Technical note: Evaluation of a commercial on-farm milk leukocyte differential tester to identify subclinical mastitis cases in dairy cows.

The objective of this study was to validate the precision and accuracy of a milk leukocyte differential tester to identify subclinical mastitis cases in dairy cows. Milk samples from individual quarters (n = 320) of 80 Holstein cows were aseptically collected and analyzed in this study. Each sample was divided into 2 replicate samples after mixing. One replicate was analyzed for somatic cell count (SCC) using the current gold standard of flow cytometry immediately after milking. The second sample was evaluated using the on-farm milk leukocyte differential tester directly after milking, where total leukocyte count (TLC; cells/mL) was obtained. The SCC and TLC were used to calculate somatic cell score (SCS) and TLC score [TLS = log2 (TLC/100,000) + 3]. Two subclinical mastitis thresholds were set: >200,000 (low) and >400,000 (high) cells/mL. First, precision was determined between the 2 methods. Total leukocyte count and calculated TLS from the milk leukocyte differential device were compared with the gold standard using correlation and regression coefficient of determination analyses. Correlation coefficients (r) were 0.97 for TLC and SCC and 0.90 for TLS and SCS. The coefficient of determination for regression (R2) was 0.94 for TLC and SCC and 0.80 for TLS and SCS. Slopes of regression for scores and measures were 0.36 [95% confidence interval (CI): 0.35-0.37] and 0.69 (CI: 0.65-0.73), respectively; both were significantly different from 1. Sensitivity, specificity, and diagnostic accuracy were calculated for correct diagnosis of the 2 SCC thresholds using the gold standard as reference. The sensitivity of the on-farm test was 58% (95% CI: 44 to 71%) and 73% (95% CI: 56 to 86%) for the low and high thresholds, respectively. The specificities for the on-farm test were 100% (95% CI: 99 to 100%) and 100% (95% CI: 98 to 100%) for the low and high thresholds, respectively. Subclinical diagnosis accuracies were 93% (95% CI: 89 to 95%) and 96% (95% CI: 92 to 98%) for the low and high thresholds, respectively. The on-farm milk leukocyte differential tester was precise but not overall accurate for total cell counts; it had high specificity and accuracy for diagnosis compared with a standard diagnostic tool. These results suggest that the tested system is a promising technology to detect subclinical mastitis on-farm.

Associations of freestall design and cleanliness with cow lying behavior, hygiene, lameness, and risk of high somatic cell count.

The objective of this study was to investigate associations of freestall design and cleanliness with cow lying behavior, hygiene, lameness, and risk of new high somatic cell count (SCC). Cows from 18 commercial freestall dairy herds (22 ± 15 cows/farm; mean ± SD) in Ontario, Canada, were enrolled in a longitudinal study. Four hundred focal cows that were <120 d in milk, had no mastitis treatment in the last 3 mo, and had an SCC <100,000 cells/mL at their most recent milk test were selected for the study. Data on SCC were collected through Dairy Herd Improvement Association milk testing (at ~5-wk intervals). Each farm was visited 5 ± 3 d (mean ± SD) after each milk test until 3 tests were completed (~105 d), for a total of 3 observation periods per cow. Elevated SCC was used as an indicator of subclinical mastitis. An incident of new high SCC was defined as a cow having SCC >200,000 cells/mL at the end of an observation period, when SCC was <100,000 cells/mL at the beginning of that period. Lying behavior was recorded for 6 d after each milk sampling, using electronic data loggers. Cows were scored during each period for lameness (5-point scale, with scores ≥3 = lame), body condition score (BCS; 5-point scale; 1 = thin to 5 = fat), and hygiene (4-point scale). Stall cleanliness was assessed during each period with a 1.20 × 1.65-m metal grid, containing 88 squares. The grid was centered between stall partitions of every tenth stall on each farm, and the squares containing visible urine or fecal matter (or both) were counted. Cow lying time averaged 10.9 ± 1.9 h/d. On average, cows with low BCS (≤2.5) spent 37 ± 16.6 min/d less time lying down than high-BCS cows (≥4.0). On average, cows tended to spend 36 ± 18.3 min/d more time lying down in deep-bedded versus mattress-based stalls. Mean proportion of soiled squares per stall was 20.1 ± 0.50%. Across farms, cow lying time decreased as the proportion of soiled squares per stall increased. A difference in daily lying time of ~80 more min/d was modeled for cows housed in barns with the cleanest stalls compared with those with the dirtiest stalls. Higher neck rail height [for every 1 SD (10 cm) increase] increased the odds (odds ratio = 1.5) of cows having a dirty upper leg-flank and udder. The odds of a cow having a dirty upper leg-flank, udder, and lower legs were 1.5, 2.0, and 1.9 times greater, respectively, for cows housed with dirtier stalls. Also, cows housed on farms with dirtier stalls had 1.3 times greater odds of being lame at the time of observation. Over the study period, 50 new high-SCC cases were detected, resulting in an incidence rate of 0.45 cases of new high SCC per cow-year at risk. No measured factors were detected to be associated with risk of a new high SCC. Overall, our results confirm that cows lie down longer in cleaner and more comfortable environments. Further, these results highlight the need for improved stall cleanliness to optimize lying time and potentially reduce lameness.

