Comparison of CellRox green fluorescence upon thawing on in vitro Bos taurus and Bos indicus embryos cryopreserved by slow freezing or vitrification.

The aim of this study was to compare the levels of reactive oxygen species (ROS) in Bos taurus and Bos indicus in vitro embryos cryopreserved using either slow freezing or vitrification. Embryos were divided into four groups based on subspecies and freezing method: Bos indicus slow freezing (BiSF; n = 8), Bos indicus vitrification (BiVT; n = 10), Bos taurus slow freezing (BtSF; n = 9), and Bos taurus vitrification (BtVT; n = 6). After thawing, the embryos were incubated with CellRox Green and images were obtained using a confocal microscope. The fluorescence intensity of each cell was measured and expressed as arbitrary units of fluorescence (auf) and compared using a multiple regression and unpaired t-test with α = 0.05. Results showed that subspecies and the freezing method significantly affected auf (P < 0.001; R2 = 0.1213). Bos indicus embryos had higher auf than Bos taurus embryos, whether frozen by slow freezing (67.05 ± 23.18 vs 51.30 ± 16.84, P < 0.001) or vitrification (64.44 ± 23.32 vs 47.86 ± 17.53, P < 0.001). Slow freezing induced higher auf than vitrification in both Bos taurus (51.30 ± 16.84 vs 47.86 ± 17.53, P < 0.001) and Bos indicus (67.05 ± 23.18 vs 64.44 ± 23.32, P < 0.014). In conclusion, Bos taurus embryos had lower ROS levels when frozen using vitrification, while Bos indicus embryos had consistent ROS patterns regardless of the freezing method. However, Bos indicus embryos frozen by slow freezing tended to have a higher number of cells with elevated ROS levels.

Multi-institutional review of non-hypospadiac penile urethral stricture management and outcomes.

OBJECTIVES: Penile urethral stricture disease not associated with hypospadias is rare, and there is a wide range of commonly used surgical repair techniques for this disease. We sought to compile a multi-institutional database of patients who had surgical correction of strictures in the penile urethra not limited to the meatus, and who had no history of hypospadias, for analysis using the Trauma and Urologic Reconstructive Network of Surgeons length, urethral segment and etiology classification system. METHODS: A retrospective database from 13 institutions was compiled of patients who had undergone surgical correction of Trauma and Urologic Reconstructive Network of Surgeons length, urethral segment and etiology urethral stricture segments S2b/S2c and excluding E5, with a minimum follow-up time of 4 months. Failure was defined as cystoscopically confirmed recurrence of a stricture measuring less than 16-Fr. RESULTS: We analyzed 222 patients with a median age of 57 years and a follow-up of 49 months. The overall surgical success rate was 80.2%. On multivariate analysis, the two variables identified that were predictive of surgical success were stricture length ≤2 cm as well as use of a buccal mucosa graft as compared to use of a fasciocutaneous flap, which had success rates of 83% and 52%, respectively (P = 0.0004). No statistically significant differences were found based on incisional approach or surgical technique, nor were outcomes different based on etiology or preoperative patient demographics. CONCLUSIONS: Surgical repair of penile urethral strictures of non-hypospadiac origin have a favorable overall success rate, at 80.2%. Regardless of incisional approach or surgical technique, all operations appear to have similar outcomes other than repairs using fasciocutaneous flap, which were statistically less successful than those using buccal mucosa graft.

Arbovirus Surveillance and First Report of Chikungunya Virus in Wild Populations of Aedes aegypti from Guerrero, Mexico.

We carried out dengue (DENV) and chikungunya virus (CHIKV) surveillance in wild populations of Aedes aegypti from Guerrero, Mexico, from 2012 to 2014 following a standard national protocol of the Mexican Dengue Control Program. A total of 284 pools (15-30 specimens/pool) of female mosquitoes were tested with real-time reverse transcriptase-polymerase chain reaction to detect DENV and CHIKV. We report for the 1st time the detection of CHIKV from field-collected mosquitoes at Acapulco and Juchitán in 2014. Results from DENV are also reported.

Natural vertical transmission of dengue-1 virus in Aedes aegypti populations in Acapulco, Mexico.

We carried out dengue virus surveillance in Aedes aegypti populations from 47 neighborhoods of Acapulco during the rainy season of 2011 following a standard national protocol and as an improvement of the entomological surveillance of the Mexican Dengue Control Program. A total of 4,146 Ae. aegypti adults collected indoors and/or emerged from eggs, larvae, or pupae from households with dengue reports (probable or confirmed cases), were grouped into pools and processed using a standardized serotype-specific 4-plex real-time reverse transcriptase-polymerase chain reaction assay. Overall, only 2 (0.9%) of 226 pools of Ae. aegypti adults (1 pool of adults emerged from field-collected larvae, and another of indoor-collected adults) were positive for dengue virus 1 (DENV-1). This is appears to be the 1st report of evidence on the vertical and transovarial transmission of DENV-1 in field-caught Ae. aegypti in Mexico.

Cryopreservation induces higher oxidative stress levels in Bos indicus embryos compared with Bos taurus.

