Month: August 2020

Indicine breeds, such as Nellore cattle, form the majority of the beef herds in tropical and subtropical countries. Zebus generally take longer to reach puberty than taurines, making the improvement of reproductive performance an impending challenge in the production systems of these regions of the world. Scrotal circumference evaluated at yearling is the most recorded reproductive trait in breeding programs for beef cattle, as the trait is inexpensive and easy to measure, is highly heritable, and is associated with testis development, quantitative and qualitative semen parameters, age at puberty in bulls and related heifers, heifer pregnancy, and body weight. Consequently, SC is used in these programs as a major indicator of precocity and fertility. Characterizing genomic regions that explain differences in SC in B. indicus can contribute to the identification of reproductive performance informative molecular markers to assist breeding, as well as to the mapping of loci implicated in reproductive biology. In this paper, we analyzed data of estimated breeding values from 861 Nellore bulls genotyped for over 777,000 single nucleotide polymorphism markers. We aimed at identifying putative genomic regions explaining differences in SC in B. indicus cattle via genome-wide mapping. The genome-wide mapping analysis detected positional candidate loci explaining approximately 4% of the dEBVs for SC. Although this represents only a fraction of the trait variance, this percentage is substantial considering that 180 loci associated with human adult height, a highly heritable and classic polygenic trait, explain only 10% of the phenotypic variance together. This is evidence that multiple loci across the genome are involved in the LEE011 abmole complex inheritance of SC, and the functional candidate genes filtered here may only scratch the surface of the molecular mechanisms underlying the trait. The dissection of the pathways regulating precocity in B. indicus cattle will require multiple studies across breeds and trait models, with intensive multidisciplinary reasoning. Nevertheless, the loci reported here excel from the genome background, and represent important data in the context of bovine reproductive biology. The region explaining the largest proportion of SC variance in the present study mapped to the beginning of chromosome 21, peaking around 1.5 Mb. The closest gene found in this region was MAGEL2. The orthologous human and murine genes regulate normal circadian output, and are highly expressed in the suprachiasmatic nucleus of the hypothalamus. The human MAGEL2 has been implicated in Prader-Willi Syndrome, a genetic disorder characterized by short stature, low muscle tone, cognitive disabilities, increased food intake, obesity, low levels of insulin and insulin-like growth factor 1, incomplete sexual development, hypogonadism, and male infertility. The disorder manifests when a segment on human chromosome 15, which encompasses seven maternally imprinted genes including MAGEL2, presents a deletion or loss of expression of the paternal alleles.

BLI made it possible to visualise disease progression including metastatic dissemination and development of distal metastasis in liver and lungs, and the intensity of the Temozolomide bioluminescence signal correlated with the tumour load. We subsequently developed a surgical procedure permitting maximum cytoreduction, confirmed by BLI. In order to standardise the methodology and surgical procedure used, at the time of surgery all mice were operated at a lower stage of disease than most human patients at their time of clinical presentation. Analogous to what has been observed clinically, surgery was the treatment modality with the greatest impact on the outcome variables when compared to chemotherapy. The group of mice treated with debulking surgery followed by adjuvant chemotherapy was, similar to observations in human studies, found to have the longest mean survival time. After maximal cytoreductive surgery, where the primary tumor is removed, there is no evidence of either local or distant metastases in patients. This was illustrated also in our model, both by the macroscopic findings after surgery and by the BLI-analysis performed after the procedure. As all mice were macroscopic tumor free after the cytoreductive surgery it was interesting to note the rather large discrepancies in disease development in the surgical cohorts with variation of 62.7 weeks. Although we cannot rule out that sufficient cytoreduction was not achieved and also the presence of occult metastases not visible to the surgical team or BLI undetectable, micro-metastasis were the most likely cause of the early relapses. To circumvent this problem clinically, imaging has become an important facet of presurgical planning and postoperative followup. More recently, the emergence of fluorescence-based imageguided surgery incorporating a fluorescently labeled biomarker of an overexpressed membrane-bound protein or receptor has been successfully translated to clinical surgery of ovarian cancer. Preclinical development of image-guided surgery has also been performed with human cell lines inoculated subcutaneously and within the peritoneal cavity. While these models are not an accurate paradigm of human ovarian cancer, the application of our surgical model will now permit the realistic evaluation of image-guided surgery techniques combined with targeted drug therapy/chemotherapeutics prior to clinical translation. Orthotopic nude mouse models have been developed for ovary carcinoma with surgical implantation of tissue but with less infiltrative and invasive growth compared to what is seen in our model. Although inclusion of primary patient cells isolated from ascites is a natural step in the evolution of our orthotopic model, a recent study demonstrates the complications of phenotypic heterogeneity and instability of human ovarian tumour cells.

