A single nucleotide polymorphism in NEUROD1 is associated with production traits in Nelore beef cattle.

Feed efficiency and carcass characteristics are late-measured traits. The detection of molecular markers associated with them can help breeding programs to select animals early in life, and to predict breeding values with high accuracy. The objective of this study was to identify polymorphisms in the functional and positional candidate gene NEUROD1 (neurogenic differentiation 1), and investigate their associations with production traits in reference families of Nelore cattle. A total of 585 steers were used, from 34 sires chosen to represent the variability of this breed. By sequencing 14 animals with extreme residual feed intake (RFI) values, seven single nucleotide polymorphisms (SNPs) in NEUROD1 were identified. The investigation of marker effects on the target traits RFI, backfat thickness (BFT), ribeye area (REA), average body weight (ABW), and metabolic body weight (MBW) was performed with a mixed model using the restricted maximum likelihood method. SNP1062, which changes cytosine for guanine, had no significant association with RFI or REA. However, we found an additive effect on ABW (P ≤ 0.05) and MBW (P ≤ 0.05), with an estimated allele substitution effect of -1.59 and -0.93 kg0.75, respectively. A dominant effect of this SNP for BFT was also found (P ≤ 0.010). Our results are the first that identify NEUROD1 as a candidate that affects BFT, ABW, and MBW. Once confirmed, the inclusion of this SNP in dense panels may improve the accuracy of genomic selection for these traits in Nelore beef cattle as this SNP is not currently represented on SNP chips.

Allele- and parent-of-origin-specific effects on expression of the KCNJ11 gene: A candidate for meat tenderness in cattle.

In contrast to the Mendelian inheritance model, parental alleles can contribute unequally to gene expression, which may result in phenotypic variance among individuals and bias in the predicted additive effect of molecular markers associated with production traits. Given the need to understand the effects of allelic variation and parent-of-origin effects on the expression of genes with a commercial interest in cattle, we analyzed the expression of KCNJ11 (potassium inwardly rectifying channel, subfamily J, member 11), which was previously described as a functional candidate gene for meat tenderness. Allele-specific and parent-of-origin-dependent expression of this gene were assessed in bovine muscle using the rs379610823 single nucleotide polymorphism as a reference. Biallelic expression was observed; however, the T allele was expressed at significantly higher levels than the C allele. Furthermore, increased expression of KCNJ11 was found in animals harboring the maternal T allele. This study is the first to describe the differential allelic expression of bovine KCNJ11. Our findings are important for understanding the mechanisms that underlie the pattern of KCNJ11 expression and its potential impact on the phenotypic variation of meat tenderness in Nelore beef cattle. This reinforces the need for further investigation of allelic- and parent-of-origin expression deviation in genetic markers eligible for the selection of target traits.

Application of fluorescence in situ hybridization (FISH) as a tool to aid cytogenetics in 1,409 fetal samples.

AIM: To evaluate the technical application of fluorescence in situ hybridization (FISH) as a support to classical cytogenetic in numerical chromosomal aneuploidies studies in samples of amniotic fluid, chorionic villus, and fetal loss. MATERIALS AND METHODS: The authois performed cytogenetic analyses in 1,409 patients (678 amniocentesis, 512 chorionic villus samples, and 219 spontaneous abortions) during one year. FISH molecular study aided traditional cytogenetic in 90 cases. These cases were indicated based on the diagnostic hypothesis of each patient or when no cellular growth was obtained. The authors standardized the FISH in discoloured slides. RESULTS: They had 85% positive FISH in amniotic fluid, 70% in chorionic villus, and 90% in abortion material using 13, 18, 21 X and Y centromeric probes. It showed 12% of altered FISH in amniotic fluid (100% trisomies), 10% in chorionic'villus (50% trisomy and 50% X - monosomy), and 22% in abortion material (50% trisomy, 25% X-monosomy, and 25% triploidy). FISH and cytogenetic analysis confirmed the results. CONCLUSION: This technique revolutionized clinical and research applications of cytogenetics. In this particular paper, FISH was a valuable and reliable technique to promptly identify rapid detection of aneuploidies in interphase cells, metaphase spread and paraffin-embedded samples. It is hoped that, in the future, the economic viability of array CGH and FISH, with the decreasing cost of testing and their genomics advantages can be incorporated as routine and customized in the approach of prenatal diagnosis.