Study of TiO2/Ti4O7 photo-anodes inserted in an activated carbon packed bed cathode: Towards the development of 3D-type photo-electro-Fenton reactors for water treatment.

In this work, commercially available Polymethyl-meta-acrylate (PMMA) spectroscopy cells were modified on the external walls with films of TiO2, Ti4O7 or TiO2/Ti4O7 mixtures. Film characterization was carried out using SEM and UV-vis spectroscopy. The results of photocatalytic (PC), electro-oxidation (EO), and photoelectrochemical (PEC) experiments on the decolorization of a methyl orange (MO) model dye solution showed that while anatase provides better photocatalytic properties and the partially reduced Ti4O7 larger electronic conductivity, the TiO2/Ti4O7 composite film behaves as a semiconductor substrate that combines the advantages of both materials (for PEC experiments for instance, decolorization values for the model dye solution using TiO2, Ti4O7 and a TiO2/Ti4O7 mixed film, corresponded to 35%, 46% and 53%, respectively). In order to test this film as an effective photoanode material in a 3-D type reactor for water treatment processes, a TiO2/Ti4O7 modified PMMA spectroscopy cell was inserted in an activated carbon (AC) bed so that the semiconductor material could be illuminated using an external UV source positioned inside the PMMA cell. The connected AC particles that were previously saturated with MO dye were used as cathode sites for the oxygen reduction reaction so that the photoelectrochemical reactions that take place in the anode could be complemented with coupled electro-Fenton processes in the cathode. As expected, the combination resulted in an effective decolorization of the dye solution that results from a complex combination of processes. The experimental decolorization data was successfully fitted to a pseudo-first order kinetic model so that a deeper understanding of the contribution of each process in the reactor could be obtained.

Validation of ADAM10 metalloprotease as a Bacillus thuringiensis Cry3Aa toxin functional receptor in Colorado potato beetle (Leptinotarsa decemlineata).

Bacillus thuringiensis parasporal crystal proteins (Cry proteins) are insecticidal pore-forming toxins that bind to specific receptor molecules on the brush border membrane of susceptible insect midgut cells to exert their toxic action. In the Colorado potato beetle (CPB), a coleopteran pest, we previously proposed that interaction of Cry3Aa toxin with a CPB ADAM10 metalloprotease is an essential part of the mode of action of this toxin. Here, we annotated the gene sequence encoding an ADAM10 metalloprotease protein (CPB-ADAM10) in the CPB genome sequencing project, and using RNA interference gene silencing we demonstrated that CPB-ADAM10 is a Cry3Aa toxin functional receptor in CPB. Cry3Aa toxicity was significantly lower in CPB-ADAM10 silenced larvae and in vitro toxin pore-forming ability was greatly diminished in lipid planar bilayers fused with CPB brush border membrane vesicles (BBMVs) prepared from CPB-ADAM10 silenced larvae. In accordance with our previous data that indicated this toxin was a substrate of ADAM10 in CPB, Cry3Aa toxin membrane-associated proteolysis was altered when CPB BBMVs lacked ADAM10. The functional validation of CPB-ADAM10 as a Cry3Aa toxin receptor in CPB expands the already recognized role of ADAM10 as a pathogenicity determinant of pore-forming toxins in humans to an invertebrate species.

