously found in different cellular systems. Gestational diabetes (GDM) is a pathophysiological state with high circulating levels of both insulin and leptin. We have previously found that these 3 signaling pathways are activated in placenta from GDM patients to promote translation, involving the activation of leptin receptor. Now, we have tested the hypothesis that both leptin and insulin receptors might contribute to this activation in a positive way that may become negative when the system is overactivated. We studied the activation of leptin and insulin receptors in placenta from GDM and healthy pregnancies. We have also performed in vitro studies with insulin and leptin stimulation of trophoblast explants from healthy placenta. We have found that both leptin and insulin receptors are activated in placenta from GDM. In vitro stimulation of trophoblast explants with both leptin and insulin at submaximal doses (0.1 nM) potentiated the activation of signaling, whereas preincubation with maximal concentrations of insulin (10 nM) and further stimulation with leptin showed negative effect. Trophoblastic explants from GDM placenta, which presented high signaling levels, had a negative signaling effect when further incubated in vitro with leptin. In conclusion, insulin and leptin receptors have positive effects on signaling, contributing to high signaling levels in GDM placenta, but insulin and leptin have negative effects upon overstimulation.

Increased Expression of Aquaporin 9 in Trophoblast From Gestational Diabetic Patients.

Gestational diabetes mellitus is the most frequent pathophysiological alteration in pregnancy, increasing the incidence of complications in both mother and fetus. The macrosomia that occurs in these fetuses may be related with some changes in nutrient transport mechanism in placenta. The presence of aquaporin 9, an aquaglyceroporin, has previously been demonstrated in placenta. We raised the question whether aquaporin 9 expression may be upregulated in placenta from gestational diabetes, thus providing a faster transport of glycerol and water through placenta. We studied 21 placentas (13 controls and 8 gestational diabetes) from cesarean delivery at term. The expression of aquaporin 9 was analyzed by quantitative PCR, immunoblot, and immunohistochemistry. The median values from quantitative PCR were compared by nonparametric tests for independent samples (Mann-Whitney U-test). We have found that trophoblast from gestational diabetes express higher amount of aquaporin 9, which was found statistically significant (p<0.05). The increase in aquaporin 9 expression was confirmed by immunoblot, and localization in the syncytiotrophoblast was checked by immunohistochemistry. The increase in aquaporin 9 expression in placenta from gestational diabetes may contribute to the higher transport rate in this pathology of pregnancy.

Expression of neurokinin B/NK3 receptor and kisspeptin/KISS1 receptor in human granulosa cells.