Selection of lactic acid bacteria to promote an efficient silage fermentation capable of inhibiting the activity of Aspergillus parasiticus and Fusarium gramineraum and mycotoxin production.

AIMS: To select lactic acid bacteria with potential silage inoculant properties. The bio-control activity against mycotoxicogenic fungi and the presence of antibiotics resistance gene were also evaluated. METHODS AND RESULTS: Lactobacillus rhamnosus RC007 and Lactobacillus plantarum RC009 were selected on the basis of growth rate and efficacy in reducing the pH of maize extract medium; therefore, they were evaluated for their bio-control ability against Fusarium graminearum and Aspergillus parasiticus. Studies on lag phase, growth rate and aflatoxin B1 (AFB1) and zearalenone (ZEA) production were carried out in vitro under different regimes of aw (0·95 and 0·99); pH (4 and 6); temperature (25 and 37°C); and oxygen availability (normal and reduced). Lactobacillus rhamnosus RC007 was able to completely inhibit the F. graminearum growth at all assayed conditions, while Lact. plantarum RC009 only did it at pH 4. Both Lactobacillus strains were able to significantly reduce the A. parasiticus growth rate mainly at 0·99 aw . A decrease in ZEA production was observed as result of Lactobacillus strains -F. graminearum interaction; however, the A. parasiticus- Lact. plantarum interaction resulted in an increased AFB1 production. Lactobacillus rhamnosus RC007 proved to have no genes for resistance to the tested antibiotics. CONCLUSIONS: The ability of Lact. rhamnosus RC007 to rapidly drop the pH and to inhibit fungal growth and mycotoxin production and the absence of antibiotic resistance genes shows the potential of its application as inoculant and bio-control agent in animal feed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the importance of selecting bacteria for silage inoculants not only for the improvement of silage fermentation but also for their effects on mycotoxicogenic fungi and the resulting mycotoxin production due to the risk that they may involve.

Communication: electronic band gaps of semiconducting zig-zag carbon nanotubes from many-body perturbation theory calculations.

Electronic band gaps for optically allowed transitions are calculated for a series of semiconducting single-walled zig-zag carbon nanotubes of increasing diameter within the many-body perturbation theory GW method. The dependence of the evaluated gaps with respect to tube diameters is then compared with those found from previous experimental data for optical gaps combined with theoretical estimations of exciton binding energies. We find that our GW gaps confirm the behavior inferred from experiment. The relationship between the electronic gap and the diameter extrapolated from the GW values is also in excellent agreement with a direct measurement recently performed through scanning tunneling spectroscopy.

In vitro culture of bovine embryos in murine ES cell conditioned media negatively affects expression of pluripotency-related markers OCT4, SOX2 and SSEA1.

Despite extensive efforts, establishment of bovine embryonic stem (ES) cell lines has not been successful. We hypothesized that culture conditions for in vitro-produced (IVP) embryos, the most used source of inner cell mass (ICM) to obtain ES cells, might affect their undifferentiated state. Therefore, the aim of this work was to improve pluripotency of IVP blastocysts to produce suitable ICM for further culturing. We tested KSR and foetal calf serum (FCS) supplements in SOF medium and ES cell conditioned medium (CM) on IVC (groups: KSR, KSR CM, FCS and FCS CM). Cleavage and blastocyst rates were similar between all groups. Also, embryonic quality, assessed by apoptosis rates (TUNEL assay), total cell number and ICM percentage did not differ between experimental groups. However, expression of pluripotency-related markers was affected. We detected down-regulation of OCT3/4, SOX2 and SSEA1 in ICM of FCS CM blastocysts (p < 0.05). SOX2 gene expression revealed lower levels (p < 0.05) on KSR CM blastocysts and a remarkable variation in SOX2 mRNA levels on FCS-supplemented blastocysts. In conclusion, pluripotency-related markers tend to decrease after supplementation with ES cell CM, suggesting different mechanisms regulating mouse and bovine pluripotency. KSR supplementation did not differ from FCS, but FCS replacement by KSR may produce blastocysts with stable SOX2 gene expression levels.