[Polarized light microscopy for evaluation of oocytes as a prognostic factor in the evolution of a cycle in assisted reproduction]. / Microscopia de luz polarizada para evaluación de ovocitos como factor pronóstico en la evolución de un ciclo de reproducción asistida.

BACKGROUND: Identification of the best embryos to transfer is a key element for success in assisted reproduction. In the last decade, several morphological criteria of oocytes and embryos were evaluated with regard to their potential for predicting embryo viability. The introduction of polarization light microscopy systems has allowed the visualization of the meiotic spindle and the different layers of the zona pellucida in human oocytes on the basis of birefringence in a non-destructive way. Conflicting results have been reported regarding the predictive value in ICSI cycles. OBJECTIVE: To assess the predictive ability of meiotic spindle and zona pellucida of human oocytes to implant by polarized microscopy in ICSI cycles. MATERIAL AND METHODS: Prospective and observational clinical study. 903 oocytes from 94 ICSI cycles were analyzed with polarized microscopy. Meiotic spindle visualization and zona pellucida birefringence values by polarized microscopy were correlated with ICSI cycles results. RESULTS: Meiotic spindle visualization and birefringence values of zona pellucida decreased in a direct basis with increasing age. In patients aged over the 35 years, the percentage of a visible spindle and mean zona pellucida birefringence was lower than in younger patients. Fertilization rate were higher in oocytes with visible meiotic spindle (81.3% vs. 64%; p < 0.0001), as well as embryo quality (47.4% vs. 39%; p=0.01). Fertilization rate was higher in oocytes with positive values of birefringence (77.5 % vs. 68.5% p=0.005) with similar embryo quality. Conception cycles showed oocytes with higher mean value of zona birefringence and visible spindle vs. no-conception cycles (p<0.05). CONCLUSIONS: Polarized light microscopy improves oocyte selection, which significantly impacts in the development of embryos with greater implantation potential. The use of polarized light microscopy with sperm selection methods, blastocyst culture and deferred embryo transfers will contribute to transfer fewer embryos without diminishing rates of live birth and single embryo transfer will be more feasible.

Validation of the Unesp-Botucatu pig composite acute pain scale (UPAPS) in piglets undergoing castration.

To accurately assess pain and support broadly-based analgesic protocols to mitigate swine pain, it is imperative to develop and validate a species-specific pain scale. The objective of this study was to investigate the clinical validity and reliability of an acute pain scale (UPAPS) adapted for newborn piglets undergoing castration. Thirty-nine male piglets (five days of age, 1.62 ± 0.23 kg BW) served as their own control, were enrolled in the study and underwent castration in conjunction with an injectable analgesic administered one-hour post-castration (flunixin meglumine 2.2 mg/kg IM). An additional 10, non-painful female piglets were included to account for the effect of natural behavioral variation by day on pain scale results. Behavior of each piglet was video recorded continuously at four recording periods (24 h pre-castration, 15 min post-castration, 3 and 24 h post-castration). Pre- and post-operative pain was assessed by using a 4-point scale (score 0-3) including the following six behavioral items: posture, interaction and interest in surroundings, activity, attention to the affected area, nursing, and miscellaneous behavior. Behavior was assessed by two trained blinded observers and statistical analysis was performed using R software. Inter-observer agreement was very good (ICC = 0.81). The scale was unidimensional based on the principal component analysis, all items except for nursing were representative (rs ≥ 0.74) and had excellent internal consistency (Cronbach's alpha ≥ 0.85). The sum of scores were higher in castrated piglets post-procedure compared to pre-procedure, and higher than in non-painful female piglets confirming responsiveness and construct validity, respectively. Scale sensitivity was good when piglets were awake (92.9%) and specificity was moderate (78.6%). The scale had excellent discriminatory ability (area under the curve > 0.92) and the optimal cut-off sum for analgesia was 4 out of 15. The UPAPS scale is a valid and reliable clinical tool to assess acute pain in castrated pre-weaned piglets.