STUDY QUESTION: Are neurokinin B (NKB), NK3 receptor (NK3R), kisspeptin (KISS1) and kisspeptin receptor (KISS1R) expressed in human ovarian granulosa cells? SUMMARY ANSWER: The NKB/NK3R and kisspeptin/KISS1R systems are co-expressed and functionally active in ovarian granulosa cells. WHAT IS KNOWN ALREADY: The NKB/NK3R and KISS1/KISS1R systems are essential for reproduction. In addition to their well-recognized role in hypothalamic neurons, these peptide systems may contribute to the control of fertility by acting directly on the gonads, but such a direct gonadal role remains largely unknown. STUDY DESIGN, SIZE, DURATION: This study analyzed matched mural granulosa cells (MGCs) and cumulus cells (CCs) collected from preovulatory follicles of oocyte donors at the time of oocyte retrieval. PARTICIPANTS/MATERIALS, SETTING, METHODS: The samples were provided by 56 oocyte donor women undergoing ovarian stimulation treatment. Follicular fluid samples containing MGCs and cumulus-oocyte complexes were collected after transvaginal ultrasound-guided oocyte retrieval. RT-PCR, quantitative real-time PCR, immunocytochemistry and western blot were used to investigate the pattern of expression of the NKB/NK3R and KISS/KISS1R systems in MGCs and CCs. Intracellular free Ca(2+) levels, [Ca(2+)]i, in MGCs after exposure to NKB or KISS1, in the presence or not of tachykinin receptor antagonists, were also measured. MAIN OUTCOME AND THE ROLE OF CHANCE: NKB/NK3R and KISS1/KISS1R systems were expressed, at the mRNA and protein levels, in MGCs and CCs, with significantly higher expression in CCs. Kisspeptin increased the [Ca(2+)]i in the cytosol of human MGCs while exposure to NKB failed to induce any change in [Ca(2+)]i. However, the [Ca(2+)]i response to kisspeptin was reduced in the presence of NKB. The inhibitory effect of NKB was only partially mimicked by the NK3R agonist, senktide and marginally suppressed by the NK3R-selective antagonist SB 222200. Yet, a cocktail of antagonists selective for the NK1, NK2 and NK3 receptors blocked the effect of NKB. LIMITATIONS, REASONS FOR CAUTION: The granulosa and cumulus cells were obtained from oocyte donors undergoing ovarian stimulation, which in comparison with natural cycles, may have affected gene and protein expression in granulosa cells. WIDER IMPLICATIONS OF THE FINDINGS: Our data demonstrate that, in addition to their indispensable effects at the central nervous system, the NKB/NK3R and kisspeptin/KISS1R systems are co-expressed and are functionally active in non-neuronal reproductive cells of the female gonads, the ovarian granulosa cells. STUDY FUNDING/ COMPETING INTERESTS: This work was supported by grants from Ministerio de Economía y Competitividad (CTQ2011-25564 and BFI2011-25021) and Junta de Andalucía (P08-CVI-04185), Spain. J.G.-O., F.M.P., M.F.-S., N.P., A.C.-R., T.A.A., M.H., M.R., M.T.-S. and L.C. have nothing to declare.

Camouflage therapy workshop for pediatric dermatology patients: a review of 6 cases.

Certain skin conditions, such as vitiligo, acne, vascular malformations, and surgical scars, can impair the quality of life of pediatric patients, especially adolescents-even to the point of hindering psychosocial development. We review the cases of 6 patients with discoloration or scarring, predominantly of the face, who attended our cosmetic camouflage workshops from January through December 2012. The quality-of-life impact of their skin disorder was assessed before and after workshop attendance. Cosmetic camouflage is an easily replicated, cheap, and noninvasive adjunctive treatment of great potential value in managing skin conditions that impair the physical and emotional well-being of pediatric patients.

Surgical treatment of giant anal condyloma in HIV patients: unanswered questions.

We are writing to comment on the article by Uribe et al., "Management on giant anal condyloma by wide local excision and anoplasty". [1] Anogenital warts are among the most common sexually transmitted diseases seen in surgical practice, they are found in up to 1.7% of the general population, but in HIV population, they range from 3 to 24.9%. It is important to realise that 78% of patients with external anogenital warts have internal lesions as well although they are unlikely to be proximal to the dentate line. [2] This article is protected by copyright. All rights reserved.

Impact of assisted reproduction treatments on Spanish newborns: report of 14,119 pregnancies.

PURPOSE: To investigate neonatal malformation, prematurity, and stillbirth in singleton and multiple pregnancies derived from different Assisted Reproductive Techniques (ART). METHODS: In this prospective cohort study data were collected, from private and public Spanish IVF units, during the years 2008 and 2009. During this period, 8,682 pregnancies were analysed from the initial 14,119 pregnancies reported. Pregnancies included in the study derived from IUI (n = 1,065), IVF (n = 838), ICSI (n = 5,080), FET (n = 1,404) and PGD (n = 295). This first analysis focuses primarily on neonatal malformation, prematurity, and stillbirth both in singleton and multiple pregnancies derived from different ART. Malformations were classified according to the WHO ICD 10 code. RESULTS: Malformations were found in 0.83 % of our newborns. No differences in malformations were observed between singletons or multiples independently of the ART used. There was a significant difference in prematurity rate among singletons depending on treatment but this association was not observed in multiple pregnancies. Stillbirth was significantly lower in singleton (0.72 %) than in multiple pregnancies (1.82 %). CONCLUSIONS: The percentage of malformations observed in ART newborns was similar to the rate observed in the normally-conceived Spanish population. Multiplicity seems to be the most important factor associated with an increased incidence of newborn complications such as prematurity or stillbirth.

