Human papilloma virus presence and its physical status in primary pterygium.

Pterygium is one of the most frequent pathologies in ophthalmology, and is a benign, overgrowth of fibrovascular tissue, often with a wing-like appearance, from the conjunctiva over the cornea. It is composed of an epithelium and highly vascular, sub-epithelial, loose connective tissue. There is much debate surround the pathogenesis of pterygium and a number of theories have been put forward including genetic instability, cellular proliferation, inflammatory influence, and degeneration of connective tissue, angiogenesis, aberrant apoptosis and viral infection. At present, the involvement of human papillomavirus (HPV) in the genesis of pterygium is controversial, as have reported that HPV is present in 58% of cases, while others have failed to detect HPV in pterygium. In this study, we evaluated the presence and viral genotype of HPV DNA in pterygia and healthy conjunctiva sample, and virus integration into the cellular genome. Forty primary pterygia samples and 12 healthy conjunctiva samples were analyzed to HPV DNA presence by polymerase chain reaction, using MY09/MY11 primers of HPV-L1 gene. Viral genotype was identified by DNA sequence analysis of this amplicon. HPV integration into the cellular genome was analyzed by western blot detecting HPV-L1 capsid protein. Presence of HPV was observed in 19 of the 40 pterygia samples. In contrast, healthy conjunctiva samples were negative. To determine virus type, sequence analyses were performed. Interestingly, 11 out of the 19-pterygium samples were identified as HPV-11 type, meanwhile, the remaining 8 pterygium samples were identified as HPV-18. HPV-L1 capsid protein were found only in 3 out of the 10 samples studied. In conclusion, our study identified the presence of HPV DNA exclusively in pterygium samples and described HPV-11 and -18 genotypes. Our results suggest that HPV may be involved in the pathogenesis of pterygium. On the other hand, the expression of the L1-HPV protein suggests viral integration into the cellular genome.

Regulation of MMP-2 by IL-8 in Vascular Endothelial Cells: Probable Mechanism for Endothelial Dysfunction in Women with Preeclampsia.

Endothelial dysfunction (ED) in preeclampsia (PE) results from the convergence of oxidative stress, inflammation, and alterations in extracellular matrix components, affecting vascular tone and permeability. The molecular network leading to ED includes IL-8 and MMP-2. In vitro, IL-8 regulates the concentration and activity of MMP-2 in the trophoblast; this interaction has not been studied in endothelial cells during PE. We isolated human umbilical vein endothelial cells (HUVECs) from women with healthy pregnancies (NP, n = 15) and PE (n = 15). We quantified the intracellular concentration of nitric oxide and reactive oxygen species with colorimetric assays, IL-8 with ELISA, and MMP-2 with zymography and using an ELISA-type system. An IL-8 inhibition assay was used to study the influence of this cytokine on MMP-2 concentration and activity. HUVECs from women with PE showed significantly higher oxidative stress than NP. IL-8 and MMP-2 were found to be significantly elevated in PE HUVECs compared to NP. Inhibition of IL-8 in HUVECs from women with PE significantly decreased the concentration of MMP-2. We demonstrate that IL-8 is involved in the mechanisms of MMP-2 expression in HUVECs from women with PE. Our findings provide new insights into the molecular mechanisms regulating the ED distinctive of PE.

Sperm Dysfunction in the Testes and Epididymides due to Overweight and Obesity Is Not Caused by Oxidative Stress.

