Standardization of a multiplex assay to identify weak D types in a mixed-race Brazilian population.

RH allele variability is caused by several types of variants, resulting in altered RhD and RhCE phenotypes. Most of the weak D phenotypes in European-derived populations are weak D types 1, 2, or 3, which are not involved in alloimmunization episodes. However, the Brazilian population is racially diverse, and the accuracy of molecular and serologic tests developed in recent years has allowed for the identification of other RH variants, that are common in the Brazilian population, such as weak D type 38 or weak partial 11, the latter involved in alloimmunization cases. Furthermore, patients with these two weak D variants must be transfused with D- red blood cell units, as do patients with weak D type 4 or DAR, which are also common D variants in Brazil. Weak D type 38 and weak partial 11 can be serologically misclassified as weak D types 1, 2, or 3 in patients, based on European experience, or as D- in donors. Additionally, pregnant women may unnecessarily be identified as requiring Rh immune globulin. RhCE phenotypes are reliable indicators of RhD variants. For individuals with the Dce phenotype, the preferred approach is to specifically search for RHD*DAR. However, when encountering DCe or DcE phenotypes, we currently lack a developed method that assists us in rapidly identifying and determining the appropriate course of action for the patient or pregnant woman. Two multiplex assays were proposed: one for the identification of RHD*weak partial 11, RHD*weak D type 38, and RHD*weak D type 3 and another for RHD*weak D type 2 and RHD*weak D type 5. The multiplex assays were considered valid if the obtained results were equivalent to those obtained from sequencing. Expected results were obtained for all tested samples. The proposed multiplex allele-specific polymerase chain reaction assays can be used in the molecular investigation of women of childbearing age, patients, and blood donors presenting a weak D phenotype with DCe or DcE haplotypes in a mixed-race population, such as Brazil.

Study of genetic variability in pigs after the traditional breeding program.

Molecular markers are tools used to improve genetic gains. The objective of this study was to analyze the security of alleles of molecular marker genes for characteristics of economic interest in a pure population of pigs. After the extraction of DNA from the hair of 272 Large White matrices, the allele and genotype frequency of single nucleotide polymorphism was performed using the ARMS-PCR Multiplex technique in the DGAT1, LEPR, H-FABP, MC4R, and SREBF1 genes using RFLP-PCR for the GH gene. After capillary electrophoresis in an automated DNA sequencing of the DGAT1, LEPR, H-FABP, and SREBF1 genes, no polymorphisms were found. Only the MC4R marker presented 100% heterozygosity. For the GH gene, 209 of the initial population samples were genotyped. The PCR product (605 bp) was digested with the restriction enzyme DdeI, with fragments being of 335, 148, and 122 bp for the D1 allele and 457 and 148 bp for the D2 allele. The genotypic frequency obtained of D1D2 was 88% and of D2D2 was 22%. The D1 allele presented a frequency of 11% and the D2 allele of 89%. The high intensity of selection for commercial breeds justifies the absence or the low number of polymorphisms for the genes studied.

Distribution of Dekkera bruxellensis in a sugarcane-based fuel ethanol fermentation plant.

