Heidge Fukumasu

Genome-wide association analysis of feed intake and residual feed intake in Nellore cattle

BackgroundFeed intake plays an important economic role in beef cattle, and is related with feed efficiency, weight gain and carcass traits. However, the phenotypes collected for dry matter intake and feed efficiency are scarce when compared with other measures such as weight gain and carcass traits. The use of genomic information can improve the power of inference of studies on these measures, identifying genomic regions that affect these phenotypes. This work performed the genome-wide association study (GWAS) for dry matter intake (DMI) and residual feed intake (RFI) of 720 Nellore cattle (Bos taurus indicus).ResultsIn general, no genomic region extremely associated with both phenotypic traits was observed, as expected for the variables that have their regulation controlled by many genes. Three SNPs surpassed the threshold for the Bonferroni multiple test for DMI and two SNPs for RFI. These markers are located on chromosomes 4, 8, 14 and 21 in regions near genes regulating appetite and ion transport and close to important QTL as previously reported to RFI and DMI, thus corroborating the literature that points these two processes as important in the physiological regulation of intake and feed efficiency.ConclusionsThis study showed the first GWAS of DMI to identify genomic regions associated with feed intake and efficiency in Nellore cattle. Some genes and QTLs previously described for DMI and RFI, in other subspecies (Bos taurus taurus), that influences these phenotypes are confirmed in this study.

Liver transcriptomic networks reveal main biological processes associated with feed efficiency in beef cattle

BackgroundThe selection of beef cattle for feed efficiency (FE) traits is very important not only for productive and economic efficiency but also for reduced environmental impact of livestock. Considering that FE is multifactorial and expensive to measure, the aim of this study was to identify biological functions and regulatory genes associated with this phenotype.ResultsEight genes were differentially expressed between high and low feed efficient animals (HFE and LFE, respectively). Co-expression analyses identified 34 gene modules of which 4 were strongly associated with FE traits. They were mainly enriched for inflammatory response or inflammation-related terms. We also identified 463 differentially co-expressed genes which were functionally enriched for immune response and lipid metabolism. A total of 8 key regulators of gene expression profiles affecting FE were found. The LFE animals had higher feed intake and increased subcutaneous and visceral fat deposition. In addition, LFE animals showed higher levels of serum cholesterol and liver injury biomarker GGT. Histopathology of the liver showed higher percentage of periportal inflammation with mononuclear infiltrate.ConclusionLiver transcriptomic network analysis coupled with other results demonstrated that LFE animals present altered lipid metabolism and increased hepatic periportal lesions associated with an inflammatory response composed mainly by mononuclear cells. We are now focusing to identify the causes of increased liver lesions in LFE animals.

Immunomodulatory effects of Pteridium aquilinum on natural killer cell activity and select aspects of the cellular immune response of mice

Pteridium aquilinum (bracken fern) is one of the most common plants. Epidemiological studies have revealed a higher risk of certain types of cancers (i.e., esophageal, gastric) in people who consume bracken fern directly (as crosiers or rhizomes) or indirectly through the consumption of milk from livestock that fed on the plant. In animals, evidence exists regarding the associations between chronic bracken fern intoxication, papilloma virus infection, and the development of carcinomas. While it is possible that some carcinogens in bracken fern could be responsible for these cancers in both humans and animals, it is equally plausible that the observed increases in cancers could be related to induction of an overall immunosuppression by the plant/its various constituents. Under the latter scenario, normal tumor surveillance responses against nascent (non-bracken-induced) cancers or responses against viral infections (specifically those linked to induction of cancers) might be adversely impacted by continuous dietary exposure to this plant. Therefore, the overall objective of this study was to evaluate the immunomodulatory effects of bracken fern following daily ingestion of its extract by a murine host over a period of 14 (or up to 30) days. In C57BL/6 mice administered (by gavage) the extract, histological analyses revealed a significant reduction in splenic white pulp area. Among a variety of immune response parameters/functions assessed in these hosts and isolated cells, both delayed-type hypersensitivity (DTH) analysis and evaluation of IFNγ production by NK cells during TH1 priming were also reduced. Lastly, the innate response in these hosts—assessed by analysis of NK cell cytotoxic functionality—was also diminished. The results here clearly showed the immunosuppressive effects of P. aquilinum and that many of the functions that were modulated could contribute to the increased risk of cancer formation in exposed hosts.