Obesity is a condition that has been linked to male infertility. The current hypothesis regarding the cause of infertility is that sperm are highly sensitive to reactive oxygen species (ROS) during spermatogenesis in the testes and transit through the epididymides, so the increase in ROS brought on by obesity could cause oxidative stress. The aim of this study was to evaluate whether the activity of the enzymes catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) is capable of counteracting oxidative stress in sperm. The male Wistar rat was used as an overweight and obesity model, and analysis of fertility in these groups was carried out including the control group. Serum testosterone levels were determined, and the scrotal fat, testes, and epididymides were extracted. The epididymides were separated ini0 3 principal parts (caput, corpus, and cauda) before evaluating sperm viability, sperm morphology, damage to desoxyribonucleic acid of the sperm, and ROS production. The protein content and specific activity of the three enzymes mentioned above were evaluated. Results showed a gain in body weight and scrotal fat in the overweight and obese groups with decreased parameters for serum testosterone levels and sperm viability and morphology. Fertility was not greatly affected and no DNA integrity damage was found, although ROS in the epididymal sperm increased markedly and Raman spectroscopy showed a disulfide bridge collapse associated with DNA. The specific activities of CAT and GPX increased in the overweight and obesity groups, but those of SOD did not change. The amounts of proteins in the testes and epididymides decreased. These findings confirm that overweight and obesity decrease concentrations of free testosterone and seem to decrease protein content, causing poor sperm quality. Implications. An increase in scrotal fat in these conditions fosters an increase of ROS, but the increase of GPX and CAT activity seems to avoid oxidative stress increase in the sperm without damaging your DNA.

A Sea Anemone Lebrunia neglecta Venom Fraction Decreases Boar Sperm Cells Capacitation: Possible Involvement of HVA Calcium Channels.

Sea anemones produce venoms characterized by a complex mixture of low molecular weight compounds, proteins and peptides acting on voltage-gated ion channels. Mammal sperm cells, like neurons, are characterized by their ion channels. Calcium channels seem to be implicated in pivotal roles such as motility and capacitation. In this study, we evaluated the effect of a low molecular weight fraction from the venom of the sea anemone Lebrunia neglecta on boar sperm cells and in HVA calcium channels from rat chromaffin cells. Spermatozoa viability seemed unaffected by the fraction whereas motility and sperm capacitation were notoriously impaired. The sea anemone fraction inhibited the HVA calcium current with partial recovery and no changes in chromaffin cells' current kinetics and current-voltage relationship. These findings might be relevant to the pharmacological characterization of cnidarian venoms and toxins on voltage-gated calcium channels.

Perfluorooctane sulfonate and perfluorooctanoic acid induce plasma membrane dysfunction in boar spermatozoa during in vitro capacitation.

Spermatozoa require the capacitation, a series of biochemical events, to perform fertilization. Many toxic compounds can interfere in this process, including perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), which belong to the perfluoroalkyl substances (PFAS). Since both substances are found in many everyday materials and are highly persistent, they accumulate in organisms where they have been associated with fertility problems. This study analyzes the effects of PFOS and PFOA on the functionality of boar spermatozoa, and changes in the plasma membrane (PM) during capacitation. The median lethal concentrations (LC50) of PFOS and PFOA were 460 and 1894 µM, respectively, while the mean inhibitory concentrations of capacitation (ICC50) were 274 µM and 1458 µM, respectively. The ICC50 of PFOA was insufficient to reduce the capacitation, but 950 µM (½ LC50) of PFOA and the ICC50 of PFOS significantly reduced the number of capacitated spermatozoa. PFOS and PFOA also impeded the progesterone (P4)-induced acrosomal reaction (iAR). These effects occur despite the accumulation of [Ca2+]i under capacitating conditions. The accumulation of [Ca2+]i produces saturation, which prevents its entry through ionophore A23187 and P4 in the presence of PFOS. Membrane potential (Emv) was deregulated. Both PFAS affected lipid membrane conductance mediated by valinomycin. The spermatozoa presented 49% and 47% of membrane dysfunction with PFOS and PFOA, respectively. By causing membrane damage, both substances prevented the release of cholesterol and altered the organization of membrane microdomains (MMDs). Data indicate that both PFAS caused alterations in PM functionality.

Beneficial and deleterious effects of sitagliptin on a methionine/choline-deficient diet-induced steatohepatitis in rats.