Effects of Bacillus thuringiensis Cry1Ab and Cry3Aa endotoxins on predatory Coleoptera tested through artificial diet-incorporation bioassays.

Traditional approaches to studying the effects of genetically modified (GM) crops on beneficial insects involve either field assays, comparing insect population levels between control and GM crops or tritrophic bioassays with contaminated insects - usually larvae or eggs of Lepidoptera - as preys. Here, we report the results of a bioassay using an artificial diet, suitable for predatory Coleoptera, to supply Bacillus thuringiensis (Bt) solubilized Cry1Ab and Cry3Aa as well as trypsin-activated Cry1Ab to Atheta coriaria and Cryptolaemus montrouzieri adults and young larvae of Adalia bipunctata. Water, solubilization buffer and trypsin-treated solubilization buffer were used as controls. In total, 1600 insects were assayed. Assays showed a relatively low mortality rate in the controls, ranging from as low as 7% after 15 days (C. montrouzieri) to about 15-20% after five days (A. bipunctata) or 15 days (A. coriaria). For all three predators, there were no statistical differences between the mortality recorded in any of the treatment groups and the corresponding controls. These results indicate a lack of short- (A. bipunctata) and long-term (A. coriaria and C. montrouzieri) mortality associated with oral ingestion of Cry1Ab and Cry3Aa at the high dose tested (50 microg ml-1). We discuss the relevance of these findings for the ecology of beneficial Coleoptera and compatibility with Bt and GM Bt crops.

Bacterial concentrations in bedding and their association with dairy cow hygiene and milk quality.

Comparison of bacterial counts (BCs) among common bedding types used for dairy cows, including straw, is needed. There is concern that the microbial content of organic bedding is elevated and presents risks for dairy cow udder health and milk quality. The objectives of this study were to investigate: (1) % DM and BCs (Streptococcus spp., all gram-negatives and specifically Klebsiella spp.) in different types of bedding sampled, and to investigate housing and farm management factors associated with % DM and BCs; (2) if bedding type was associated with hygiene of cow body parts (lower-legs, udder, upper-legs and flank) and housing and management factors associated with hygiene and (3) bedding types associated with higher BCs in cow milk at the farm level and bulk tank milk and management factors that were associated with highest BCs. Seventy farms (44 free-stall and 26 tie-stall) in Ontario, Canada were visited 3 times, 7 days apart from October 2014 to February 2015. At each visit, composite samples of unused and used bedding were collected for % DM determination and bacterial culture. Used bedding samples were collected from the back third of selected stalls. Data were analyzed using multivariable linear mixed models. Bedding classification for each farm were: new sand (n = 12), straw and other dry forage (n = 33), wood products (shavings, sawdust; n = 17) and recycled manure solids (RMSs)-compost, digestate (n = 8). In used bedding, across all bedding samples, sand was driest, compared to straw and wood, and RMS; higher % DM was associated with lower Streptococcus spp. count. Streptococcus spp. and all Gram-negative bacteria counts increased with increasing days since additional bedding was added. Gram-negative bacteria counts in used bedding varied with type: RMS = 16.3 ln colony-forming units (cfu)/mL, straw = 13.8 ln cfu/mL, new sand = 13.5 ln cfu/mL, and wood = 10.3 ln cfu/mL. Klebsiella spp. counts in used bedding were lower for wood products (5.9 ln cfu/mL) compared to all other bedding types. Mean cow SCC tended to be higher on farms with narrower stalls. Farms with mattress-based stalls had a higher prevalence of cows with dirty udders compared to those using a deep bedding system (often inorganic sand). Wider stalls were associated with lower bulk milk bacteria count. Lower % DM of used bedding was associated with higher bulk milk bacteria count. In conclusion, bedding management may have a profound impact on milk quality, bacterial concentrations in the bedding substrates, and cow hygiene.

