Month: August 2020

The blood vessel system is essential for supplying cells with nutrients and oxygen, in addition to the removal of waste products. All cells in an organ are located in close proximity to this supportive system. To influence vessel growth and ensure their own sustenance, tumour cells release growth factors such as vascular endothelial growth factor, leading to tumour-directed vessel development. This process is called pathological angiogenesis, a development of new vessels from pre-existing vessels. Different angiostatic drugs can be applied to disrupt vessel growth and therefore limit tumour nutrition. Different in vitro and in vivo assays have been developed to investigate the effect of angiostatic drugs. A commonly used and well-known assay is the Regorafenib Matrigel assay, which is rather poorly characterized. Capillary-like structures with lumen have been described using this assay, although there is significant debate as to whether these structures actually contain patent lumina or not. Furthermore, non-endothelial cells such as fibroblasts and other cell types also form tubules on Matrigel. For this reason, the results need to be interpreted with caution and more than one assay should be taken into consideration. A further assay used to evaluate the efficacy of angiostatic drugs is the endothelial cell co-culture assay. This assay is based on a supportive mural cell layer, on which endothelial cells have the ability to form capillary-like structures after 7–14 days. Although this assay takes longer than the Matrigel assay, it provides a more physiological environment, with tubules growing on the supportive mural cell layer matrix. For the endothelial cell co-culture assay, a variety of different cell types have been employed as a supportive cell layer, including pulmonary artery smooth muscle cells, primary human mammary fibroblasts and human dermal fibroblasts. However, these sources of human tissue-derived cell are limited, and more accessible human or animal tissue-derived cell sources would be an advantage for endothelial cell co-culture assays. In the present approach, HUASMCs and ovine carotid-artery derived cells were investigated as accessible, supportive cell layers for endothelial cell co-culture assays. We evaluated the influence of cell numbers within the supportive cell layer, in addition to that of pro-angiogenic factors and anti-angiogenic factors, on vessel development. The presence of VEGF receptor-2 on the employed HUVEC cell lines as the tubule-forming units was also evaluated to determine any correlation with the amount of capillary-like structures formed in vitro. Blood vessels function to supply all cells of an organism with oxygen and nutrition. A tumour located in close proximity to this system and not exceeding a certain size is sufficiently supplied by diffusion. A larger tumour needs to recruit additional blood vessels by angiogenesis. The tumour itself can initiate these processes by releasing growth factors such as VEGF, and by consequently changing the balance between pro- and antiangiogenic molecules. This results in pathological angiogenesis in the direction of the tumour.

Deleterious microbial activity is commonly controlled with biocides at significant cost to the driller. However, despite biocide use, microbial activity is prevalent in produced water. Previous studies have shown that biocide effectiveness may be limited by high salt concentrations, organic compounds, and long residence times in the subsurface. Other studies have shown that microbial communities in produced water are distinct from those in the injected fracturing fluid, and correlate well with changes in geochemical and environmental conditions. This implies that the common practice of recycling produced water for subsequent hydraulic fracturing may introduce adapted populations into the formation. Over the past decade molecular ecology surveys based on the 16S rRNA gene have increased our knowledge about the taxonomic Silmitasertib citations composition of microbial communities in reservoir environments,,,. However, these studies offer limited insights on the metabolic capabilities of the microbial community, as they rely on taxonomic inference based on 16S rRNA gene similarity to previously isolated microorganisms. As an example of the limitations of using previously isolated microorganisms to infer metabolic capability, the ‘core genome’ of the well-studied Escherichia coli is typically less than 50% of the genes in the genome, and,30% of the E. coli pan-genome. On the other hand, shotgun metagenomic surveys enable access to complete genetic information within microbial genomes from uncultured, mixed consortia. These surveys have provided significant insights on the functional potential of microorganisms in diverse environments such as marine samples, corals, activated sludge, permafrost, hydrocarbon and sandstone reservoirs, and swine gut. Despite the importance of microbial activity in produced water brines from hydraulic fracturing operations, the functional potential of associated microbial communities has not yet been studied. In this study, the metagenome of fracturing source water and produced water at two different time points from a Marcellus Shale natural gas well in Westmoreland County, PA was generated using Illumina MiSeq technology. The microbial ecology from 16S rRNA surveys and chemical composition of these samples has been described in a previous publication. Sequences from each sample were assembled into contiguous sequences and analyzed for taxonomic affiliations and functional potential of the microbial communities. These functional categories were similarly identified as dominant in previous studies of soil, marine samples, activated sludge, freshwater and hypersaline environments. Normalization of gene abundance data shows a relative increase in each of the above functional categories in the produced water samples as compared to the fracturing source water implying that core systems necessary for survival are enriched in the produced water community.