Non-alcoholic fat liver disease (NAFLD) is the most common chronic liver disease in the world. NAFLD is a spectrum of diseases ranging from simple steatosis to hepatic carcinoma. The complexity of pathomechanisms makes treatment difficult. The oral antidiabetic agents, dipeptidyl peptidase four inhibitors (DPP-4i) have been proposed as possible therapeutic agents. This study was performed using a well-established NAFLD model in rats to elucidate whether sitagliptin could prevent steatohepatitis. Rats were fed a methionine/choline-deficient (MCD) diet with or without sitagliptin treatment for six weeks. Liver tissue was examined to estimate sitagliptin's effect on the development of NASH. The MCD diet decreased the SAM/SAH ratio, and increased plasma levels of homocysteine, free fatty acids, and long-chain acylcarnitines in the MCD rats. MMP2 and Col1A2 expression also increased under the MCD diet. Sitagliptin treatment did not reverse these effects and increased steatosis and long-chain acylcarnitines. In conclusion, sitagliptin was ineffective to prevent from NAFLD in the MCD rat model. This result challenges previous data reporting beneficial effects and is consistent with the clinical trials' negative results.

Boar sperm incubation with reduced glutathione (GSH) differentially modulates protein tyrosine phosphorylation patterns and reorganization of calcium in sperm, in vitro fertilization, and embryo development depending on concentrations.

The sperm in the female's reproductive tract undergo changes to fertilize the oocyte (sperm capacitation). These changes are regulated by redox system. However, some assisted reproductive technologies require sperm capacitation under in vitro conditions, though this increases the generation of ROS. Therefore, the aim of this study was to evaluate the effect of GSH as an antioxidant agent during the capacitation of boar sperm [evaluated by calcium compartmentalization, tyrosine phosphorylation (Tyr-P), motility, viability, and acrosomal integrity], in vitro fertilization (evaluated by penetration, monospermy, and efficiency %), and later embryo development (evaluated by cleavage and blastocyst rates, total number of cells per blastocyst and blastocyst diameter). Four experimental groups with different GSH concentrations (0-control, 0.5, 1, and 5 mM) were formed. When 1-GSH was added to the medium, the percentage of capacitated sperm increased after 4 h of incubation; the localization of Tyr-P was modified at 1 h and 4 h of incubation depending on the GSH concentration. Percentages of total and progressive sperm motility also increased at 4 h of incubation, but only in the 5-GSH group compared to control. Viability, acrosomal integrity, and general Tyr-P (Western blot) not differ among the experimental groups. The addition of GSH during gamete interaction increased penetration, monospermy, and efficiency rates in the 1-GSH group compared to the others. However, the effect of GSH was not observed in cleavage and blastocyst rates compared to the control. In conclusion, adding GSH modulates sperm capacitation (by means of calcium compartmentalization and tyrosine phosphorilation pattern) depending on its concentration, and improves IVF output at 1-GSH during gamete interaction.

Cystathionine ß-synthase and methylenetetrahydrofolate reductase mutations in Mexican individuals with hyperhomocysteinemia.

BACKGROUND: Hyperhomocysteinemia, a thrombotic risk factor, may have several causes. Among the genetic causes of hyperhomocysteinemia, there are polymorphisms in the enzymes methylenetetrahydrofolate reductase (C677T) and cystathionine ß-synthase (C699T, C1080T, and 844ins68). Although the frequency of hyperhomocysteinemia in our country is high, there is no evidence about the frequencies of these polymorphisms. METHODS: We analyzed 80 healthy individuals from several regions in our country. We evaluated the fasting and post-oral methionine load plasma Hcy and the genotypes in order to obtain the allele frequencies of the polymorphisms C677T of methylenetetrahydrofolate reductase and C699T, C1080T, and 844ins68 of the cystathionine ß-synthase. RESULTS: No individual had deficiency of folic acid, vitamins B12, or B6, but 80% had post-oral methionine load hyperhomocysteinemia. We found a significant increase in the Hcy plasma concentration associated with age and gender. Only the polymorphism C1080T was significantly associated with hyperhomocysteinemia. CONCLUSION: There is an association between fasting and post-oral methionine load plasma Hcy concentrations with the allelic frequencies of the polymorphisms C669T, 844ins68, and C1080T of the cystathionine ß-synthase and C667T of the methylenetetrahydrofolate reductase in healthy Mexican individuals. As compared with individuals with normal fasting or post-oral methionine load Hcy plasma levels, only C1080T was significantly associated with hyperhomocysteinemia.