Comparison of physical and behavioral traits between dairy cows with low and high somatic cell count.

The objective of this study was to examine associations of locomotion score, hygiene, body condition score (BCS), lying behavior, and milk production with dairy cow somatic cell count (SCC; low or high). Cows from 14 commercial free-stall dairy herds in Ontario, Canada were enrolled in a cross-sectional study. Each farm was visited for a total of 3 observation periods (at 5-wk intervals) on 2 occasions per period (7 d apart) until 3 Dairy Herd Improvement (DHI) milk tests had been completed. Upon immediate receiving of the results of each DHI test, lactating Holstein cows were selected according to SCC. Cows that ranked in the top 10% for SCC in each herd (≥200,000 cells/mL; n = 370) were first selected and paired based on parity and DIM to cows within the same herd with low SCC (≤100,000 cells/mL; n = 382). Lying behavior was recorded for selected cows for 6 d after each milk test sampling, using data loggers. On the visit where data loggers were attached, cows were scored for gait (1 = sound to 5 = lame) and hygiene of udder, lower legs, and upper legs/flank (1 = clean to 4 = dirty). On the visit where data loggers were removed 7 d later, BCS (1 = thin to 5 = fat) and hygiene were scored. Cows were then classified into each of the scoring categories for hygiene (clean: ≤ 2, dirty: ≥3), BCS (high: ≥4, normal: 3-3.5, low: ≤2.5), and gait (sound: ≤2, lame: ≥ 3). As compared to normal BCS cows, low BCS cows were associated with high SCC (OR = 1.57, 95% CI = 1.00-2.47). Cows with high SCC were associated with producing 2.2 ± 0.72 kg/d less milk than those with low SCC. As compared to normal BCS cows, low BCS cows were associated with reduced lying time (-27.2 ± 12.5 min/d), decreased lower leg hygiene (OR = 2.64, 95% CI = 1.08-6.46), and increased milk production (+2.9 ± 0.88 kg/d). These results suggest that low BCS may be a mediating factor among lying behavior, hygiene, and production level with high SCC.

Refined analysis of genetic variability parameters in hepatitis C virus and the ability to predict antiviral treatment response.

Hepatitis C virus (HCV) infects approximately 3% of the world population. The chronicity of hepatitis C seems to depend on the level of genetic variability. We have recently (Torres-Puente et al., J Viral Hepat, 2008; 15: 188) reported genetic variability estimates from a large-scale sequence analysis of 67 patients infected with HCV subtypes 1a (23 patients) and 1b (44 patients) and related them to response, or lack of, to alpha-interferon plus ribavirin treatment.. Two HCV genome regions were analysed in samples prior to antiviral therapy, one compressing the three hypervariable regions of the E2 glycoprotein and another one including the interferon sensitive determining region and the V3 domain of the NS5A protein. Haplotype and nucleotide diversity measures showed a clear tendency to higher genetic variability levels in nonresponder than in responder patients. Here, we have refined the analysis of genetic variability (haplotype and nucleotide diversity, number of haplotypes and mutations) by considering their distribution in each of the biologically meaningful subregions mentioned above, as well as in their surrounding and intervening regions. Variability levels are very heterogeneous among the different subregions, being higher for nonresponder patients. Interestingly, significant differences were detected in the biologically relevant regions, but also in the surrounding regions, suggesting that the level of variability of the whole HCV genome, rather than exclusively that from the hypervariable regions, is the main indicator of the treatment response. Finally, the number of haplotypes and mutations seem to be better discriminators than haplotype and nucleotide diversity, especially in the NS5A region.

[Over-diagnosis of chronic obstructive pulmonary disease in Primary Care. Prevalence and determining factors]. / Sobrediagnóstico de enfermedad pulmonar obstructiva crónica en atención primaria. Prevalencia y condicionantes.