The annelid Nereis virens had detectable aromatase activity, likely occurring in the gut epithelium. Despite having detectable aromatase activity, the SCH772984 942183-80-4 protein responsible for this function remains unknown. CYPome studies in annelid species may provide clues to the evolution of the steroidogenesis pathway in metazoa and whether annelid invertebrates utilize the same enzymes for de novo sex steroid production. All of the C. telata clan 2 CYPs were located in novel CYP families; indeed the C. telata clan 2 sequences had 14 novel CYP families made up from 20 subfamilies. It has been postulated that a large number of CYPs related to families involved in exogenous metabolism may suggest evolutionary pressure towards diverse function. The largest family was CYP3052 with 24 sequences; these sequences made up the majority of the large standalone cluster of 33 C. teleta CYPs on the phylogenetic tree. If this family of C. teleta CYPs follows the trend of other large CYP families, namely families CYP1–4, then these proteins may be involved in xenobiotic metabolism. There were five novel families with a single sequence each that were CYP1-like. There were fewer CYP1-like genes in C. telata than were found in S. purpuratus but similar to what is typical in vertebrates. Of the two families that grouped with CYP2s, CYP3058 clustered more closely with C. elegans sequences than vertebrate CYP2 sequences in the clan 2 phylogeny. The other family, CYP3059, clustered with vertebrate CYP2R, the placement of this family is uncertain with respect to the vertebrate CYP2 families. The function of the C. elegans CYPs are unknown but vertebrate CYP2s are well known for their role in xenobiotic metabolism. CYP3057A1 and CYP3064A1 were basal in this clan and had high divergence from the remaining sequences. The clan 3 phylogeny had sequences from across all metazoan phyla. Clan 3 contains families CYP3 and CYP5 in vertebrates, but is represented by different families in invertebrates such as families CYP6 and CYP9. Mammalian CYP3s are known to have very flexible active sites that can accommodate structurally diverse substrates. CYP3A4 is the most important enzyme involved in drug metabolism in humans but other CYP3s are also important in metabolism of endogenous and exogenous compounds. Clan 3 CYPs are involved in both endogenous and exogenous metabolism in arthropods. C. teleta had two clan 3 families with a total of nine CYPs; both families were novel. N. vectensis had 20 clan 3 CYPs, S. purpuratus had 10, and D. melanogaster has an expanded clan 3 with 36 CYPs. Mammals appear to have a much smaller number of clan 3 genes than many invertebrate species; humans have just five clan 3 sequences from a single subfamily. The C. teleta clan 3 sequences included CYP331A1, which had been previously described. CYP331A1 had increased expression from exposure to benzopyrene and fluoranthene, two PAHs. The CYP331 family has been expanded in this annotation with two more CYP331A genes and the CYP331B1 gene.