Early-Life Exposure of Pigs to Topsoil Alters miRNA and mRNA Expression in Peripheral Blood Mononuclear Cells.

Exposure to less-hygienic conditions during early childhood has been associated with stimulation and development of the immune system. A recent study indicated that exposure of piglets to soil-borne microbes during lactation was related with modulation of gut microbiota and immune function. To identify the potential molecular mechanisms and pathways impacted by early-life topsoil exposure, we analyzed the messenger RNA (mRNA) and micro-RNA (miRNA) expression in peripheral blood mononuclear cells (PBMCs) from these piglets. Total RNA was extracted from the PBMCs of piglets exposed to topsoil only from d 4-d 21 of life (mRNA n = 6; miRNA n = 5) or unexposed control pigs (mRNA n = 6; miRNA n = 8) at 11, 20, and 56 days of age. Small RNA and mRNA were sequenced with 50-bp single-end reads using Illumina chemistry. Sequence data were quality checked with FASTQC software and aligned to the Sscrofa 11.1 genome with the STAR aligner for mRNA and mirDeep2 for miRNA. Differential expression (DE) analysis was performed using PROC Glimmix of SAS to evaluate changes in expression due to topsoil exposure over time with genes declared DE at a false discovery rate (FDR) of q < 0.10. A total of 138 mRNA and 21 miRNAs were identified as DE for the treatment by age interaction. Ontology enrichment analysis of DE mRNA revealed Gene ontology (GO) terms directly involved in the connection between T-cell and antigen-presenting cells that are associated with T-cell activation. Key regulatory genes identified include PTPRJ, ITGB3, TRBV30, CD3D, mir-143, mir-29, and mir-148a. While these results require validation, this study provides data supporting the hypothesis that less-hygienic environments during early life may contribute to the development of the immune system.

Anomalous n-type electrical behaviour of Pd-contacted CNTFET fabricated on small-diameter nanotube.

A Pd-contacted dopant-free CNTFET with small-diameter (0.57 nm) carbon nanotube showing an anomalous n-type electrical characteristic is reported for the first time. This observed behaviour is attributed to a carbon nanotube work function higher than (or close to) palladium as well as a large hole-to-electron effective mass ratio of approximately 2.5 predicted by hybridization in small-diameter nanotubes. A variation of the conduction type with temperature is also observed and is attributed to an increase of the palladium work function and decrease of the CNT work function with increasing temperature.

Effects of immunosuppressant FK-506 on tooth movement.

OBJECTIVE: To test the hypothesis that immunosuppressant tacrolimus treatment can interfere with bone turnover and rate of tooth movement. MATERIAL AND METHODS: One-hundred twenty Wistar male rats were divided into four groups: Group 1 (rats subjected to orthodontic movement plus treatment with saline solution vehicle), Group 2 (rats subjected to orthodontic movement plus treatment with FK506), Group 3 (rats treated with FK506 only), and Group 4 (rats treated with saline solution vehicle). The maxillary incisors were laterally moved with a reciprocal load of 35 cN. The dosage of FK506 was 2 mg/kg/day. Howship's lacunae, osteoclasts, and macrophages were counted. RESULTS: Tooth movement was found to be greater in Group 1 than in Group 2 for all time periods (on days 3, 7, and 14), although a significant difference was observed only on days 7 and 14 (p < 0.05). The number of osteoclasts was smaller in Group 1 than in Group 2, whereas the number of Howship's lacunae was greater. CONCLUSION: FK506 has the capacity of promoting osteoclasts inhibition with probable osteoclastic apoptosis of alveolar bone following tooth movement.

Common dysregulation of Wnt/Frizzled receptor elements in human hepatocellular carcinoma.