UNLABELLED: We investigated the presence of the yeast Dekkera bruxellensis in samples collected at three points surrounding the industrial alcoholic fermentation plants of two distilleries where there are often cases of contamination caused by this yeast: this involved sugar cane wash water, feeding sugar cane juice and vinasse from the treatment pond. Total yeast was isolated in WLN medium with bromocresol green and cycloheximide and further selected on the basis of its ability to grow in synthetic medium containing nitrate. Following this, colonies were selected from the distribution on nitrate plates and identified by amplification with species-specific primers and DNA sequencing of the 26S-D1/D2 locus. The results showed that D. bruxellensis is introduced through the feeding substrate, which suggests that its cells originated with the harvested cane. Subsequently, its population circulates as a result of the reuse of water for washing the cane, in a continuous re-inoculation of the plant with yeasts. Furthermore, the yeast population is formed in the vinasse by the addition of wash water into the treatment ponds and then reintroduced to the culture fields by fertigation, so that the process can be renewed in the following season. It is now possible to adopt sanitation procedures that can prevent the entry of the contamination to the fermentation process. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of the yeast Dekkera bruxellensis is sometimes attributed to a decline in the industrial productivity of ethanol since it has a more limited fermentation capacity than Saccharomyces cerevisiae. Although its adaptability to the industrial environment has been noted, so far, there has been no evidence to determine the source of this contamination. In this study, we provide evidence to show that D. bruxellensis comes from the fields together with the harvested cane and is then accumulated and recirculated. It might be possible to prevent the accumulation of this yeast by carrying out sanitation controls during the harvesting season.

Effects of lactic acid bacteria with bacteriocinogenic potential on the fermentation profile and chemical composition of alfalfa silage in tropical conditions.

The fermentation profile, chemical composition, and microbial populations of alfalfa silages treated with microbial inoculants (MI) at different fermentation periods (T) were evaluated in tropical conditions. A 4×6 factorial arrangement was used in a randomized design with 3 replicates. Fresh alfalfa was treated with (1) no treatment (CTRL), (2) commercial inoculant (CIN), (3) Pediococcus acidilactici (strain 10.6, S1), and (4) Pediococcus pentosaceus (strain 6.16, S2). An inoculant application rate of 10(6) cfu/g of fresh forage was used. The fermentation periods were 1, 3, 7, 14, 28, and 56 d. Alfalfa was harvested 82 d after sowing at the early flowering stage, chopped into 1.5-cm particle size, and ensiled in 25 × 35 cm vacuum-sealed plastic bags. The numbers of lactic acid bacteria, enterobacteria, mold, and yeast in alfalfa before ensiling were 5.42, 5.58, 4.82, and 4.8 log cfu/g, respectively. Silage chemical composition was evaluated only at 56 d. All parameters were affected by the interaction MI × T, except the concentrations of lactic and propionic acids. Alfalfa silage treated with S1 or S2 had lower pH values than CTRL from the first day until 28 d. However, the inoculants resulted in similar pH after 56 d, and these values were lower than the CTRL. The highest concentration of lactic acid was observed in the silage treated with S1 and S2 at 7 and 14 d of ensiling. The concentration of acetic acid was lower in the silages treated with S1 and S2 than the CTRL and CIN at 3 and 28 d of fermentation. There was no effect of MI or MI × T interaction on the microbial populations. However, the number of enterobacteria decreased over the fermentation period until 14 d and increased slightly after this time point. The chemical composition of alfalfa silage was not affected by MI at 56 d of ensiling. The strain P. pentosaceus 6.16 was the most efficient in dominating the fermentation process by decreasing the pH more quickly and increasing the concentration of lactic acid, suggesting its potential use as a silage inoculant.

Short Communication Quick method for identifying horse (Equus caballus) and donkey (Equus asinus) hybrids.

The domestication of the Equus genus 5000-6000 years ago has influenced the history of human civilization. As soon as horse and donkey species had been domesticated, they were crossbred, producing humanity's first documented attempt at animal genome manipulation. Since then, the mule (male donkey x female horse) and the reciprocal cross (the hinny, male horse x female donkey) have been the most common equine hybrids in the world. Due to their hybrid vigor, mules and hinnies have been intensively used for carrying loads and people and for tilling the land. Despite their importance, visual distinction of mules and hinnies is difficult due to high phenotypic resemblance. However, the distinction between these two hybrids is of pivotal importance for equid breeders and ranchers. In this study, an easy, low-cost, effective, and fast multiplex-polymerase chain reaction method was developed to distinguish the maternal origin of mules and hinnies, targeting the hyper-variable mitochondrial DNA D-loop region. This methodology can help breeders, ranchers, animal science professionals, and researchers manage their equine herds with more confidence and precision.