Certification of protein biomarker standards using element MS and generic standards: Application to human cytokines.

The availability of protein standards and methods for their characterization, quantification, and purity assessment are currently a bottleneck in absolute quantitative proteomics. In this work, we introduce an absolute quantitative analytical strategy based on ICP-MS sulfur detection that uses sulfate as generic standard to quantify and certify the mass purity of protein standards. The methodology combines capillary chromatographic separation with parallel detection with ICP-MS and ESI-MS to determine proteoforms concentration and identity, respectively. The workability of the methodology was demonstrated using recombinant human cytokine standards IP-10 and Flt3L (2 batches), which are relevant biomarkers for carcinoma or inflammatory diseases. Every key factor (transport efficiency, column recovery, signal stability and internal standard suitability) was taken into account and certified BSA standard was used as quality control for validation purposes. Protein quantification values and resulting mass purity certification of IP-10 and one batch of Flt3L were very high (100 and 86%, respectively). Lower mass purity obtained for another batch of Flt3L (<70%) concurred with the finding of significant proteoforms resulted from oxidation processes as observed by parallel ESI-MS.

Characterization of the kisspeptin system in human spermatozoa.

Kisspeptin, the product of the KISS1 gene, plays an essential role in the regulation of spermatogenesis acting primarily at the hypothalamic level of the gonadotropic axis. However, the presence of kisspeptin and its canonical receptor, KISS1R, in spermatozoa has not been explored nor the direct effects of kisspeptin on sperm function have been studied so far. In the present study, we analysed the expression of kisspeptin and its receptor in sperm cells by western blot and immunocytochemistry assays and evaluated the effects of exposure to kisspeptin on sperm intracellular Ca(2+) concentration, [Ca(2+)]i, sperm motility, sperm hyperactivation and the acrosome reaction. Changes in [Ca(2+)]i were monitored using Fura-2, sperm kinematic parameters were measured using computer-assisted sperm analysis (CASA), and the acrosome reaction was measured using fluorescein isothiocyanate-coupled Pisum sativum agglutinin lectin (FITC-PSA method). We found that kisspeptin and its receptor are present in sperm cells, where both are mainly localized in the sperm head, around the neck and in the flagellum midpiece. Exposure to kisspeptin caused a slow, progressive increase in [Ca(2+)]i, which reached a plateau about 3-6 min after kisspeptin exposure. In addition, kisspeptin modulated sperm progressive motility causing a biphasic (stimulatory and inhibitory) response and also induced transient sperm hyperactivation. The effects of kisspeptin on sperm motility and hyperactivation were inhibited by the antagonist of KISS1R, peptide 234. Kisspeptin did not induce the acrosome reaction in human spermatozoa. These data show for the first time that kisspeptin and its receptor are present in human spermatozoa and modulate key parameters of sperm function. This may represent an additional mechanism for their crucial function in the control of male fertility.

[Models of public care for the population with acquired brain injury in Spain: a study of the situation by Spanish autonomous communities]. / Modelos de atención pública a la población con daño cerebral adquirido en España: un estudio de la situación por comunidades autónomas.