Dysregulation of growth factors and their receptors is central to human hepatocellular carcinoma (HCC). We previously demonstrated that the Frizzled-7 membrane receptor mediating the Wnt signalling can activate the beta-catenin pathway and promotes malignancy in human hepatitis B virus-related HCCs. Expression patterns of all the 10 Frizzled receptors, and their extracellular soluble autoparacrine regulators (19 Wnt activators and 4 sFRP inhibitors) were assessed by real-time RT-PCR in 62 human HCC of different etiologies and their matched peritumorous areas. Immunostaining was performed to localise Frizzled on cell types in liver tissues. Regulation of three known Frizzled-dependent pathways (beta-catenin, protein kinase C, and C-Jun NH(2)-terminal kinase) was measured in tissues by western blot. We found that eight Frizzled-potentially activating events were pleiotropically dysregulated in 95% HCC and 68% peritumours as compared to normal livers (upregulations of Frizzled-3/6/7 and Wnt3/4/5a, or downregulation of sFRP1/5), accumulating gradually with severity of fibrosis in peritumours and loss of differentiation status in tumours. The hepatocytes supported the Wnt/Frizzled signalling since specifically overexpressing Frizzled receptors in liver tissues. Dysregulation of the eight Frizzled-potentially activating events was associated with differential activation of the three known Frizzled-dependent pathways. This study provides an extensive analysis of the Wnt/Frizzled receptor elements and reveals that the dysregulation may be one of the most common and earliest events described thus far during hepatocarcinogenesis.

Phosphorylated cAMP response element-binding protein as a molecular marker of memory processing in rat hippocampus: effect of novelty.

From mollusks to mammals the activation of cAMP response element-binding protein (CREB) appears to be an important step in the formation of long-term memory (LTM). Here we show that a 5 min exposure to a novel environment (open field) 1 hr after acquisition of a one-trial inhibitory avoidance training hinders both the formation of LTM for the avoidance task and the increase in the phosphorylation state of hippocampal Ser 133 CREB [phosphorylated CREB (pCREB)] associated with the avoidance training. To determine whether this LTM deficit is attributable to the reduced pCREB level, rats were bilaterally cannulated to deliver Sp-adenosine 3', 5'-cyclic monophosphothioate (Sp-cAMPS), an activator of PKA. Infusion of Sp-Adenosine 3',5'-cyclic monophosphothioate Sp-cAMPS to CA1 region increased hippocampal pCREB levels and restored normal LTM of avoidance learning in rats exposed to novelty. Moreover, a 5 min exposure to the open field 10 min before the avoidance training interferes with the amnesic effect of a second 5 min exposure to the open field 1 hr after avoidance training and restores the hippocampal levels of pCREB. In contrast, the avoidance training-associated activation of extracellular signal-regulated kinases (p42 and p44 mitogen-activated protein kinases) in the hippocampus is not altered by novelty. Together, these findings suggest that novelty regulates LTM formation by modulating the phosphorylation state of CREB in the hippocampus.

Comparative histopathology of endomyocardial biopsies in chagasic and non-chagasic heart transplant recipients.

BACKGROUND: Heart transplantation has been an option for the treatment of chagasic (C) cardiomyopathy despite difficulties concerning the control of rejection and reactivation. The parasite-host interaction under the influence of immunosuppressive therapy may affect the immunological response to the graft in a pattern different from that in non-chagasic (NC) patients. The aim of this study was to compare the major histopathological features in heart transplantation in C and NC patients. METHODS: We studied 293 endomyocardial biopsies from two groups of heart transplanted patients, including 18 C and 15 NC. Both groups had identical surgical and clinical procedure except immunosuppressive therapy was lower in C patients. The histopathological parameters evaluated were the Quilty effect, rejection, C myocarditis reactivation, fibrosis, hypertrophy, and ischemia. In addition, lymphocytic cellular infiltration of myocarditis due to rejection or reactivation was immunophenotyped in the biopsies of both groups with rejection grades 3 to 4, in biopsies with signs of reactivation, and in fragments of the receptor heart with chronic C myocarditis. A search for Trypanosoma cruzi was performed in all biopsies in the C group in which lymphocyte immunophenotyping was done. We used immunofluorescence and confocal microscopy. RESULTS: The Quilty effect was present in 23% of the biopsies, involving 69.7% of the patients without a significant difference between groups (p = 0.509). Rejection was frequently observed in biopsies with the Quilty effect and the effect often recurred in the same patient. Rejection grades 3 to 4 was more frequent in the C group (p = 0.023). There were 5 episodes of Chagas' disease reactivation with myocarditis in 2 cases. The mean numbers of CD8+ and CD4+ T cells, and the CD4+-to-CD8+ ratio were similar for rejection in both groups (p > 0.05), while the CD4+-to-CD8+ ratio was significantly lower in chronic C myocarditis compared to rejection in the C group (p = 0.043). There was no significant difference in ischemic damage or interstitial fibrosis in the groups but there was a higher frequency of hypertrophy in the NC group (p = 0.007). CONCLUSIONS: The histopathological features of heart transplantation in C patients did not differ from that in NC patients in regard to the Quilty effect, development of myocardial fibrosis and ischemia. However, the higher involvement of the C group for rejection grades 3 to 4 suggested higher susceptibility to this event. The similarity of the lymphocytic cellular composition for rejection in both groups indicates that C patients respond to immunological stimulus in a similar pattern as NC patients.