Perfluorooctane Sulfonate (PFOS) and Perfluorohexane Sulfonate (PFHxS) Alters Protein Phosphorylation, Increase ROS Levels and DNA Fragmentation during In Vitro Capacitation of Boar Spermatozoa.

Perfluorooctane sulfonate (PFOS) and perfluorohexane sulfonate (PFHxS) are toxic and bioaccumulative, included in the Stockholm Convention's list as persistent organic pollutants. Due to their toxicity, worldwide distribution, and lack of information in spermatozoa physiology during pre-fertilization processes, the present study seeks to analyze the toxic effects and possible alterations caused by the presence of these compounds in boar sperm during the in vitro capacitation. The spermatozoa capacitation was performed in supplemented TALP-Hepes media and mean lethal concentration values of 460.55 µM for PFOS, and 1930.60 µM for PFHxS were obtained. Results by chlortetracycline staining showed that intracellular Ca2+ patterns bound to membrane proteins were scarcely affected by PFOS. The spontaneous acrosome reaction determined by FITC-PNA was significantly reduced by PFOS and slightly increased by PFHxS. Both toxic compounds significantly alter the normal capacitation process from 30 min of exposure. An increase in ROS production was observed by flow cytometry and considerable DNA fragmentation by the comet assay. The immunocytochemistry showed a decrease of tyrosine phosphorylation in proteins of the equatorial and acrosomal zone of the spermatozoa head. In conclusion, PFOS and PFHxS have toxic effects on the sperm, causing mortality and altering vital parameters for proper sperm capacitation.

Identification of lipid raft glycoproteins obtained from boar spermatozoa.

The surface of the spermatozoa is coated with glycoproteins the redistribution of which during in vitro capacitation plays a key role in the subsequent fertilization process. Lipid rafts are membrane microdomains involved in signal transduction through receptors and include or recruit specific types of proteins and glycoproteins. Few studies have focused on identifying glycoproteins resident in the lipid rafts of spermatozoa. Proteins associated with lipid rafts modify their localization during capacitation. The objective of the study was to identify the glycoproteins associated with lipid rafts of capacitated boar spermatozoa through a lectin-binding assay coupled to mass spectrometry approach. From the proteomic profiles generated by the raft proteins extractions, we observed that after capacitation the intensity of some bands increased while that of others decreased. To determine whether the proteins obtained from lipid rafts are glycosylated, lectin blot assays were performed. Protein bands with a good resolution and showing significant glycosylation modifications after capacitation were analyzed by mass spectrometry. The bands of interest had an apparent molecular weight of 64, 45, 36, 34, 24, 18 and 15 kDa. We sequenced the 7 bands and 20 known or potential glycoproteins were identified. According to us, for ten of them this is the first time that their association with sperm lipid rafts is described (ADAM5, SPMI, SPACA1, Seminal plasma protein pB1, PSP-I, MFGE8, tACE, PGK2, SUCLA2, MDH1). Moreover, LYDP4, SPAM-1, HSP60, ZPBP1, AK1 were previously reported in lipid rafts of mouse and human spermatozoa but not in boar spermatozoa. We also found and confirmed the presence of ACR, ACRBP, AWN, AQN3 and PRDX5 in lipid rafts of boar spermatozoa. This paper provides an overview of the glycosylation pattern in lipid rafts of boar spermatozoa before and after capacitation. Further glycomic analysis is needed to determine the type and the variation of glycan chains of the lipid rafts glycoproteins on the surface of spermatozoa during capacitation and acrosome reaction.

Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa.