The effect of a chemical additive on the fermentation and aerobic stability of high-moisture corn.

The objective of this experiment was to evaluate the effect of a chemical additive on the fermentation and aerobic stability of high-moisture corn (HMC). Ground HMC (~63% dry matter) was untreated, or treated with an additive containing sodium benzoate, potassium sorbate, and sodium nitrite as active ingredients, at 0, 2, 3, or 4 L/t of fresh matter. Laboratory silos (7.5 L) were prepared and ensiled for 21 and 90d (4 silos/treatment per d of ensiling). Small bag silos were prepared for untreated HMC and HMC treated with 4 L/t of the additive and analyzed for nitrate-N and nitrite-N after 0, 3, and 7d of ensiling. The concentration of nitrate-N was similar between these 2 treatments and was below levels considered problematic for ruminants. Nitrite-N was greater in HMC treated with the high level of additive but was also very low for both treatments. Numbers of yeasts were similar among treatments in fresh HMC and decreased substantially after ensiling. Numbers of yeasts were similar among treatments after 21d of ensiling but after 90d they were lower in treated versus untreated HMC. Concentrations of organic acids (lactic, acetic, and propionic) and pH were not different among treatments at any time of ensiling. In contrast, treatment with the additive markedly decreased the concentration of ethanol in HMC after 21 and 90d when compared with untreated HMC. Treatment with all levels of the additive markedly improved the aerobic stability and improved the recovery of dry matter compared with untreated HMC. Overall, our findings suggest that the chemical additive used in this study has the potential to improve the fermentation and aerobic stability of HMC after a relatively short period (21d) and after a moderate length (90d) of ensiling.

Effect of regrowth interval and a microbial inoculant on the fermentation profile and dry matter recovery of guinea grass silages.

The objectives of this study were to characterize and quantify the microbial populations in guinea grass (Panicum maximum Jacq. cultivar Mombasa) harvested at different regrowth intervals (35, 45, 55, and 65 d). The chemical composition and fermentation profile of silages (after 60 d) with or without the addition of a microbial inoculant were also analyzed. Before ensiling, samples of the plants were used for the isolation and identification of lactic acid bacteria (LAB) in the epiphytic microbiota. A 4 × 2 factorial arrangement of treatments (4 regrowth intervals × with/without inoculant) was used in a completely randomized design with 3 replications. Based on the morphological and biochemical characteristics and the carbohydrate fermentation profile, Lactobacillus plantarum was found to be the predominant specie of LAB in guinea grass forage. Linear increases were detected in the dry matter (DM) content and concentrations of neutral detergent fiber, acid detergent fiber, acid detergent insoluble nitrogen, and DM recovery as well as linear reductions in the concentrations of crude protein and NH3-N with regrowth interval. Additionally, linear reductions for gas and effluent losses in silages were detected with increasing regrowth interval. These results demonstrate that guinea grass plants harvested after 55 d of regrowth contain a LAB population sufficiently large to ensure good fermentation and increase the DM recovery. The use of microbial inoculant further enhanced the fermentation of guinea grass at all stages of regrowth by improving the DM recovery.

Functional capacity, lung function, and muscle strength in patients undergoing hematopoietic stem cell transplantation: A prospective cohort study.