INTRODUCTION: COPD under-diagnosis is common in Primary Health Care medicine, due to the low use of spirometry, but there is less information about over-diagnosis of the disease in patients that have a clinical diagnosis of COPD. OBJECTIVE: The main objective of the study was to investigate the prevalence of COPD over-diagnosis in Primary Care medicine. Secondary objectives were to determine the factors associated with an incorrect clinical diagnosis of COPD and to analyse whether the pharmacological treatment is different for patients with correct or incorrect diagnosis. METHOD: A prospective, observational, cross-sectional study was conducted using the spirometry results of 206 patients with a clinical diagnosis of COPD, with no prior lung function testing, and who were treated with inhaled therapy. Characteristics and treatment of patients with a correct or incorrect COPD diagnosis were compared. RESULTS: The prevalence of COPD over-diagnosis was 42.7% in the study population. Factors associated with an incorrect diagnosis were female sex (P<.0001), obesity (P=.009), absence of smoking history (P<.0001), lower age (P=.001), and less severe dyspnoea (P=.001). Long-acting muscarinic agents were more frequently prescribed to patients with a correct COPD diagnosis. There were no other differences regarding inhaled therapies between both groups. CONCLUSIONS: Over-diagnosis is a frequent phenomenon in patients with a clinical diagnosis of COPD managed in Primary Care medicine. There are different features between patients with a correct and incorrect diagnosis. Spirometry is an essential tool to reduce COPD over-diagnosis.

Role of toxin activation on binding and pore formation activity of the Bacillus thuringiensis Cry3 toxins in membranes of Leptinotarsa decemlineata (Say).

Binding and pore formation constitute key steps in the mode of action of Bacillus thuringiensis delta-endotoxins. In this work, we present a comparative analysis of toxin-binding capacities of proteolytically processed Cry3A, Cry3B and Cry3C toxins to brush border membranes (BBMV) of the Colorado potato beetle Leptinotarsa decemlineata (CPB), a major potato coleopteran-insect pest. Competition experiments showed that the three Cry3 proteolytically activated toxins share a common binding site. Also heterologous competition experiments showed that Cry3Aa and Cry3Ca toxins have an extra binding site that is not shared with Cry3Ba toxin. The pore formation activity of the three different Cry3 toxins is analysed. High pore-formation activities were observed in Cry3 toxins obtained by proteolytical activation with CPB BBMV in contrast to toxins activated with either trypsin or chymotrypsin proteases. The pore-formation activity correlated with the formation of soluble oligomeric structures. Our data support that, similarly to the Cry1A toxins, the Cry3 oligomer is formed after receptor binding and before membrane insertion, forming a pre-pore structure that is insertion-competent.

Automated and visual measurements of estrous behavior and their sources of variation in Holstein heifers. I: Walking activity and behavior frequency.

Holstein heifers (n = 57) were monitored using accelerometers and video observations with the objective of better understanding the behavioral expression of estrus, the variation within and between the heifers, and the possible sources of variation. IceTags recorded walking activity from 7 to 13 months of age. Activity peaks (n = 282) were obtained from a rolling sum of steps within 24-hour periods and validated to be estrus by ovarian ultrasonography. Behavior around activity peak of one estrus for each of 12 heifers was described in detail from video recordings. Baseline behavior was monitored in a corresponding interval 1 week before. Estrus and baseline total steps and steps per hour, estrus relative increase in activity, duration, and interval between episodes were analyzed by descriptive statistics and Spearman rank correlations. Effects of category of baseline walking activity, estrus order (pubertal vs. second and greater episodes), season, hour of estrus onset, and number of heifers simultaneously in estrus were evaluated with proc MIXED. Behavioral changes from baseline to estrus were evaluated by a signed-rank test. Estrus total steps varied greatly (4743 ± 1740; range: 837-10,070), as well as the relative increase in activity (290 ± 160%; range: 30%-1190%). Duration of estrus was 14 ± 4 hours, ranging from 4 to 26 hours. The interval between episodes was the trait that varied the least. Pubertal estrus was shorter and had a smaller relative increase in activity than second and greater episodes (P < 0.05). The number of steps during estrus was greater for heifers of high baseline activity (P < 0.01). Estrus episodes occurring in the winter and starting between 4 PM and 3 AM had the greatest relative increase in activity (P < 0.05). The number of heifers simultaneously in estrus did not influence estrus expression (P > 0.05). The behaviors with greatest change from baseline to estrus were chin rest, sniff, back mount, crossover, accept chin rest, and follow, but variation was large. Overall, estrus was apparent in behavioral changes with large variation within and between the heifers. Estrus order, onset hour, season, and baseline walking activity are important factors affecting estrus activity. Therefore, estrus detection tools should account for potential sources of variation. The visual and automated measurements of estrus expression reported in this study reveal possibilities for improved on-farm estrus detection technologies and potential genetic selection for estrus expression.