It is of interest to examine whether silencing Cad11 in Bo-786-O cells can decrease RCC bone metastasis. Our attempts to address this question were inconclusive as a majority of animals injected with Bo-786-O cells with or without knockdown of Cad11 did not survive long enough for further analysis of tumors in bone. We have performed x-ray, microCT, and histology on mice injected with 786-O cells in order to determine whether an osteolytic or osteoblastic reaction occurs, and did not detect obvious osteolytic lesions due to insufficient tumor growth in bone. This problem may be unique to 786-O cells, as we did not encounter such a problem when injecting mice with PC3-mm2 prostate cancer cells. Thus, whether an increase in Cad11 expression alone is sufficient to increase RCC bone RG7204 Raf inhibitor metastasis requires further study. CXCR4 is another adhesion molecule that has been implicated in the acquisition of invasive and metastatic phenotypes in several cancer types, such as breast cancer, melanoma, prostate cancer and renal cancer. Studies have shown that higher CXCR4 expression is strongly associated with advanced RCC and in RCC metastasis. Our observations that CXCR4 expression was elevated in metastatic cell lines from bone and other organs, suggesting that CXCR4 may play a role in 786-O cells metastasis, but not specifically to the bone. The hypervascularity of RCC is attributed to the mutation of the VHL tumor suppressor gene. Indeed, 786-O harbors an inactivating mutation in one VHL allele, while the second allele is deleted. Our study revealed that the gene expression levels of angiogenic molecules such as HIF-1a and VEGF in 786-O cell lines were relatively high. However, we did not detect significant differences in the gene expression among metastatic cell lines derived from organs. These results indicate that although angiogenesis plays an important role in the development and metastasis of RCC due to the loss of VHL function, it is not specific to bone metastasis. The angiopoietinTie-2 signaling axis is an alternative pathway to promote angiogenesis. However, the role of Ang-1 in tumor angiogenesis remains controversial. Some studies suggested that Ang-1 is angiogenic, whereas, others indicated that it inhibits angiogenesis, tumor growth and vascular permeability. We found that Ang1 message is decreased in organ-derived 786-O RCC cells. However, whether this leads to a decrease in protein expression was not examined. The significance of Ang1 in 786-O bone metastasis is not clear and therefore requires further study. Bone lesions in patients with RCC are exclusively osteolytic. In many cancers, like breast and prostate cancers, tumorproduced growth factors or cytokines like PTHrP, RANKL, and IL-6 play important roles in bone osteolysis. Contrasting evidence has been found.