OBJECTIVE/BACKGROUND: Hematopoietic stem cell transplantation (HSCT) is a treatment for benign and malignant hematological diseases. These aggressive treatments cause reduced levels of physical activity, decreased lung function, and worse quality of life. Alterations in pulmonary function tests before HSCT are associated with the risk of respiratory failure and early mortality. The objective of this study was to evaluate functional capacity and lung function before and after HSCT and identify the predictors of mortality after 2 years. METHODS: A prospective cohort study was carried out with individuals with oncohematological diseases. The evaluations were carried out in two moments during hospitalization and at hospital discharge. Follow-up was carried out after 48 months. Assessments were carried out on 34 adults, using spirometry, manovacuometry, 6-Minute Walk Test (6MWT), Handgrip Strength Test, and 30-Second Chair Stand Test (30-s CST). RESULTS: There was a statistically significant reduction for the variables in forced vital capacity, forced expiratory volume predicted in the 1st second, Tiffeneau index, handgrip strength, and distance covered (% predicted) on the 6MWT (p < .05). There was a significant difference in the 30-s CST when individuals were compared according to the type of transplant. We found that a 10% reduction in the values of maximum inspiratory pressure (MIP) can predict an increased risk for mortality. CONCLUSIONS: Individuals undergoing HSCT have reduced functional capacity, lung function, and muscle strength during the hospitalization phase. Reduction in the values of MIP increases the risk of nonrelapse mortality.

Effects of vitamin D administration on nociception and spinal cord pro-oxidant and antioxidant markers in a rat model of neuropathic pain.

Reactive oxygen species (ROS) are involved in neuropathic pain, a complicated condition after nerve tissue lesion. Vitamin D appears to improve symptoms of pain and exhibits antioxidant properties. We investigated the effects of oral administration of vitamin D3, the active form of vitamin D, on nociception, the sciatic functional index (SFI), and spinal cord pro-oxidant and antioxidant markers in rats with chronic constriction injury (CCI) of the sciatic nerve, a model of neuropathic pain. Vitamin D3 (500 IU/kg per day) attenuated the CCI-induced decrease in mechanical withdrawal threshold and thermal withdrawal latency (indicators of antinociception) and SFI. The vitamin prevented increased lipid hydroperoxide levels in injured sciatic nerve without change to total antioxidant capacity (TAC). Vitamin D3 prevented increased lipid hydroperoxide, superoxide anion generation (SAG), and hydrogen peroxide (H2O2) levels in the spinal cord, which were found in rats without treatment at 7 and 28 days post-CCI. A significant negative correlation was found between mechanical threshold and SAG and between mechanical threshold and H2O2 at day 7. Vitamin D3 also prevented decreased spinal cord total thiols content. There was an increase in TAC in the spinal cord of vitamin-treated CCI rats, compared to CCI rats without treatment only at 28 days. No significant changes were found in body weight and blood parameters of hepatic and renal function. These findings demonstrated, for first time, that vitamin D modulated pro-oxidant and antioxidant markers in the spinal cord. Since antinociception occurred in parallel with oxidative changes in the spinal cord, the oxidative changes may have contributed to vitamin D-induced antinociception.

Oxidative stress is involved in LLLT mechanism of action on skin healing in rats.

The skin injury healing process involves the main phases of homoeostasis, inflammation, proliferation, and remodeling. The present study aimed to analyze the effects of low-level laser therapy (LLLT) on hematological dynamics, oxidative stress markers, and its relation with tissue healing following skin injury. Wistar rats were divided into control, sham, skin injury, and skin injury LLLT. The biochemical and morphological analyses were performed in the inflammatory (1 and 3 days) and regenerative phases (7, 14, and 21 days) following injury. The skin injury was performed in the dorsal region, between the intrascapular lines, using a surgical punch. LLLT (Al-Ga-In-P, λ=660 nm, energy density of 20 J/cm2, 30 mW power, and a time of 40 s) was applied at the area immediately after injury and on every following day according to the experimental subgroups. LLLT maintained hematocrit and hemoglobin levels until the 3rd day of treatment. Surprisingly, LLLT increased total leukocytes levels compared to control until the 3rd day. The effects of LLLT on mitochondrial activity were demonstrated by the significant increase in MTT levels in both inflammatory and regenerative phases (from the 1st to the 7th day), but only when associated with skin injury. The results indicated that LLLT modulated the inflammatory response intensity and accelerated skin tissue healing by a mechanism that involved oxidative damage reduction mostly at early stages of skin healing (inflammatory phase).

