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.

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.

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.

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.

Antinociceptive, anti-inflammatory and wound healing features in animal models treated with a semisolid herbal medicine based on Aleurites moluccana L. Willd. Euforbiaceae standardized leaf extract: semisolid herbal.

ETHNOPHARMACOLOGICAL RELEVANCE: Aleurites moluccana L. (Willd) Euforbiaceae is a native tree of Indonesia and India that has become acclimatized and well-adapted to the South and Southwest of Brazil. It is commonly used in traditional medicine to treat pain, fever, inflammation, asthma, hepatitis, headache, gastric ulcer, cuts, skin sores and other ailments. The oral antinociceptive effects of standardized 70:30 (v/v) ethanol:water spray dried extract of A. moluccana leaf, as well as its flavonoids 2"-O-rhamnosylswertisin (I) and swertisin (II), have previously been reported. AIM: The aim of this study was to develop a stable and effective semisolid herbal medicine for topical use in the treatment of pain, inflammation and wound healing, containing 0.5 and 1.0% of standardized dried extract of A. moluccana. MATERIALS AND METHODS: The chemical markers I and II were assayed by HPLC-UV analysis after extraction by matrix solid dispersion phase (MSDP) followed analytical validation as ICH Guidelines. The semisolid preparations of Hostacerin CG(®) vehicle containing 0.5 and 1.0% of dried extract of A. moluccana were submitted to stability studies (180 day of accelerated and long-term studies). The phytomedicine semisolid was analysed in croton oil-induced ear oedema model in mice, in the healing process, using the excisional wound model in rats, and to prevent mechanical sensitization following plantar incision in rats in the postoperative model of pain. RESULTS: The MSDP method showed average recovery of 101.6 and 105.7% for I and II, respectively, with good precision (RSD<2.0%) and selectivity, without interference of the excipients. The formulations were approved in the stability studies, maintaining conformity after 180 day of accelerated and long-term studies, with variation<10% in the analytical parameters. The phytomedicine reduced the ear oedema in 37.6±5.7% and 64.8±6.2%, for 0.5 and 1.0% of dried extract, respectively. The formulation also accelerated the healing process by up to 50.8±4.1% and 46.0±4.0% at 0.5 and 1.0% of extract, respectively, and both amounts were capable of preventing the development of mechanical sensitization following plantar incision in rats. CONCLUSIONS: The MSDP followed by HPLC-UV analytical method was appropriate for the quality control of the topical phytomedicine based on A. moluccana. The formulation developed at 0.5 and 1.0% of A. moluccana dried extract proved to be effective as an analgesic, anti-inflammatory and wound healing in the pre-clinical studies, which is in agreement with the ethnopharmacological data.

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.

Comparative evaluation of three commercially available complement fixation test antigens for the diagnosis of glanders.

The sensitivity and specificity of three commercially available complement fixation test (CFT) antigens from c.c.pro (c.c.pro), Central Veterinary Institute of Wageningen UR (CIDC) and the United States Department of Agriculture (USDA) were comparatively evaluated by testing 410 sera collected from glanders-endemic and non-endemic areas (200 true-negative randomly collected sera and 210 sera collected from experimentally immunised animals (12 rabbits, 19 horses), clinically positive (135) and culture-positive (44) horses, donkeys and mules). Immunoblotting (IB) was used as the gold standard test. Highest sensitivity was shown for the CIDC antigen (100 per cent) followed by the c.c.pro antigen (99.39 per cent). However, the USDA antigen showed substantially less (p<0.05) sensitivity (62.19 per cent). Highest specificity was found for the USDA antigen (100 per cent) followed by the CIDC (97.5 per cent) and c.c.pro antigen (96.5 per cent). Positive and negative predictive values (assumed glanders prevalence of <0.1 per cent) for each antigen were calculated to be 95.88 and 99.48 (c.c.pro), 97.04 and 100 (CIDC), 100 and 76.33 per cent (USDA), respectively. Almost perfect agreement (0.96) was found between CFT using either c.c.pro or CIDC and IB.

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.

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.

Filicene obtained from Adiantum cuneatum interacts with the cholinergic, dopaminergic, glutamatergic, GABAergic, and tachykinergic systems to exert antinociceptive effect in mice.

In the present study, we describe the antinociceptive effect of filicene, a triterpene isolated from Adiantum cuneatum (Adiantaceae) leaves, in several models of pain in mice. When evaluated against acetic acid-induced abdominal constrictions, filicene (10, 30 and 60 mg/kg, i.p.) produced dose-related inhibition of the number of constrictions, being several times more potent [ID(50)=9.17 (6.27-13.18) mg/kg] than acetaminophen [ID(50)=18.8 (15.7-22.6) mg/kg], diclofenac [ID(50)=12.1(9.40-15.6) mg/kg] and acetylsalicylic acid [ID(50)=24.0(13.1-43.8) mg/kg] in the same doses as those used for the standard drugs. Filicene also produced dose-related inhibition of the pain caused by capsaicin and glutamate, with mean ID(50) values of 11.7 (8.51-16.0) mg/kg and <10 mg/kg, respectively. Its antinociceptive action was significantly reversed by atropine, haloperidol, GABA(A) and GABA(B) antagonists (bicuculline and phaclofen, respectively), but was not affected by L-arginine-nitric oxide, serotonin, adrenergic and the opioid systems. Together, these results indicate that the mechanisms involved in its action are not completely understood, but seem to involve interaction with the cholinergic, dopaminergic, glutamatergic, GABAergic and tachykinergic systems.

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.

Experimental evidence for exciton scaling effects in self-assembled molecular wires.

Resonant Rayleigh scattering from self-assembled one-dimensional molecular J-aggregate wires reveals a distinct dependence of the exciton energy on the width of lateral extension. For the J aggregates used in this study, strong in-line dipole coupling leads to a delocalization of the exciton wave function over several molecular units. Polarization dependent measurements of resonantly scattered light from the wires show that the exciton dipole moment is oriented perpendicular to the long axis. The experimental observations can be described by applying a quantization condition to the center of mass motion of the J-band exciton in the wires.

Phytochemical analysis and analgesic properties of Curcuma zedoaria grown in Brazil.

The present study describes the phytochemical analysis and analgesic activity of Curcuma zedoaria rhizomes grown in Brazil. The results showed that the hydroalcoholic extract, fractions, specially dichloromethane, and a pure compound, denoted as curcumenol (1), exhibited potent and dose-related analgesic activity when evaluated in several models of pain in mice, including writhing, formalin and capsaicin. Compound (1), which seems to be the main active principle from this plant, presented promising analgesic effects, being several times more potent than different reference drugs evaluated in the same experimental models. The calculated ID50 values (micromol/kg, i.p) were 22 and 12 when evaluated in writhing and capsaicin tests, respectively, and 29 micromol/kg in relation to the second phase of the formalin model. The lack of effect in the hot plate test suggests that (1) act by a mechanism which do not involves the participation of the opioid system. The phytochemical analysis indicated that the chemical composition of the plant grown in Brazil is similar to that grown in other countries. The results confirm and justify the popular use of this plant for the treatment of dolorous processes.

Growth of precursors in silicon using pseudopotential calculations.

Based on the results of pseudopotential calculations, the progression of a single interstitial into small compact clusters and ultimately into chain-like defects is examined. For clusters that consist of more than eight interstitials, the capture of bond-centered interstitials reveals a change in the growth mechanism leading to enhanced stability of clusters. The five-interstitial model is proposed to be a plausible candidate for optically active W centers, observed in ion irradiated silicon.