Freezing and thawing of Bos indicus embryos affect their quality for embryo transfer. The objective of this study was to compare the levels of reactive oxygen species between Bos indicus and Bos taurus embryos produced in vivo, before and after conventional freezing, as well as to analyze damage caused by apoptosis and lipid peroxidation. Bos indicus has higher levels of reactive oxygen species than Bos taurus embryos, both fresh (14.32 ± 1.41 auf vs 8.07 ± 1.15 auf (arbitrary units of fluorescence), P < 0.05) and after freezing (20.91 ± 1.21 auf vs 14.39 ± 0.58 auf, P < 0.05). The number of apoptotic nuclei is also significantly higher in Bos indicus embryos than Bos taurus (8.28 ± 0.80 vs 1 ± 0.57, P < 0.05) but highlighting a notable increase after the freeze-thaw process in both subspecies (Bos indicus from 8.28 ± 0.80 to 10.71 ± 0.42, P < 0.05; Bos taurus from 1 ± 0.57 to 5.5 ± 1.15, P < 0.05). Finally, although lipid peroxidation is lower in Bos indicus embryos before freezing in comparison with Bos taurus (2.46 ± 0.14 vs 4.20 ± 0.51), the effect after the freeze-thaw process showed an increase of 4.34 in Bos indicus than Bos taurus embryos (51.45 ± 5.52 auf vs 11.85 ± 2.88 auf, P < 0.05). In conclusion, in comparison with Bos taurus, Bos indicus embryos undergo greater oxidative stress causing increases in the cryopreservation process, promoting major cell damage and lowering embryonic viability.

IgTM: an algorithm to predict transmembrane domains and topology in proteins.

BACKGROUND: Due to their role of receptors or transporters, membrane proteins play a key role in many important biological functions. In our work we used Grammatical Inference (GI) to localize transmembrane segments. Our GI process is based specifically on the inference of Even Linear Languages. RESULTS: We obtained values close to 80% in both specificity and sensitivity. Six datasets have been used for the experiments, considering different encodings for the input sequences. An encoding that includes the topology changes in the sequence (from inside and outside the membrane to it and vice versa) allowed us to obtain the best results. This software is publicly available at: http://www.dsic.upv.es/users/tlcc/bio/bio.html CONCLUSION: We compared our results with other well-known methods, that obtain a slightly better precision. However, this work shows that it is possible to apply Grammatical Inference techniques in an effective way to bioinformatics problems.

Lipid droplets in clusters negatively affect Bos indicus embryos during cryopreservation.

Embryo transfer using cryopreserved B. indicus embryos results in low pregnancy rates. The low viability of B. indicus embryos is likely a result of their low freezability relative to embryos from other species, notably B. taurus. Freezability is probably related to the quantity of lipid droplets in the embryonic cells. However, the mechanism underlying this association is unknown. Using multiphoton and transmission electron microscopy, the proportion, volume, morphology and composition of lipid droplets in fresh and cryopreserved B. indicus and B. taurus embryos were studied. B. indicus embryos have more droplets than B. taurus and a greater percentage of lipid droplets in clusters (34% vs. 24%; p < 0.05). Also, B. indicus individual droplets are of greater volume than those of B. taurus (47 µm3 vs. 34 µm3 ; p < 0.05). After freezing, the percentage of clusters decreased in B. indicus (11% vs. 5%; p < 0.05), while in B. taurus they increased (2% vs. 7%; p < 0.05). Freezing influenced the lipid droplet morphology, increasing the number of cracked droplets in B. indicus embryos (33% fresh vs. 62% frozen; p < 0.05), but not in B. taurus embryos (35% fresh vs. 34% frozen; ns). Regarding composition, saturated lipid droplets prevail in B. indicus embryos, contrary to B. taurus embryos. We observed differences in lipid droplet organization, proportion, volume, morphology and composition between B. indicus and B. taurus embryos which are altered in opposite directions following freezing.

Evidence of vertical transmission and co-circulation of chikungunya and dengue viruses in field populations of Aedes aegypti (L.) from Guerrero, Mexico.

BACKGROUND: We report results of the entomo-virological surveillance system in Aedes aegypti local populations performed by the Ministry of Health of Guerrero. METHODS: Indoor-adult Ae. aegypti collected at Acapulco, Zihuatanejo, Coyuca de Benitez and Atoyac de Alvarez (dry season, 2015) were processed for dengue virus (DENV) and chikungunya virus (CHIKV) using RT-PCR. RESULTS: We identified different seroptypes of DENV (2, 3 and 4), CHIKV and their co-circulation in field-caught mosquitoes across a significant geographic area. Pools of males were positive for CHIKV and DENV 3 and 4 suggesting vertical transmission. CONCLUSIONS: Entomo-virological surveillance in Guerrero has identified early circulation of CHIKV and DENV and provided a trigger for timely and focalized vector control actions.

Inference of reversible tree languages.

In this paper, we study the notion of k-reversibility and k-testability when regular tree languages are involved. We present an inference algorithm for learning a k-testable tree language that runs in polynomial time with respect to the size of the sample used. We also study the tree language classes in relation to other well known ones, and some properties of these languages are proven.

Antioxidant N-acetyl-cysteine protects retinal pigmented epithelial cells from long-term hypoxia changes in gene expression.

PURPOSE: To further know the signaling pathways involved in hypoxia-induced apoptosis in retinal pigmented epithelial (RPE) cells and to improve the understanding of the antioxidant N-acetyl-cysteine (NAC) treatment effect. METHODS: We analyzed the expression levels of several apoptosis-related genes by semiquantitative reverse transcriptase-polymerase chain reaction in RPE after 72 h of maintained hypoxia, with or without 10 mM NAC treatment. RESULTS: Under hypoxic conditions, we detected a higher expression level of p53 and CASP8. Cell treatment with NAC 10 mM prevented this increase. Other apoptosis-related genes such as bax, CASP3, CASP4, CASP7, and fas did not show an increase in expression levels in hypoxia. CONCLUSIONS: NAC prevents the increased expression levels of p53 and CASP8 induced by long-term maintained hypoxia. The supply of antioxidants could be a useful preventive approach in protecting RPE from the effects of chronic oxygen stress, which is of great interest in oxygen stress-related diseases such as age-related macular degeneration and other senescence-associated pathologies.