Paullinia cupana Mart var. sorbilis, guaraná, reduces cell proliferation and increases apoptosis of B16/F10 melanoma lung metastases in mice

We showed that guaraná (Paullinia cupana Mart var. sorbilis) had a chemopreventive effect on mouse hepatocarcinogenesis and reduced diethylnitrosamine-induced DNA damage. In the present experiment, we evaluated the effects of guaraná in an experimental metastasis model. Cultured B16/F10 melanoma cells (5 x 10(5) cells/animal) were injected into the tail vein of mice on the 7th day of guaraná treatment (2.0 mg P. cupana/g body weight, per gavage) and the animals were treated with guaraná daily up to 14 days until euthanasia (total treatment time: 21 days). Lung sections were obtained for morphometric analysis, apoptotic bodies were counted to calculate the apoptotic index and proliferating cell nuclear antigen-positive cells were counted to determine the proliferation index. Guaraná-treated (GUA) animals presented a 68.6% reduction in tumor burden area compared to control (CO) animals which were not treated with guaraná (CO: 0.84 +/- 0.26, N = 6; GUA: 0.27 +/- 0.24, N = 6; P = 0.0043), a 57.9% reduction in tumor proliferation index (CO: 23.75 +/- 20.54, N = 6; GUA: 9.99 +/- 3.93, N = 6; P = 0.026) and a 4.85-fold increase in apoptotic index (CO: 66.95 +/- 22.95, N = 6; GUA: 324.37 +/- 266.74 AB/mm(2), N = 6; P = 0.0152). In this mouse model, guaraná treatment decreased proliferation and increased apoptosis of tumor cells, consequently reducing the tumor burden area. We are currently investigating the molecular pathways of the effects of guaraná in cultured melanoma cells, regarding principally the cell cycle inhibitors and cyclins.

Paullinia cupana Mart. var. sorbilis, guarana, increases survival of Ehrlich ascites carcinoma (EAC) bearing mice by decreasing cyclin‐D1 expression and inducing a G0/G1 cell cycle arrest in EAC cells

The objective of this work is to report the antiproliferative effect of P. cupana treatment in Ehrlich Ascites Carcinoma (EAC)‐bearing animals. Female mice were treated with three doses of powdered P. cupana (100, 1000 and 2000 mg/kg) for 7 days, injected with 105 EAC cells and treated up to day 21. In addition, a survival experiment was carried out with the same protocol. P. cupana decreased the ascites volume (p = 0.0120), cell number (p = 0.0004) and hemorrhage (p = 0.0054). This occurred through a G1‐phase arrest (p < 0.01) induced by a decreased gene expression of Cyclin D1 in EAC cells. Furthermore, P. cupana significantly increased the survival of EAC‐bearing animals (p = 0.0012). In conclusion, the P. cupana growth control effect in this model was correlated with a decreased expression of cyclin D1 and a G1 phase arrest. These results reinforce the cancer therapeutic potential of this Brazilian plant. Copyright

Vascular Endothelial Growth Factor Expression and Microvascular Density in Soft Tissue Sarcomas in Dogs

The aim of the current study was to evaluate the expression of vascular endothelial growth factor (VEGF) and the microvascular density in canine soft-tissue sarcomas. Immunohistochemistry for VEGF expression was performed on 20 canine neoplasms by the streptavidin–biotin–peroxidase method using an anti-VEGF mouse monoclonal antibody (ab-119). The volume fraction of microvessels in the sarcomas was quantified in hematoxylin and eosin–stained tissue sections. At least 10 fields of view (40x magnification) per neoplasm were analyzed by positioning a grid with 100 points and counting the microvessels that fell into the intersection points. This percentage was considered the volume fraction of these microvessels in the tumor section. VEGF expression was detected in 65% of the neoplasms. In 92.3% of the neoplasms, the expression occurred in the peritumor region; in 46.15%, in the intratumor region; and in 38.46%, the expression was present in both regions. The cells responsible for VEGF expression were fibroblasts and macrophages in the peritumor region or in the pseudocapsule and neoplastic cells in the intratumor region. Greater intratumoral VEGF was expressed in hemangiopericytomas (P = 0.04). No difference was present in the volume fraction of tumor microvessels between VEGF-positive and VEGF-negative neoplasms (P = 0.3416) or for the different types of neoplasms (P = 0.5). The results of this study suggest that VEGF participates in the angiogenesis of soft-tissue sarcoma in dogs. Additional research will be necessary to elucidate the contribution of VEGF to the progression of malignancy.