INTRODUCTION: Rehabilitation after Acquired Brain Injury (ABI) is a dynamic, progressive process aimed at functional improvement of the affected person and their family. There is an international consensus on how the model that guides this process should be; however, in our country there are numerous territorial discrepancies regarding its application. Therefore, the objective of this work is to analyze and update the knowledge about the practices developed in the field of rehabilitation and the regulations available in each autonomous community. SUBJECTS AND METHODS: 62 participants (71.7% women), including both direct care professionals and public administration positions from the 17 Spanish Autonomous Communities and the autonomous cities of Ceuta and Melilla. A questionnaire composed of 31 questions on the functioning of rehabilitation throughout the three phases of care that often follow ABI was developed. This questionnaire was applied to these professionals in each of the Autonomous Communities, through semi-structured interviews. RESULTS: The results show the great territorial inequality existing in the functioning of rehabilitation during the recovery process of a person with ABI and the significant deficiencies of specific resources, specialized professionals such as neuropsychologists and necessary aspects such as socio-sanitary coordination. CONCLUSIONS: It is necessary to develop state regulations that guarantee the minimum basic aspects of the rehabilitation process in the DCA, in its different phases and in all the Autonomous Communities.

TITLE: Modelos de atención pública a la población con daño cerebral adquirido en España: un estudio de la situación por comunidades autónomas.Introducción. La rehabilitación tras el daño cerebral adquirido (DCA) es un proceso dinámico, progresivo y orientado a la mejora funcional de la persona afectada y de su núcleo familiar. Existe un consenso internacional sobre cómo debe ser el modelo que guíe dicho proceso; sin embargo, en nuestro país, se producen numerosas discrepancias territoriales en lo que respecta a su aplicación. Por ello, el objetivo del presente trabajo es analizar y actualizar el conocimiento sobre las prácticas desarrolladas en materia de rehabilitación y la normativa disponible en cada comunidad autónoma. Sujetos y métodos. Los participantes fueron 62 personas (71,7% mujeres), entre las cuales se encontraban tanto profesionales de atención directa como cargos de la administración pública de las 17 comunidades autónomas españolas y de las ciudades autónomas de Ceuta y Melilla. Se elaboró un cuestionario compuesto de 31 preguntas sobre el funcionamiento de la rehabilitación a lo largo de las tres fases asistenciales que suceden al DCA y que fue respondido por dichos profesionales en cada una de las comunidades autónomas a través de entrevistas semiestructuradas. La información obtenida se sintetizó por medio de análisis descriptivos. Resultados. Los resultados ponen de manifiesto la gran desigualdad territorial existente en el funcionamiento de la rehabilitación durante el proceso de recuperación tras un DCA y las carencias significativas de recursos específicos, profesionales especializados, como los neuropsicólogos, y aspectos necesarios, como la coordinación sociosanitaria. Conclusiones. Resulta necesario elaborar una normativa estatal que garantice los aspectos básicos mínimos sobre el proceso rehabilitador en el DCA, en sus distintas fases y en todas las comunidades autónomas.

Frailty as a predictor of clinical problems and events that require elderly patients with heart failure to use health resources.

BACKGROUND: The clinical management of elderly patients with heart failure (HF) is not firmly established. Decision-making should be individualized depending on the biological deterioration of each patient, from aggressive management to a palliative approach. Frailty can serve as the basis for this comprehensive individualized management. Our objective was to evaluate the importance of the main clinical problems, as well as the events that required the use of health resources, based the degree of frailty, in elderly patients with HF. METHODS AND RESULTS: Retrospective observational cohort study. Frailty was defined according to the deficit accumulation construct. A total of 546 patients hospitalized for acute HF were included. The median age (Q1-Q3) was 82 (78-86) years. A total of 454 patients (83%) showed some degree of frailty: 221 (48.7%) mild, 207 (45.6%) moderate and 26 (5.7%) advanced. There was a significant tendency towards polypharmacy from no to severe frailty. Hospital events were recorded for 4 (1-6) patients with mild frailty, 4 (2-6) patients with moderate frailty and 2 ((1-4) patients with advanced frailty (p = 0.045). A total of 204 patients (37.4%) died during follow-up. The median time to death was 11.4 (4-16.8), 6.7 (3.3-11.6), 6.5 (3.4-12.2) and 4.1 (0.8-7.7) months for patients with no, mild, moderate, or advanced frailty, respectively (p = 0.006). CONCLUSIONS: Frailty due to deficit accumulation is a good predictor of clinical problems and events that require the use of health resources; therefore, it can serve as a basis for the management of HF in the elderly.