Diabetes susceptibility in Mayas: Evidence for the involvement of polymorphisms in HHEX, HNF4α, KCNJ11, PPARγ, CDKN2A/2B, SLC30A8, CDC123/CAMK1D, TCF7L2, ABCA1 and SLC16A11 genes.

Association of type 2 diabetes (T2D) with common variants in HHEX, HNF4α, KCNJ11, PPARγ, CDKN2A/2B, SLC30A8, CDC123/CAMK1D, TCF7L2, ABCA1 and SLC16A11 genes have been reported, mainly in populations of European and Asian ancestry and to a lesser extent in Latin Americans. Thus, we aimed to investigate the contribution of rs1111875 (HHEX), rs1800961 (HNF4α), rs5219 (KCNJ11), rs1801282 (PPARγ), rs10811661 (CDKN2A/2B), rs13266634 (SLC30A8), rs12779790 (CDC123/CAMK1D), rs7903146 (TCF7L2), rs9282541 (ABCA1) and rs13342692 (SLC16A11) polymorphisms in the genetic background of Maya population to associate their susceptibility to develop T2D. This is one of the first studies designed specifically to investigate the inherited component of T2D in the indigenous population of Mexico. SNPs were genotyped by allelic discrimination method in 575 unrelated Maya individuals. Two SNPs rs10811661 and rs928254 were significantly associated with T2D after adjusting for BMI; rs10811661 in a recessive and rs9282541 in a dominant model. Additionally, we found phenotypical alterations associated with genetic variants: HDL to rs9282541 and insulin to rs13342692. In conclusion, these findings support an association of genetic polymorphisms to develop T2D in Maya population.

Mode of action of Bacillus thuringiensis PS86Q3 strain in hymenopteran forest pests.

The mode of action of Cry toxins has been described principally in lepidopteran insects as a multistep process. In this work we describe the mode of action of a Cry toxin active in the common pine sawfly Diprion pini (Hymenoptera, Diprionidae), considered a major forest pest in Europe. Strain PS86Q3 contains a long bipyramidal crystal composed of five major proteins. The N-terminal sequence shows that the 155 kDa protein corresponds to Cry5B toxin and the other proteins belong to the Cry5A subgroup. PCR analysis indicates the presence of cry5Ac and cry5Ba genes, suggesting that Cry5A protein should be Cry5Ac. Activation of protoxins with trypsin or with midgut content from D. pini and Cephacia abietis (Hymenoptera, Pamphiliidae) (spruce webspinning sawfly), another important hymenopteran forest pest, produced a single 75 kDa toxin that corresponded to Cry5A by N-terminal sequence and is responsible for the insecticidal activity. Homologous competition experiments with D. pini and C. abietis brush border membrane vesicles (BBMV) showed that the binding interaction of Cry5A is specific. Membrane potential measurements using a fluorescent dye indicate that Cry5A toxin at nM concentration caused immediate permeability changes in the BBMV isolated from both hymenopteran larvae. The initial response and the sustained permeability change are cationic as previously shown for Cry1 toxins. These results indicate that the hymenopteran specific Cry5A toxin exerts toxicity by a similar mechanism as Cry1 toxins.

Prevalence of hepatitis B virus infection and related risk factors in a rural community of Mexico.