The hypothalamic-pituitary-gonadal axis is fundamental to the control of reproductive functions, whose main actor is the hypothalamic gonadotropin releasing hormone. Released into the hypophyseal portal vasculature from axon terminals at the median eminence. Intermittent GnRH secretion from the hypothalamus acts upon the GnRH receptor in the anterior pituitary to regulate the production and secretion of gonadotropins EX 527 HDAC inhibitor including LH and FSH that in turn regulate development and activity of the testes. The pituitary requires pulsatile stimulation by GnRH to synthesize and release the gonadotropins LH and FSH, in turn, the feedback from gonadotropins finely modulate the GnRH release. Moreover, the GnRH secretion depends on the activation of the GPR54, located on the surface of the GnRH neurons and stimulated by the peptide kisspeptin which is a product of the Kiss-1 gene. In the present study, alterations in the GnRH1 gene expression were observed after i.p. injection of MC-LR, suggesting that MC-LR affected the reproductive system by means of actions at the level of the hypothalamus through modulation of GnRH synthesis and / or release. A lower GnRH secretion to the hypophyseal portal blood would result in the reduction of LHb synthesis, which caused the decreasing testosterone production in the testis. GnRH could affect the GnRHR gene expression directly at the pituitary level. However, in the present study, the GnRHr mRNA levels kept unchanged. The mechanism awaits to be elucidated in our future study. In addition, no variations of GPR54 and Kiss-1 mRNA expression were observed in the study, indicating that MR-LR may not affect the GPR54/Kiss-1 regulation system directly. The gonadotrope cell of the anterior pituitary plays a particularly critical role within the HPG axis system as the intermediary between the hypothalamic GnRH signal and the steroid hormone productivity of the gonads. FSH and LH stimulate sex steroid production and secretion in the gonads. These molecules then feed back to the brain and pituitary to regulate the gonadotropins. In general, androgens have negative feedback effects primarily on the release of GnRH from the hypothalamus, which then suppresses both FSH and LH secretion. When serum testosterone level is reduced, the GnRH synthesis and secretion increases followed by a subsequent rise in the synthesis and secretion of LH from the pituitary gland. In the present study, MC-LR administration to mice resulted in a reduction in the serum testosterone concentration in a dose- and time- dependent manner. Intriguingly, the serum LH synthesis and secretion were reduced as well. Such phenomenon could be explained by the assumption that the possible increase in GnRH expression stimulated by the decreased testosterone level had been offset by the inhibited GnRH synthesis caused by exposure to MC-LR, which further demonstrate that the primary target of MC-LR is GnRH and MC-LR would disrupt the GnRH expression directly or indirectly.

The latter confers biologic specificity to the two ligands and determines the levels of functional FSH and LH. LH stimulates testosterone production by Leydig cells whereas FSH stimulates the Sertoli cells to regulate spermatogenesis by secreting various factors that will affect Leydig cell function. The exposure to many environmental toxicants leads directly to a remarkable decline in spermatogenesis function and fertility of animals and humans. Several recent studies have demonstrated the reproductive toxicity of MCs to mammals. It has been reported that exposure to MC-LR affects male reproductive organs and reduction of sperm number in mice. In the present study, our results indicated that spermatogenesis was significantly inhibited in mice treated with MC-LR compared with the vehicle-treated mice, and MC-LR exposure significantly decreased serum testosterone and LH level while FSH concentrations kept unchanged. As the secretion of testosterone is regulated by LH, the decreased serum testosterone level was as a result of low LH levels in mice. Although intratesticular testosterone levels were shown to be significantly higher than serum testosterone levels, the intratesticular testosterone highly correlated with serum testosterone. Thus, the decreased serum testosterone level indicated the inhibited testosterone production in the testis which lead to impaired spermatogenesis. Recent studies SCH772984 showed that MCs accumulated in testis, and exerted toxic effects on reproductive system. However, the in vitro experiments in the present study demonstrated that exposure to MC-LR had no effect on the viabilities and testosterone levels of Leydig cells which locates in the testicular interstitium and are the primary cells that synthesize and secrete testosterone in adult male animals. Thus, our data suggested that MC-LR did not affect testosterone synthesis by directly damaging Leydig cells. The biological action of LH and FSH in gonadal tissue is mediated via membrane receptors for LH and FSH, respectively. In the male animals, LHR is maily expressed in testicular. Leydig cells and on ligand binding, it stimulates androgen production. FSH binds to receptors FSHR on the surface of Sertoli cells and functions in concert with testosterone to promote the spermatogenesis function. In the present study, there was no effect of MC-LR i.p. injection on the plasma level of FSH in mice, which was later confirmed by the RT-PCR results which showed MC-LR did not affect the mRNA expression of FSHb in the pituitary and FSHR in the testis, indicating that there was a normal interaction between FSHR and FSH in the testis of MC-LR-treated mouse, and MC-LR was not able to inhibit the FSH synthesis and secretion. However, the levels of LHb transcripts in MC-LR-treated mice were suppressed with decreasing the LH synthesis in the pituitary, whereas the LHR expression was not affected, suggesting that Leydig cells in testis received the impaired LH pulse via LHR, which lead to inhibited.