[Surgical treatment of a perianal giant condyloma acuminata in a HIV patient]. / Tratamiento quirúrgico de un condiloma gigante perianal en una paciente con síndrome de inmunodeficiencia adquirida humana.

Buschke-Löwenstein tumor is a slowly growing neoplasm with high potential of local invasion. We described a 29 year-old female with acquired immunodeficiency syndrome who was surgically treated for a Buschke- Löwenstein tumor with a wide local excision, bilateral gluteal flaps and loop ileostomy. At 12 months follow-up, there was no evidence of recurrence. Despite it does not metastasize, Buschke-Löwenstein tumor has a high recurrence rate and a 50% risk of malignant transformation into squamous cell carcinoma. Surgery is considered the treatment of choice for this disease. Podophyllin, immunotherapy, interferon and radiotherapy are other treatments with a limited therapeutic response.

Early prediction of developing spontaneous activity in cultured neuronal networks.

Synchronization and bursting activity are intrinsic electrophysiological properties of in vivo and in vitro neural networks. During early development, cortical cultures exhibit a wide repertoire of synchronous bursting dynamics whose characterization may help to understand the parameters governing the transition from immature to mature networks. Here we used machine learning techniques to characterize and predict the developing spontaneous activity in mouse cortical neurons on microelectrode arrays (MEAs) during the first three weeks in vitro. Network activity at three stages of early development was defined by 18 electrophysiological features of spikes, bursts, synchrony, and connectivity. The variability of neuronal network activity during early development was investigated by applying k-means and self-organizing map (SOM) clustering analysis to features of bursts and synchrony. These electrophysiological features were predicted at the third week in vitro with high accuracy from those at earlier times using three machine learning models: Multivariate Adaptive Regression Splines, Support Vector Machines, and Random Forest. Our results indicate that initial patterns of electrical activity during the first week in vitro may already predetermine the final development of the neuronal network activity. The methodological approach used here may be applied to explore the biological mechanisms underlying the complex dynamics of spontaneous activity in developing neuronal cultures.

Prorocentroic Acid, a Neuroactive Super-Carbon-Chain Compound from the Dinoflagellate Prorocentrum hoffmannianum.

Prorocentroic acid (PA) was isolated from the dinoflagellate Prorocentrum hoffmannianum. Relative configurations for its 35 asymmetric centers were determined by analysis of NMR data including heteronuclear couplings and quantum mechanical calculations. PA was tested by using murine cortical neurons grown on microelectrode arrays. Long-term exposure to subtoxic concentrations induced a significant reorganization of neuronal signaling, mainly by changes in the bursting activity. The observed effects could be due to the activation of a plasticity process.

Na+/K+-ATPase inhibitor palytoxin enhances vulnerability of cultured cerebellar neurons to domoic acid via sodium-dependent mechanisms.

Dysfunction or deficiency of the Na(+)/K(+)-ATPase appears to be a common event in a variety of pathological conditions in the central nervous system. Studies on neurotoxicity associated to impaired Na(+)/K(+)-ATPase activity have focused on NMDA receptors, while the involvement of non-NMDA receptors has been much less explored. We show that mild, non-toxic, exposures to the Na(+)/K(+)-ATPase inhibitor palytoxin (PTX) synergistically sensitized the vulnerability of neurons to normally non-toxic concentrations of domoic acid, leaving NMDA receptor-mediated excitotoxic response unaltered. Enhancement of excitotoxicity required at least 1 h pre-exposure to PTX, was not observed after longer exposures to PTX, and did not require RNA synthesis. PTX caused a voltage-sensitive Na(+) channel-independent increase in intracellular Na(+). Both intracellular Na(+) increase and potentiation of excitotoxicity depended upon the external concentrations of Na(+) and Cl(-), and were suppressed by the anion exchanger blocker 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid in a dose-dependent manner. Other stilbene derivatives, chloride channel antagonists or Na(+) cotransporter inhibitors proved ineffective. Our results demonstrate a crucial role for Na(+)/K(+)-ATPase activity in determining neuronal vulnerability to domoic acid-mediated excitotoxicity. They also raise reasonable concern about possible risks for human health associated to the ingestion of low amounts of phycotoxins PTX and domoic acid in food.