Interstitial dendritic cells of the heart harbor Trypanosoma cruzi antigens in experimentally infected dogs: importance for the pathogenesis of chagasic myocarditis.

Heart sections from 16 mongrel dogs, two normal controls and 14 infected with Trypanosoma cruzi, were submitted to immunohistochemical staining with either rabbit anti-cow S100 Protein monoclonal antibody or rabbit anti-T. cruzi purified specific antibody, using the peroxidase technique to investigate the participation of the interstitial dendritic cells of the heart (IDCs) in myocarditis of Chagas disease. Trypanosoma cruzi antigens were revealed as granular and dense deposits in IDC membrane in the heart of infected dogs both during acute and chronic myocarditis, but not in normal controls. Anti-S100 Protein labeled the IDCs, both in normal and infected dogs and a significant increase in the numbers of IDCs occurred in the myocardium, proportionally to the intensity of the inflammatory infiltration. These findings suggest that IDCs, probably by presenting T. cruzi antigens to immune-competent cells, play an important role in the pathogenesis of Chagas disease.

Role of the spleen on the pathogenesis of schistosomal periportal (pipestem) fibrosis of the liver: an experimental approach.

Splenectomized and sham-operated mice with chronic Schistosoma mansoni infection were compared for the development of periportal fibrosis (pipestem fibrosis). Although this lesion appeared less frequently in splenectomized mice, it also developed in the absence of the spleen. The time of splenectomy, spleen weight, presence of anti-idiotypic antibodies, the number of eggs in the liver, and the type and size of periovular granulomas in the liver as evaluated by computerized morphometry did not show statistically significant differences between the two groups. It is concluded that the role of the spleen in the development of pipestem fibrosis seems ancillary and that multifactorial influences, including worm burden, hepatic vascular adjustment, and factors associated with the biology of extracellular matrix of the liver, probably play a more significant role.

Melanin-concentrating hormone (MCH) modifies memory retention in rats.

The purpose of the present study was to evaluate the possible effect of melanin-concentrating hormone (MCH) on learning and memory by using the one-trial step-down inhibitory avoidance test in rats. The peptide was infused into hippocampus, amygdala, and entorhinal cortex. MCH caused retrograde facilitation when given at 0 or 4 h post-training into hippocampus, but only at 0 h into amygdala. From these results, it seems that MCH modulates memory early after training by acting on both the amygdala and hippocampus and, 4 h after training, on the hippocampus.

NMDA-coupled periaqueductal gray glycine receptors modulate anxioselective drug effects on plus-maze performance.

The present study was carried out to investigate a possible interaction between the effects of anxiety modulating drugs which act at the GABA-A receptor complex and selective N-methyl-D-aspartic acid (NMDA) coupled glycine receptor (GLY-B receptor) ligands within the dorsal periaqueductal gray (DPAG). The plus-maze performance of rats pretreated with diazepam (0.37 and 0.75 mg/kg, i.p.) or pentylenetetrazole (15 and 30 mg/kg, i.p.), standard anxiolytic and anxiogenic drugs respectively, was assessed following intra-periaqueductal injections of either glycine (0.2 M, 0.4 microl/30 s, i.c.) or its competitive antagonist, 7-chlorokynurenic acid (7ClKYN, 0.02 M, 0.4 microl/30 s, i.c.). Whilst diazepam produced a typical anxiolytic effect in intracranially-injected CSF rats, increasing open arm exploration, pentylenetetrazole displayed an opposite anxiogenic profile. Either anxiogenic or anxiolytic effects were seen in peripherally-injected vehicle rats following intra-periaqueductal injections of glycine or 7ClKYN, respectively. Intra-periaqueductal injection of glycine markedly attenuated the anxiolytic effect of diazepam. Moreover, while the anxiogenic effects of pentylenetetrazole were barely changed by glycine, they were markedly attenuated by intra-periaqueductal injection of 7ClKYN. Interaction of diazepam and 7ClKYN produced non-selective sedative-like effects which masked any possible anxiolytic action. Accordingly, the present results suggest that the NMDA-coupled glycine receptors located in the DPAG interfere with anxioselective effects of GABA-A acting drugs on the elevated plus-maze. In spite of the prevailing notion that the NMDA coupled glycine receptor is saturated at in vivo brain concentrations of glycine, our results also suggest that either unoccupied or low-affinity GLY-B receptors are likely to be activated by glycine injection into DPAG.