Actin cytoskeleton remodeling is a critical process for the acquisition of fertilizing capacity by spermatozoa during capacitation. However, the molecular mechanism that regulates this process has not been fully elucidated. In somatic cells, Ras-related C3 botulinum toxin substrate 1 protein (Rac1) promotes the polymerization of actin by participating in the modeling of two structures: lamellipodia and adhesion complexes linked with the plasma membrane. Rac1 is expressed in mammalian spermatozoa; however, the role of Rac1 in sperm physiology is unknown. This study aimed to elucidate the participation of Rac1 in capacitation and acrosome reaction (AR). Rac1 was found to be dispersed throughout the acrosome and without changes in the middle piece. After 60 minutes of capacitation, Rac1 was found in the apical region of the acrosome only, which concurred with an increase in Rac1-GTP. Rac1 inhibition prevented such changes. In the middle piece, Rac1 localization remained unchanged. Besides, Rac1 inhibition blocked capacitation and AR. The present study demonstrates that Rac1 participates only in the actin cytoskeleton remodeling that occurs in the acrosomal apical region during capacitation, a region where a large amount of actin is polymerized and shaped in a diadem-like structure. Our data also show that this actin cytoskeleton organized by Rac1 interacts with filamin-1, and such interaction was blocked by the inhibition of Rac1, which led to a different organization of the actin cytoskeleton. All these outcomes imply that the formation of an F-actin cytoskeleton in the acrosomal apical region is a necessary event for capacitation and AR, and which is Rac1 driven.

Novel transglycosylation activity of ß-N-acetylglucosaminidase of Lecanicillium lecanii produced by submerged culture.

N-acetylglucosaminidase produced from Lecanicillium lecanii on submerged culture displayed hydrolytic and transglycosylation activities. The highest specific activity for the enzyme was 1.87 U/mg after 120 h of culture. The chromatographic purification for a single protein fraction showed a molecular weight of 50.4 kDa and hydrolytic N-acetylglucosaminidase activity of 17.59 U/mg at 37 °C and pH 6. This enzyme was able to transglycosylate and to synthesize oligosaccharides from 2 to 6 units with a degree of acetylation between 100 and 26% employing glucose, mannose, N-acetyl-D-glucosamine and N-acetyl-D-lactosamine as donor substrates. Optimal conditions of temperature and pH were determined for both types of enzymatic activities.

Potential of the HSP70 protein family as biomarker of Crassostrea virginica under natural conditions (Ostreoida: Ostreidae)

The HSP70 proteins are used as exposure biomarkers, and the oyster Crassostrea virginica is considered as a bioindicator organism in environmental assessment. According to the season, the level of expression of the HSP70 family in wild oysters has not been characterized before. The aim of this work was to analyze the expression of the HSP70 family as an exposure biomarker using C. virginica gills from individuals and groups of oysters from the Tampamachoco Lagoon under natural conditions. Ninety oyster samples were collected at locations from the Tampamachoco Lagoon during the dry and "north winds" seasons. One group of oysters was maintained under laboratory conditions and exposed to thermal stress. The pH, temperature, salinity, and dissolved oxygen (DO2) were measured inside the brackish Tuxpam-Tampamachoco system. Salinity and DO2 were outside the limits recommended for brackish systems in both seasons. The electrophoresis and immunodetection assay of HSP70 were performed using proteins from oyster gills. The HSP73, HSP72 and HSP69 isoforms were expressed in dry season tissue samples, while only the HSP73 and HSP72 isoforms were detected in north winds season tissue samples and positive control tissue samples. The HSP73 isoform has not been previously reported in C. virginica. To evaluate the expression of HSP70 protein family at individual and group levels from wild animals, it is also important to determine a seasonal baseline expression.