Aging and low-intensity exercise change oxidative biomarkers in brain regions and radiographic measures of femur of Wistar rats.

We investigated changes in oxidative biomarkers in brain regions such as brainstem, cerebellum, and cerebral cortex of 3-, 6-, 18-, 24-, and 30-month-old rats. We also assessed the effects of low-intensity exercise on these biomarkers in these regions of 6-, 18-, and 24-month-old rats that started exercise on a treadmill at 3, 15, and 21 months of age, respectively. Radiographic images of the femur were taken for all rats. A total of 25 rats (age: twelve 6-, ten 18-, ten 24-, and three 30-month-old rats) were used. Lipid hydroperoxide levels increased in cerebellum at 18 months. Total antioxidant activity exhibited lowest values in brainstem at 3 months. Superoxide dismutase activity did not exhibit significant changes during aging. Total thiol content exhibited lowest values in brain regions of 24- and 30-month-old rats. Exercise reduced total thiol content in brainstem at 6 months, but no change occurred in other regions and other ages. Femur increased its length and width and cortical thickness with advancing age. No change occurred in medullary width. Radiolucency increased and sclerosis was found in cortical and medullary bone with advancing age. Exercise reduced radiolucency and medullary sclerosis. Therefore, aging differentially changed oxidative biomarkers in different brain regions and radiographic measures of the femur. Low-intensity exercise only ameliorated some radiographic measurements of femur. Since the present study possessed limitations (small number of rats per group), a beneficial effect of regular low-intensity exercise on oxidative markers in brain cannot be ruled out.

Cell cycle arrest induced by pisosterol in HL60 cells with gene amplification.

The leukemia cell line HL60 is widely used in studies of the cell cycle, apoptosis, and adhesion mechanisms in cancer cells. We conducted a focused cytogenetic study in an HL60 cell line, by analyzing GTG-banded chromosomes before and after treatment with pisosterol (at 0.5, 1.0, and 1.8 microg/ml), a triterpene isolated from Pisolithus tinctorius, a fungus collected in the Northeast of Brazil. Before treatment, 99% of the cells showed the homogeneously staining region (HSR) 8q24 aberration. After treatment with 1.8 microg/ml pisosterol, 90% of the analyzed cells lacked this aberration. We further performed a pulse test, in which the cells treated with pisosterol (0.5, 1.0, and 1.8 microg/ml) were washed and re-incubated in the absence of pisosterol. Only 30% of the analyzed cells lacked the HSR 8q24 aberration, suggesting that pisosterol probably blocks the cells with HSRs at interphase. No effects were detected at lower concentrations. At the highest concentration examined (1.8 microg/ml), pisosterol also inhibited cell growth, but this effect was not observed in the pulse test, reinforcing our hypothesis that, at the concentrations tested, pisosterol probably does not induce cell death in the HL60 line. The results found for pisosterol were compared with those for doxorubicin. Cells that do not show a high degree of gene amplification (HSRs and double-minute chromosomes) have a less aggressive and invasive behavior and are easy targets for chemotherapy. Therefore, further studies are needed to examine the use of pisosterol in combination with conventional anti-cancer therapy.

Age-related changes and effects of regular low-intensity exercise on gait, balance, and oxidative biomarkers in the spinal cord of Wistar rats.