An in vivo culture system for human embryos using an encapsulation technology: a pilot study.

BACKGROUND: Animal studies have demonstrated better embryo development in vivo than in vitro. This pilot study tested the feasibility of using a novel in utero culture system (IUCS) to obtain normal human fertilization and embryo development. METHODS: The IUCS device comprised a perforated silicone hollow tube. The study included 13 patients (<36 years) undergoing a first intracytoplasmic sperm injection (ICSI) treatment and 167 metaphase II oocytes in three groups. In Group 1, 1-2 h after ICSI, sibling oocytes were assigned to IUCS or conventional in vitro culture. The device was retrieved on Day 1, and all zygotes were cultured in vitro till Day 5. In Group 2, fertilized oocytes were assigned on Day 1, embryos retrieved on Day 3 and all embryos cultured till Day 5. In Group 3, after Day 0 assignment, embryos were retrieved on Day 3 for blastomere biopsy and fluorescence in situ hybridization (FISH) and cultured until Day 5. The highest quality blastocysts were transferred on Day 5. RESULTS: Fertilization and embryo development were comparable in the in vitro and IUCS arms, with a tendency towards better embryo quality in the IUCS. FISH analysis in Group 3 revealed more normal embryos using the IUCS (P = 0.049). Three clinical pregnancies and live births were obtained: two from the IUCS arm and one from the in vitro arm. CONCLUSIONS: Our pilot study shows that this new IUCS appears to be feasible and safe, supporting normal fertilization, embryo development and normal chromosomal segregation. Furthermore, live births are possible after the transient presence of a silicone device in the uterus. Clinicaltrials.gov: NCT00480103.

Tyrosine kinase A, C and fibroblast growth factor-2 receptors in bovine embryos cultured in vitro.

Neurotrophins and basic fibroblast growth factor are ligands of tyrosine kinase receptors, though they bind to different tyrosine kinase receptor classes. Neurotrophins bind to receptor tyrosine kinase class VII, Trk receptor family, while basic fibroblast growth factor binds to receptor tyrosine kinase class IV, FGF receptor family. The mammalian uterine tract immunolocalizes neurotrophins and bFGF; therefore their cognate receptors might exert a role during embryonic development. Using RT-PCR, we found mRNA for p75(NTR) TrkA, TrkC and FGFr2 throughout the early bovine embryonic development in vitro. Immunofluorescent staining, assessed by confocal microscopy, showed the expression of TrkA and TrkC proteins in oocytes and all embryonic stages analyzed. We have provided a novel description of TrkA and TrkC proteins, and TrkA, TrkC, p75(NTR) and FGFr2 mRNA expression throughout mammalian embryonic development. This work may help to design future research with neurotrophins in bovine embryo culture and embryonic stem cells.

Inhibition of protein phosphatases impairs the ability of astrocytes to detoxify hydrogen peroxide.

We have used protein phosphatase (PP) inhibitors and rat cerebellar glial cells in primary culture to investigate the role of PP activity in the ability of glial cells to detoxify exogenously applied hydrogen peroxide (H2O2). The marine toxin okadaic acid (OKA), a potent PP1 and PP2A inhibitor, caused a concentration-dependent degeneration of astrocytes and increased the formation of hydroperoxide radicals significantly. Subtoxic exposures to OKA significantly potentiated toxicity by exogenous H2O2. The concentration of H2O2 that reduced by 50% the survival of astrocytes after 3 h was estimated at 720+/-40 microM in the absence and 85+/-30 microM in the presence of the toxin. The PP inhibitors calyculin A and endothall also potentiated H2O2 toxicity in cerebellar astrocytes. OKA caused a time-dependent inhibition of both glial catalase and glutathione peroxidase, reducing by approximately 50% the activity of these enzymes after 3 h, whereas other enzymatic activities remained unaffected. Also, OKA reduced the cellular content of total glutathione and elevated oxidized glutathione to about 25% of total glutathione. OKA-treated astrocytes cleared H2O2 from the incubation medium approximately two times more slowly than control cultures. Our results suggest a prominent role for PP activity in the antioxidant mechanisms protecting astrocytes against damage by H2O2.