Las proteínas HSP70 se utilizan como biomarcadores de exposición y el ostión Crasostrea virginica es considerado como un organismo bioindicador en evaluaciones ambientales. De acuerdo con la estación, el nivel de expresión de la familia de las HSP70 en ostiones silvestres no ha sido caracterizado. El objetivo de este trabajo fue analizar la expresión de la familia de las proteínas HSP70 como un biomarcador de exposición utilizando branquias de C. virginica de manera individual y grupal bajo condiciones naturales. Noventa muestras de ostión se recolectaron en un sitio dentro de la Laguna Tampamachoco durante las estaciones de secas y "nortes". Un grupo se mantuvo bajo condiciones de laboratorio y expuesto a estrés térmico. El pH, la temperatura, la salinidad y el oxígeno disuelto (DO2) se midieron dentro del sistema salobre Tuxpan-Tampamachoco. La salinidad y el DO2 estuvieron fuera de los límites recomendados para sistemas salobres en ambas estaciones. La electroforesis y el ensayo de inmunodetección se llevaron a cabo con branquias de ostiones. Las isoformas HSP73, HSP72 y HSP69 se expresaron en tejido de las muestras de la época de secas, mientras que solamente las isoformas HSP73 y HSP72 se detectaron en tejido de las muestras de época de nortes y control positivo. La isoforma HSP73 no ha sido reportada previamente en C. virginica. Para evaluar la expresión de la familia de las proteínas HSP70 a nivel individual y grupal de organismos silvestres es importante determinar una línea de base de expresión estacional.

Exfoliated Human Olfactory Neuroepithelium: A Source of Neural Progenitor Cells.

Neural progenitor cells (NPC) contained in the human adult olfactory neuroepithelium (ONE) possess an undifferentiated state, the capability of self-renewal, the ability to generate neural and glial cells as well as being kept as neurospheres in cell culture conditions. Recently, NPC have been isolated from human or animal models using high-risk surgical methods. Therefore, it was necessary to improve methodologies to obtain and maintain human NPC as well as to achieve better knowledge of brain disorders. In this study, we propose the establishment and characterization of NPC cultures derived from the human olfactory neuroepithelium, using non-invasive procedures. Twenty-two healthy individuals (29.7 ± 4.5 years of age) were subjected to nasal exfoliation. Cells were recovered and kept as neurospheres under serum-free conditions. The neural progenitor origin of these neurospheres was determined by immunocytochemistry and qPCR. Their ability for self-renewal and multipotency was analyzed by clonogenic and differentiation assays, respectively. In the cultures, the ONE cells preserved the phenotype of the neurospheres. The expression levels of Nestin, Musashi, Sox2, and ßIII-tubulin demonstrated the neural origin of the neurospheres; 48% of the cells separated could generate neurospheres, determining that they retained their self-renewal capacity. Neurospheres were differentiated in the absence of growth factors (EGF and FGF), and their multipotency ability was maintained as well. We were also able to isolate and grow human neural progenitor cells (neurospheres) through nasal exfoliates (non-invasive method) of the ONE from healthy adults, which is an extremely important contribution for the study of brain disorders and for the development of new therapies.

The proximal segment of the embryonic outflow (conus) does not participate in aortic vestibule development.

OBJECTIVE: There is no consensus on the embryonic components or morphogenetic processes involved in mature ventricular outflow tract development. Our goal was to use in vivo labelling to investigate the prospective fate of the myocardium of each conal wall. The conal and atrioventricular cushion mesenchyme changes during transformation into mature structures and their role in apoptosis were also investigated. METHODS: Plastic labels were placed at the cephalic and caudal conal limits of chicken embryo hearts (stage 22HH) and traced up to stage 36HH. Histological analyses, scanning electron microscopy and apoptotic detection using Lysotracker-Red were performed. The conal longitudinal length and medial displacement were registered. Muscle myosin was identified by immunofluorescence. RESULTS: Labels positioned in the myocardium of each conal wall moved to the right ventricle (RV), shifting from the arterial subvalvular myocardial zone to the apex. No labels were found in the aortic vestibule. At stage 22HH, the conus was a tubular structure composed of myocardium and endocardium with scarce mesenchyme. The dorso-left conal myocardial wall gradually lost continuity and the free ends separated, while the myocardium was distributed to the RV free wall (24HH-28HH). At stage 22HH, conal crests were not observed, but they were apparent at the dorsal zone of the conus at stage 26HH; towards stage 30HH, they fused to form the supraventricular crest, and the pulmonary infundibulum was evident. The ventro-superior cushion of the AV canal was reorganized into the fibrous and muscular structures lined the aortic vestibule. CONCLUSIONS: The posterior conus is an erroneous concept. The conal myocardium is reorganized in the free wall of the RV. Internally, the conal lumen is transformed into the pulmonary infundibulum. The aortic vestibule is formed from the ventro-superior cushion of the AV canal. Thus, the ventricular outflow tracts have different embryonic origins.