The present study aimed to analyze age-related changes to motor coordination, balance, spinal cord oxidative biomarkers in 3-, 6-, 18-, 24-, and 30-month-old rats. The effects of low-intensity exercise on these parameters were also analyzed in 6-, 18-, and 24-month-old rats. Body weight, blood glucose, total cholesterol, and high-density lipoprotein (HDL) cholesterol were assessed for all rats. The soleus muscle weight/body weight ratio was used to estimate skeletal muscle mass loss. Body weight increased until 24 months; only 30-month-old rats exhibited decreased blood glucose and increased total cholesterol and HDL cholesterol. The soleus muscle weight/body weight ratio increased until 18 months, followed by a small decrease in old rats. Exercise did not change any of these parameters. Stride length and step length increased from adult to middle age, but decreased at old age. Stride width increased while the sciatic functional index decreased in old rats. Performance in the balance beam test declined with age. While gait did not change, balance improved after exercise. Aging increased superoxide anion generation, hydrogen peroxide levels, total antioxidant capacity, and superoxide dismutase activity while total thiol decreased and lipid hydroperoxides did not change. Exercise did not significantly change this scenario. Thus, aging increased oxidative stress in the spinal cord, which may be associated with age-induced changes in gait and balance. Regular low-intensity exercise is a good alternative for improving age-induced changes in balance, while beneficial effects on gait and spinal cord oxidative biomarkers cannot be ruled out because of the small number of rats investigated (n=5 or 6/group).

Bonding Interaction and Shrinkage Stress of Low-viscosity Bulk Fill Resin Composites With High-viscosity Bulk Fill or Conventional Resin Composites.

OBJECTIVE: To analyze the shrinkage stress, bonding interaction, and failure modes between different low-viscosity bulk fill resin composites and conventional resin composites produced by the same manufacturer or a high-viscosity bulk fill resin composite used to restore the occlusal layer in posterior teeth. METHODS & MATERIALS: Three low-viscosity bulk fill resin composites were associated with the conventional resin composites made by the same manufacturers or with a high-viscosity bulk fill resin composite, resulting in six groups (n=10). The bonding interaction between resin composites was tested by assessing the microshear bond strength (µSBS). The samples were thermocycled and were tested with 1-mm/min crosshead speed, and the failure mode was evaluated. The post-gel shrinkage (Shr) of all the resin composites was measured using a strain gauge (n=10). The modulus of elasticity (E) and the hardness (KHN) were measured using the Knoop hardness test. Two-dimensional finite element models were created for analyzing the stress caused by shrinkage and contact loading. The µSBS, Shr, E, and KHN data were analyzed using the Student t-test and one-way analysis of variance. The failure mode data were subjected to chi-square analysis (α=0.05). The stress distribution was analyzed qualitatively. RESULTS: No significant difference was verified for µSBS between low-viscosity bulk fill resin composites and conventional or high-viscosity bulk fill composites in terms of restoring the occlusal layer (p=0.349). Cohesive failure of the low-viscosity bulk fill resin composites was the most frequent failure mode. The Shr, E, and KHN varied between low-viscosity and high-viscosity resin composites. The use of high-viscosity bulk fill resin composites on the occlusal layer reduced the stress at the enamel interface on the occlusal surface. CONCLUSIONS: The use of high-viscosity bulk fill resin composites as an occlusal layer for low-viscosity bulk fill resin composites to restore the posterior teeth can be a viable alternative, as it shows a similar bonding interaction to conventional resin composites as well as lower shrinkage stress at the enamel margin.

Obesity, estrone, and coronary artery disease in postmenopausal women.

OBJECTIVES: The link between obesity and endogenous estrogen with coronary artery disease (CAD) in postmenopausal women is uncertain. In this prospective study we analyzed the association of body mass index (BMI) and blood levels of estrone in postmenopausal women with known CAD or with a high risk factor score for CAD. METHODS: Participants were 251 female clinic patients aged 50-90 years who were postmenopausal and not using estrogen therapy. Clinical and behavioral characteristics and fasting blood for estrone and heart disease risk factors were collected at baseline, and again at 1 and 2 years. Women were grouped according to their BMI (kg/m2) as normal (18.5< or =BMI<25), overweight (25< or =BMI<30) or obese (BMI > or =30), and by low and high estrone levels (<15 and > or =15pg/mL, respectively). Fatal and nonfatal events were recorded for 2 years after baseline. RESULTS: Women with a low estrone level were older, thinner, and had less hypertension, diabetes, and lower triglyceride and glucose levels. BMI was positively associated with estrone levels, hypertension, and diabetes and inversely associated with HDL cholesterol. There were 14 deaths, 8 attributed to CAD. The Kaplan-Meier survival curve showed a nonsignificant trend (p=0.074) of greater all cause mortality in women with low estrone levels (<15mL). In this model, adjusted for BMI, age [OR=1.08; p=0.03], C-reactive protein [OR=1.24; p=0.024] and hypertension [OR=6.22; p=0.003] were independent predictors of all cause mortality. CONCLUSIONS: Postmenopausal women with low estrone levels (<15pg/mL) had a trend for increased mortality over the next 2 years. Larger, longer studies are needed.