Total selenium and selenomethionine in pharmaceutical yeast tablets: assessment of the state of the art of measurement capabilities through international intercomparison CCQM-P86.

Results of an international intercomparison study (CCQM-P86) to assess the analytical capabilities of national metrology institutes (NMIs) and selected expert laboratories worldwide to accurately quantitate the mass fraction of selenomethionine (SeMet) and total Se in pharmaceutical tablets of selenised-yeast supplements (produced by Pharma Nord, Denmark) are presented. The study, jointly coordinated by LGC Ltd., UK, and the Institute for National Measurement Standards, National Research Council of Canada (NRCC), was conducted under the auspices of the Comité Consultatif pour la Quantité de Matière (CCQM) Inorganic Analysis Working Group and involved 15 laboratories (from 12 countries), of which ten were NMIs. Apart from a protocol for determination of moisture content and the provision of the certified reference material (CRM) SELM-1 to be used as the quality control sample, no sample preparation/extraction method was prescribed. A variety of approaches was thus used, including single-step and multiple-step enzymatic hydrolysis, enzymatic probe sonication and hydrolysis with methanesulfonic acid for SeMet, as well as microwave-assisted acid digestion and enzymatic probe sonication for total Se. For total Se, detection techniques included inductively coupled plasma (ICP) mass spectrometry (MS) with external calibration, standard additions or isotope dilution MS (IDMS), inductively coupled plasma optical emission spectrometry , flame atomic absorption spectrometry and instrumental neutron activation analysis. For determination of SeMet in the tablets, five NMIs and three academic/institute laboratories (of a total of five) relied upon measurements using IDMS. For species-specific IDMS measurements, an isotopically enriched standard of SeMet (76Se-enriched SeMet) was made available. A novel aspect of this study relies on the approach used to distinguish any errors which arise during analysis of a SeMet calibration solution from those which occur during analysis of the matrix. To help those participants undertaking SeMet analysis to do this, a blind sample in the form of a standard solution of natural abundance SeMet in 0.1 M HCl (with an expected value of 956 mg kg(-1) SeMet) was provided. Both high-performance liquid chromatography (HPLC)-ICP-MS or gas chromatography (GC)-ICP-MS and GC-MS techniques were used for quantitation of SeMet. Several advances in analytical methods for determination of SeMet were identified, including the combined use of double IDMS with HPLC-ICP-MS following extraction with methanesulfonic acid and simplified two-step enzymatic hydrolysis with protease/lipase/driselase followed by HPLC-ICP-IDMS, both using a species-specific IDMS approach. Overall, satisfactory agreement amongst participants was achieved; results averaged 337.6 mg kg(-1) (n = 13, with a standard deviation of 9.7 mg kg(-1)) and 561.5 mg kg(-1) (n = 11, with a standard deviation of 44.3 mg kg(-1)) with median values of 337.6 and 575.0 mg kg(-1) for total Se and SeMet, respectively. Recovery of SeMet from SELM-1 averaged 95.0% (n = 9). The ability of NMIs and expert laboratories worldwide to deliver accurate results for total Se and SeMet in such materials (selensied-yeast tablets containing approximately 300 mg kg(-1) Se) with 10% expanded uncertainty was demonstrated. The problems addressed in achieving accurate quantitation of SeMet in this product are representative of those encountered with a wide range of organometallic species in a number of common matrices.