Colony stimulating factor-1 and leukemia inhibitor factor expression from current-cycle cannula isolated endometrial cells are associated with increased endometrial receptivity and pregnancy.

BACKGROUND: Poor endometrial quality is associated with more than a third of embryo implantation failures. Current ultrasonography technology lacks the capacity to determine efficiently the endometrial receptivity during ongoing cycle transfers. We analyzed the relationship between the gene expression profile associated with implantation and clinical pregnancy from endometrial cells taken during embryo transfer. METHODS: Seventy-six patients submitted to a standard ovarian stimulation protocol, in vitro fertilization, and good quality embryos were collected (morphological assessment). Endometrial samples were taken with ultrasonography guidance and cells were Hematoxylin and Eosin stained for morphological identification. Total RNA was extracted and the expression of Mucin 1 (MUC1), Homeobox A10 (HOXA-10), Leukemia Inhibitor Factor (LIF), Colony Stimulating Factor-1 (CSF-1), and ribosomal 18 s (endogenous control) were analyzed using RT-qPCR. Presence of a gestational sac, ß-hGC (≥10 mIU/mL on Day 20), and a fetal heartbeat were used to determine a positive embryo implantation and pregnancy. RESULTS: Samples collected from same cycle embryo transfer showed clear morphological staining for endometrial cells (80-90% of the cells). Cells in the sample were molecularly identified as the endometrium (HOXA-10 positive and MUC-1 negative). CSF-1 expression was 4.55-fold and LIF expression was 12.25-fold higher in patients who became pregnant. Both increases were statistically significant (p < 0.05). CONCLUSIONS: Here, we provide evidence of a new method to assess endometrial receptivity. Furthermore, we demonstrate that the expression profile, based on LIF and CSF-1, showed a difference between a receptive and a non-receptive endometrium.

Oxidative stress as a damage mechanism in porcine cumulus-oocyte complexes exposed to malathion during in vitro maturation.

Malathion is one of the most commonly used insecticides. Recent findings have demonstrated that it induces oxidative stress in somatic cells, but there are not enough studies that have demonstrated this effect in germ cells. Malathion impairs porcine oocyte viability and maturation, but studies have not shown how oxidative stress damages maturation and which biochemical mechanisms are affected in this process in cumulus-oocyte complexes (COCs). The aims of the present study were to determine the amount of oxidative stress produced by malathion in porcine COCs matured in vitro, to define how biochemical mechanisms affect this process, and determine whether trolox can attenuate oxidative damage. Sublethal concentrations 0, 750, and 1000 µM were used to evaluate antioxidant enzyme expressions, reactive oxygen species (ROS production), protein oxidation, and lipid peroxidation, among other oxidation products. COCs viability and oocyte maturation decreased in a concentration-dependent manner. Malathion increased Cu, Zn superoxide dismutase (SOD1), glutathione-S-transferase (GST), and glucose 6 phosphate dehydrogenase (G6PD) protein level and decreased glutathione peroxidase (GSH-Px) and catalase (CAT) protein level. Species reactives of oxygen (ROS), protein oxidation and Thiobarbituric acid reactive substances (TBARS) levels increased in COCs exposed to the insecticide, but when COCs were pre-treated with the trolox (50 µM) 30 min before and during malathion exposure, these parameters decreased down to control levels. This study showed that malathion has a detrimental effect on COCs during in vitro maturation, inducing oxidative stress, while trolox attenuated malathion toxicity by decreasing oxidative damage.