Mechanisms involved in anti-aging effects of guarana (Paullinia cupana) in Caenorhabditis elegans.

Guarana (Paullinia cupana) is habitually ingested by people in the Amazon region and is a key ingredient in various energy drinks consumed worldwide. Extension in longevity and low prevalence of chronic age-related diseases have been associated to habitual intake of guarana. Anti-aging potential of guarana was also demonstrated in Caenorhabditis elegans; however, the mechanisms involved in its effects are not clear. Herein, we investigated the putative pathways that regulate the effects of guarana ethanolic extract (GEE) on lifespan using C. elegans. The major known longevity pathways were analyzed through mutant worms and RT-qPCR assay (DAF-2, DAF-16, SKN-1, SIR-2.1, HSF-1). The possible involvement of purinergic signaling was also investigated. This study demonstrated that GEE acts through antioxidant activity, DAF-16, HSF-1, and SKN-1 pathways, and human adenosine receptor ortholog (ADOR-1) to extend lifespan. GEE also downregulated skn-1, daf-16, sir-2.1 and hsp-16.2 in 9-day-old C. elegans, which might reflect less need to activate these protective genes due to direct antioxidant effects. Our results contribute to the comprehension of guarana effects in vivo, which might be helpful to prevent or treat aging-associated disorders, and also suggest purinergic signaling as a plausible therapeutic target for longevity studies.

Deletion of a single allele of Cx43 is associated with a reduction in the gap junctional intercellular communication and increased cell proliferation of mouse lung pneumocytes type II.

OBJECTIVES: Connexins (Cx) are proteins that form the gap junctional channels at neighbouring plasma membranes between adjacent cells. Cxs are involved in cell communication, which is reportedly correlated with cell proliferation and differentiation. Alterations in connexin expression and/or gap junctional intercellular communication (GJIC) capacity have long been postulated to be important in a number of pathological conditions including cancer. This study was performed to determine the consequences of the deletion of a single allele of Gja1 (Cx43 gene) in Alveolar Type II cells (APTIIs), and its impact on GJIC and cell proliferation. MATERIAL AND METHODS: In order to do so, APTIIs from wild type (Cx43(+/+)) and heterozygous (Cx43(+/-)) mice were harvested and cultured for 4 days. The GJIC capacity was evaluated by scrape-loading method, with the transfer of lucifer yellow dye. The expression of Cx43 was evaluated by immunofluorescence method and Western blotting. Cell proliferation was evaluated by 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS: It was observed that GJIC capacity was significantly reduced and cell proliferation index was significantly higher in Cx43(+/-) cells compared to Cx43(+/+) cells. CONCLUSIONS: These results show that knocking out one allele of Cx43 leads to a lower cell to cell communication capacity, and consequently induces a higher cell proliferation. Because chemically induced lung adenomas in mice are known to originate from APTIIs, these alterations may play a critical role in their susceptibility to lung carcinogenesis.

Psychiatric comorbidity in young heroin users.