Protective action of indole-3-acetic acid on induced hepatocarcinoma in mice.

In this study, we report the protective effects of IAA on diethylnitrosamine (DEN)‐induced hepatocarcinogenesis. BALB/c mice received daily IAA at 50 (T50), 250 (T250), and 500 (T500) mg Kg−1 per body mass by gavage for 15 days. At day 15, animals were administered DEN and sacrificed 4 h later. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed in sera. In addition, hepatomorphologic alterations, activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR), gene expression of antioxidant enzymes and DNA integrity were evaluated in the liver. IAA administration did not show any alterations in any of the parameters available, except for a reduction of the gene expression for antioxidant enzymes by 55, 56, 27, and 28% for SOD, CAT, GPx, and GR upon T500, respectively compared with the control. Several hepatic alterations were observed by DEN exposure. Moreover, IAA administration at 3 doses was shown to provide a total prevention of the active reduction of CAT and GR induced by DEN exposure compared with the control. IAA at T500 was shown to give partial protection (87, 71, 57, and 90% for respectively SOD, CAT, GPx, and GR) on the down‐regulation of the enzymes induced by DEN and this auxin showed a partial protection (50%) on DEN‐induced DNA fragmentation for both parameters when compared to DEN alone. This work showed IAA hepatocarcinogenesis protection for the first time by means of a DEN‐protective effect on CAT and GR activity, and by affecting antioxidant gene expression and DNA fragmentation. Copyright

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

Abstract.  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.

Higher susceptibility of spontaneous and NNK-induced lung neoplasms in connexin 43 deficient CD1 × AJ F1 mice: Paradoxical expression of connexin 43 during lung carcinogenesis

Connexins (Cxs) are proteins that form the communicating gap junctions, and reportedly have a role in carcinogenesis. Here, we evaluated the importance of Connexin43 (Cx43) in spontaneous and 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK)‐induced lung carcinogenesis. Male wild‐type (Cx43+/+) and hemizygote (Cx43+/−) CD1 × AJ F1 mice were injected with NNK or saline. After 60 weeks mice were euthanized; lung nodules were counted, measured, and fixed in formalin or snap frozen. Immunohistochemistry for Cx43 and Beta‐catenin (β‐catenin) was performed and Cx43 mRNA expression was evaluated by real‐time PCR. Cx43 deletion significantly increased the incidence and number of spontaneous nodules in the CD1 × AJ F1 mice and the number of gross lesions and the aggressiveness of lesions in NNK‐treated mice. Cx43 mRNA increased significantly and was correlated with the aggressiveness of tumors, although lesions from Cx43+/− mice expressed less Cx43 RNAm than their counterparts. Lung parenchyma presented a Cx43 immunostaining pattern with points or plaques between cells. In hyperplasias and adenomas, Cx43 was found in the membrane and in cytoplasm. Malignant lesions presented increased Cx43 in cytoplasm and a few membrane spots of immunostaining. β‐catenin was weakly expressed in lung parenchyma. Though hyperplasias presented some cells with nuclear β‐catenin, NNK‐induced tumors contained a higher number of this staining pattern. Also, no difference in β‐catenin occurred between both genotypes independently of the histological grade. In summary, our results indicate that Cx43 acts as a tumor suppressor gene in early lung tumorigenesis and loses this property in advanced carcinogenesis. Therefore, Cxs are better classified as conditional tumor suppressors.

A genomewide association mapping study using ultrasound-scanned information identifies potential genomic regions and candidate genes affecting carcass traits in Nellore cattle.

The aim of this study was to identify candidate genes and genomic regions associated with ultrasound-derived measurements of the rib-eye area (REA), backfat thickness (BFT) and rumpfat thickness (RFT) in Nellore cattle. Data from 640 Nellore steers and young bulls with genotypes for 290 863 single nucleotide polymorphisms (SNPs) were used for genomewide association mapping. Significant SNP associations were explored to find possible candidate genes related to physiological processes. Several of the significant markers detected were mapped onto functional candidate genes including ARFGAP3, CLSTN2 and DPYD for REA; OSBPL3 and SUDS3 for BFT; and RARRES1 and VEPH1 for RFT. The physiological pathway related to lipid metabolism (CLSTN2, OSBPL3, RARRES1 and VEPH1) was identified. The significant markers within previously reported QTLs reinforce the importance of the genomic regions, and the other loci offer candidate genes that have not been related to carcass traits in previous investigations.