Focal adhesion kinase is required for actin polymerization and remodeling of the cytoskeleton during sperm capacitation.

Several focal adhesion proteins are known to cooperate with integrins to link the extracellular matrix to the actin cytoskeleton; as a result, many intracellular signaling pathways are activated and several focal adhesion complexes are formed. However, how these proteins function in mammalian spermatozoa remains unknown. We confirm the presence of focal adhesion proteins in guinea pig spermatozoa, and we explore their role during capacitation and the acrosome reaction, and their relationship with the actin cytoskeleton. Our results suggest the presence of a focal adhesion complex formed by ß1-integrin, focal adhesion kinase (FAK), paxillin, vinculin, talin, and α-actinin in the acrosomal region. Inhibition of FAK during capacitation affected the protein tyrosine phosphorylation associated with capacitation that occurs within the first few minutes of capacitation, which caused the acrosome reaction to become increasingly Ca(2+) dependent and inhibited the polymerization of actin. The integration of vinculin and talin into the complex, and the activation of FAK and paxillin during capacitation, suggests that the complex assembles at this time. We identify that vinculin and α-actinin increase their interaction with F-actin while it remodels during capacitation, and that during capacitation focal adhesion complexes are structured. FAK contributes to acrosome integrity, likely by regulating the polymerization and the remodeling of the actin cytoskeleton.

Enzymatic hydrolysis of chitin pretreated by rapid depressurization from supercritical 1,1,1,2-tetrafluoroethane toward highly acetylated oligosaccharides.

The hydrolysis of chitin treated under supercritical conditions was successfully carried out using chitinases obtained by an optimized fermentation of the fungus Lecanicillium lecanii. The biopolymer was subjected to a pretreatment based on suspension in supercritical 1,1,1,2-tetrafluoroethane (scR134a), which possesses a critical temperature and pressure of 101°C and 40bar, respectively, followed by rapid depressurization to atmospheric pressure and further fibrillation. This methodology was compared to control untreated chitins and chitin subjected to steam explosion showing improved production of reducing sugars (0.18mg/mL), enzymatic hydrolysis and high acetylation (FA of 0.45) in products with degrees of polymerization between 2 and 5.

In vivo analysis of effects of venom from the jellyfish Chrysaora sp. in zebrafish (Danio rerio).

The jellyfishes of the genus Chrysaora are present in all of the world's oceans, but the toxicity of their venoms has not yet been thoroughly characterized. The zebrafish as a toxicology model can be used for general toxicity testing of drugs and the investigation of toxicological mechanisms. The aim of this study was to evaluate the effect of crude venom from jellyfish Chrysaora sp., a species of jellyfish observed in the tropical lagoons of the Gulf of Mexico, on the zebrafish Danio rerio. Juvenile zebrafish were injected with different concentrations of venom from Chrysaora sp. via intraperitoneal and subcutaneous injections. The effects of the venom were determined by histopathological analysis and through the measurement of hemolytic and phospholipase A2 activities. The crude venom was examined by SDS-PAGE. The effect of sublethal concentrations of crude venom from Chrysaora sp. on D. rerio was hemorrhaging in the eyes, while the histopathological analysis demonstrated that the primary organs targeted were the pseudobranch, which displayed hyperemia, and the gill, which displayed hyperplasia and hypertrophy. The blood analysis exhibited hemolysis, nuclear abnormalities, and echinocytes by the action of phospholipase A2, which was determined to have 596 units of activity/mg of protein in the venom. The crude venom has proteins with molecular weights ranging from 250 to 6 kDa, with more density in the bands corresponding to 70, 20 and 15 kDa. The venom of Chysaora sp. caused disturbances in circulation associated with vascular dilation due to the localized release of inflammatory mediators. The hemolysis of erythrocytes was caused by the action of phospholipase A2. These findings not only provide an excellent study model but also have a great pharmacological potential for designing new drugs and for the elucidation of the mechanisms of action of and treatment against stings.