Hepatic capillariasis in rats: a new model for testing antifibrotic drugs.

Rats infected with the helminth Capillaria hepatica regularly develop septal hepatic fibrosis that may progress to cirrhosis in a relatively short time. Because of such characteristics, this experimental model was selected for testing drugs exhibiting antifibrosis potential, such as pentoxifylline, gadolinium chloride and vitamin A. Hepatic fibrosis was qualitatively and quantitatively evaluated in liver samples obtained by partial hepatectomy and at autopsy. The material was submitted to histological, biochemical and morphometric methods. A statistically significant reduction of fibrosis was obtained with pentoxifylline when administered intraperitoneally rather than intravenously. Gadolinium chloride showed moderate activity when administered prophylactically (before fibrosis had started), but showed a poor effect when fibrosis was well advanced. No modification of fibrosis was seen after vitamin A administration. Hydroxyproline content was correlated with morphometric measurements. The model appears to be adequate, since few animals die of the infection, fibrosis develops regularly in all animals, and the effects of different antifibrotic drugs and administration protocols can be easily detected.

Pharmacological differences between memory consolidation of habituation to an open field and inhibitory avoidance learning.

Rats implanted bilaterally with cannulae in the CA1 region of the dorsal hippocampus or the entorhinal cortex were submitted to either a one-trial inhibitory avoidance task, or to 5 min of habituation to an open field. Immediately after training, they received intrahippocampal or intraentorhinal 0.5-microl infusions of saline, of a vehicle (2% dimethylsulfoxide in saline), of the glutamatergic N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphono pentanoic acid (AP5), of the protein kinase A inhibitor Rp-cAMPs (0.5 microg/side), of the calcium-calmodulin protein kinase II inhibitor KN-62, of the dopaminergic D1 antagonist SCH23390, or of the mitogen-activated protein kinase kinase inhibitor PD098059. Animals were tested in each task 24 h after training. Intrahippocampal KN-62 was amnestic for habituation; none of the other treatments had any effect on the retention of this task. In contrast, all of them strongly affected memory of the avoidance task. Intrahippocampal Rp-cAMPs, KN-62 and AP5, and intraentorhinal Rp-cAMPs, KN-62, PD098059 and SCH23390 caused retrograde amnesia. In view of the known actions of the treatments used, the present findings point to important biochemical differences in memory consolidation processes of the two tasks.

Antinociceptive properties of the methanolic extract obtained from Ipomoea pes-caprae (L.) R. Br.

Ipomoea pes-caprae is a medicinal plant used in many countries for the treatment of several ailments, including inflammatory and algesic processes. The present study describes the antinociceptive effects of the methanolic extract and two fractions obtained from aerial parts of this plant. The results indicated that both methanolic extract and two fractions (ethyl acetate and aqueous) exhibited considerable antinociceptive activity against two classical models of pain in mice. Methanolic extract presented a calculated ID50 value of 33.8 mg/kg, i.p. against writhing test and also inhibited both phases of pain (neurogenic and inflammatory) of the formalin test with ID50 of 37.7 and 12.5 mg/kg, i.p. for the first and second phase, respectively. Preliminary phytochemical analysis suggested the presence of steroids, terpenoids, alkaloids and flavonoids. These findings support, at least in part, the popular use of I. pes-caprae to treat dolorous processes.