Whereas, in the study of Strube et al., PTHrP was highly expressed in metastatic cell lines suggesting that PTHrP might play a role in tumor-induced osteolysis similar to breast cancer bone metastasis. Additionally, it has also shown that RANKL did not substantially contribute to RANK-induced bone resorption. In the current study, we found that gene expression of PTHrP and IL-6 was significantly lower in bone-derived RCC 786-O cells than that in parental 786-O cells, and that RANKL gene expression in the 786-O RCC cells was too low to be detected. Our results agree with previous Erlotinib reports indicating that no RANKL mRNA expression was detected in human clear cell RCC cell lines, such as ACHN and Caki-1 cells. From these observations, we conclude that these tumor-produced factors may not play a critical role in affecting the metastasis of 786-O cells to bone. However, the possibility that these factors may be secreted as a result of interactions between 786-O RCC cells and bone marrow mesenchymal cells, and therefore may play a role in supporting the growth of RCC 786-O cells in bone, cannot be excluded. Strube et al. has also reported the selection of bone-derived metastatic 786-O cell lines through multiple cycles of in vivo selection. The highly selected cells showed strong osteolytic property with high levels of PTHrP. As tumor cells are heterogeneous with ability to metastasize to various organ sites, we chose to use first generation of metastatic tumor 786-O RCC cell lines to determine the very initial factors that may involve in homing, retention and proliferation at bone site. Whether repeated in vivo selection enriched for the cells that express high levels of PTHrP is not clear. In conclusion, among the several candidate factors examined, including angiogenic and osteolytic factors, we found that only one membrane protein, Cad11, was involved in organ-specific metastasis in bone using the 786-O cell line. Additional membrane proteins that are important for organ-specific targeting of metastatic RCC cells may be identified by using other RCC cell lines, and by other methods such as proteomics. We recently report that living in an enriched housing environment that provides physical, social, and cognitive stimuli reduces tumor growth and increases remission in mouse models of melanoma and colon cancer. Our mechanistic studies have elucidated one key mechanism underlying the anti-cancer effect of environmental enrichment : the activation of a previously poorly understood neuroendocrine hypothalamic-sympathoneural-adipocyte axis. The complex environmental stimuli induce the expression of brain-derived neurotrophic factor in the hypothalamus and the ensuing increase in sympathetic tone to white adipose tissue. The preferential sympathetic activation of white adipose tissue suppresses leptin expression and release via action on b-adrenergic receptors leading to a robust drop of leptin level in circulation.

In addition, although glucose metabolism appears to be disrupted, excess glucose accumulation as a result of reduced glycolysis leads to the production of sorbitol, and, consequently, the potential formation of glycation products that can generate free radicals and trigger tissue damage. Lactate levels did not significantly change, suggesting that excess glucose is used instead by the sorbitol pathway or pentose phosphate pathway. Based on our metabolomics and microarray data, we tentatively suggest that the human lung with advanced PAH does not produce high levels of lactate that are typically a signature trait of the Warburg effect in the earlier developing stages of PAH. Further experimentation based on the radioactive targeted approach on the human PAH lung will clarify this issue. Our study suggests that the process of vascular remodeling in PAH involves alterations in glycolysis in multiple cells, limited not only to SMCs but also includes endothelial cells and other tissues such as collagen fibers around the peri-vascular tissue. Lung samples from PAH patients exhibited higher levels of glucose, sorbitol, and fructose. By gene array and immunostaining, we showed that genes in vascular smooth muscle cells encoding the key enzymes for glycolysis, such as LDH-B, were significantly increased, whereas genetic expression of other key enzymes in the glycolytic pathway, specifically glucose-6-phosphatase subunit C3 was significantly downregulated. Glucose-6-phosphate, a key rate-limiting metabolite in normal glycolysis and a substrate for G6PC3, can enter many pathways, including gluconeogenesis to produce glucose, glycogenesis for storing glucose, anaerobic glycolysis to convert to pyruvate, or entrance to the pentose phosphate pathway for generating ribose5-phsophate for the synthesis of nucleotides and erythrose-4- phosphate for the biosynthesis of aromatic amino acids. In particular, the enzyme glucose-6-phosphatase plays a major role in the gluconeogenesis process of dephosphorylating glucose-6- phsophate to generate glucose. Our studies showed that G6PC3 was down-regulated in PAH at both the transcriptional and translational level, suggesting that decreased expression of G6PC3 may be due to a decrease of G6P as a result of glucose being shuttled towards the sorbitol fructose pathway. Despite a decrease in glycolytic key intermediates and enzymes, PFKFB2, an enzyme responsible for irreversibly converting fructose-6-phosphate to fructose-1,6-bisphosphate in the committed step of glycolysis was increased, perhaps in response to Tubacin increased F6P levels, yet there was a decrease in the product fructose 1,6-bisphosphate in PAH lungs. An increase in PFKFB2 may be a feedback mechanism of decreased fructose 1,6- bisphosphate in an attempt to restore normal glycolysis, although protein levels of PFKB2 did not display significant changes.