In order to determine the prevalence of psychiatric comorbidity in a population of young heroin users recruited from outside of the healthcare context, a sample was assembled by targeted sampling and nomination techniques; it was comprised of regular current users of heroin aged between 18 and 30 years and resident in Barcelona, Spain. Psychiatric evaluation was done with the Psychiatric Research Interview for Substance and Mental Disorders (PRISM) semi-structured interview. Of 149 individuals evaluated, 33% were women, whose mean age was 25.1 years; 93% received a diagnosis of heroin dependence and 71% of cocaine dependence. Thirty-two percent of the subjects had never been treated for substance use. Around two-thirds (67.1%, 95% CI: 59.6-74.7%) of the sample had lifetime psychiatric comorbidity, with antisocial personality and mood disorders being the most frequent conditions (33% and 26%, respectively). Mood, anxiety and eating disorders were more common among women than men. There were no differences in ever having been in treatment for drug use according to the presence of psychiatric comorbidity, although comorbidity was lower among those currently in treatment. Young heroin users recruited on the street presented a high prevalence of psychiatric comorbidity which was unrelated to past treatment history.

Protective effects of guarana (Paullinia cupana Mart. var. Sorbilis) against DEN-induced DNA damage on mouse liver.

Guarana (Paullinia cupana Mart. var. Sorbilis) is a plant originally from Brazil, which is rich in tannins. Some tannins are known to present protective effects against DNA damage. This study was performed to investigate the anti-genotoxic/cytotoxic properties of guarana in hepatocytes of mice injected with N-nitrosodiethylamine (DEN). The protective effect of guarana was evaluated both by comet assay and DNA smear fragmentation technique in two month-old female BALB/c mice. These were treated previously with 2.0 mg/g bw of guarana for 16 days and then injected with DEN (160 microg/g body weight) to induce DNA damage. The DEN-only treated group presented higher comet image length than the guarana plus DEN and untreated groups (116.06+/-5.0 microm, 104.09+/-3.3 microm and 93.28+/-14.4 microm, respectively; p<0.01). Guarana treatment presented a 52.54% reduction in comet image length when animals were exposed to DEN (p<0.05). DNA samples from the guarana plus DEN group clearly showed less EtBr fluorescence intensity when compared to the DEN-only group, reinforcing the comet assay data. These results show, for the first time, that guarana has a protective effect against DEN-induced DNA damage in mouse liver.

Effects of vitamin D administration on nociception and spinal cord pro-oxidant and antioxidant markers in a rat model of neuropathic pain

Reactive oxygen species (ROS) are involved in neuropathic pain, a complicated condition after nerve tissue lesion. Vitamin D appears to improve symptoms of pain and exhibits antioxidant properties. We investigated the effects of oral administration of vitamin D3, the active form of vitamin D, on nociception, the sciatic functional index (SFI), and spinal cord pro-oxidant and antioxidant markers in rats with chronic constriction injury (CCI) of the sciatic nerve, a model of neuropathic pain. Vitamin D3 (500 IU/kg per day) attenuated the CCI-induced decrease in mechanical withdrawal threshold and thermal withdrawal latency (indicators of antinociception) and SFI. The vitamin prevented increased lipid hydroperoxide levels in injured sciatic nerve without change to total antioxidant capacity (TAC). Vitamin D3 prevented increased lipid hydroperoxide, superoxide anion generation (SAG), and hydrogen peroxide (H2O2) levels in the spinal cord, which were found in rats without treatment at 7 and 28 days post-CCI. A significant negative correlation was found between mechanical threshold and SAG and between mechanical threshold and H2O2 at day 7. Vitamin D3 also prevented decreased spinal cord total thiols content. There was an increase in TAC in the spinal cord of vitamin-treated CCI rats, compared to CCI rats without treatment only at 28 days. No significant changes were found in body weight and blood parameters of hepatic and renal function. These findings demonstrated, for first time, that vitamin D modulated pro-oxidant and antioxidant markers in the spinal cord. Since antinociception occurred in parallel with oxidative changes in the spinal cord, the oxidative changes may have contributed to vitamin D-induced antinociception.