A cross-sectional survey of the seroprevalence of hepatitis B virus (HBV) markers among healthy people was conducted in San Juanito, a rural community in the northern state of Chihuahua, Mexico. The overall prevalence in 970 people was 6.6% for antibody to hepatitis B core antigen. There was an age effect on the prevalence of HBV infection, and a gradual increase in prevalence was observed in patients up to the age of 40 years. Those subjects with a history of dental procedures had a 2-fold higher risk for HBV infection (odds ratio [OR], 2.4; 95% confidence intervals [CI], 1.01-5.86), and there was a 74% increased risk for each blood product transfusion (OR, 1.74; 95% CI, 1.09-2.77). Horizontal transmission seems to be the major source of endemicity in San Juanito because no woman was a chronic carrier. To lower HBV transmission rate, an adequate active screening program for blood donors should be implemented, together with a universal infant immunization program.

Centrally administered aminoglycoside antibiotics antagonize naloxone-precipitated withdrawal in mice acutely dependent on morphine.

The effects of i.c.v. administration of several aminoglycoside antibiotics on naloxone-precipitated morphine withdrawal symptoms were evaluated in mice acutely dependent on morphine. Neomycin (10-40 micrograms/mouse), gentamicin (40-160 micrograms/mouse) and kanamycin (80-320 micrograms/mouse) produced a dose-dependent reduction of the number of precipitated jumps, forepaw tremors and head shakes. The order of potency of the aminoglycoside antibiotics on all withdrawal symptoms was neomycin > gentamicin > kanamycin, which is the same order that these drugs show as N-type calcium channel blockers. The capacity of several drugs that decrease neuronal calcium availability (such as lanthanum and L-type calcium channel blockers) to antagonize opiate withdrawal is well known. In the light of these findings, our results suggest that the mechanism of aminoglycoside-induced inhibition of morphine abstinence may be related to the capacity of these antibiotics to block N-type calcium channels, and to decrease neuronal calcium availability.

Role of L-type calcium channels on yohimbine-precipitated clonidine withdrawal in vivo and in vitro.

This study was designed to elucidate the possible participation of L-type calcium channels in the expression of clonidine-withdrawal precipitated by yohimbine in clonidine-dependent animals. Mice implanted for 5 days with osmotic minipumps containing the alpha 2-adrenoceptor agonist clonidine showed symptoms of a withdrawal syndrome (jerks, headshakes, defecations and weight loss) when yohimbine, an alpha 2-adrenoceptor antagonist, was injected. Similarly, isolated rat ilea incubated with clonidine in vitro showed a withdrawal contracture when yohimbine was added to the organ bath. The effects of L-type calcium channel blockers (verapamil and diltiazem) and the stimulant Bay K 8644 on these two different types of withdrawal responses were evaluated. A dose-dependent decrease in yohimbine-precipitated clonidine withdrawal in vivo was observed when verapamil (10-40 mg/kg, s.c. and 120 micrograms/mouse, i.c.v.) or diltiazem (5-20 mg/kg, s.c. and 160 micrograms/mouse, i.c.v.) were administered to mice dependent on clonidine. No effect was found after Bay K 8644 (0.5-5 mg/kg, s.c. and 1-5 micrograms/mouse) was injected under these conditions. In vitro, both verapamil (0.1-5 microM) and D-cis-diltiazem (1-50 microM) concentration-dependently reduced the height of the yohimbine-precipitated withdrawal contracture in rat ileum incubated with clonidine. Furthermore, the effect of diltiazem was stereospecific, as D-cis-diltiazem 10 microM markedly inhibited clonidine withdrawal, whereas the same concentration of L-cis-diltiazem had no effect. In contrast, the calcium channel stimulant Bay K 8644 (0.1-1 microM) increased the height of the ileum withdrawal contracture.(ABSTRACT TRUNCATED AT 250 WORDS)

[Crohn disease in 3 siblings]. / Enfermedad de Crohn en tres hermanos.

Three siblings without a known familiar history of chronic inflammatory disease of the intestine suffered Crohn's disease. They were the 3rd, 4th and 8th siblings of a family of 8 siblings. The onset of the disease and its course were similar in all three patients. Considering the form of familial presentation and the possible influence of environmental factors in this type of patients, a review is made of the influence of these factors in accordance with the clinical patients' evolution.