Month: January 2020

Technology affords scientists unparalleled opportunities to explore the transcriptome of practically any species in multiple ways such as comparative genomics, development of genotyping markers, and digital gene expression. In this study, we constructed and analyzed the first de novo transcriptome for female Culicoides sonorensis during non-feeding, blood feeding, and sucrose feeding using the Illumina HiSeq2000 platform. The objectives of this study were to perform a comprehensive comparison of digital gene expression profiles during these different feeding conditions, and to identify transcripts that may be relevant to key biological processes such as digestion, growth, and reproduction. The results of this study may be useful to further elucidating how midges function on a cellular and molecular level from a whole transcriptome perspective, and provide a rich data set to augment the ongoing genome-sequencing project. Comparative analysis of differential transcriptome responses between non-feeding, early blood meal, late blood meal, early sucrose meal, and late sucrose meal to the Culicoides unigene was performed using the Tuxedo software package, where reads were mapped to the unigene assembly with the Bowtie2 software. Cufflinks was used to generate a transcriptome assembly for each condition and replicate, and Cuffmerge was used to merge transcriptome assemblies into one file for statistical analysis by the Cuffdiff software to identify genes whose expression profiles were statistically increased or decreased in abundance across the various feeding conditions over time. Differentially expressed genes were categorized into functional groups with the Agbase functional classification tool to observe trends in molecular response to the different feeding conditions. Volcano and heat map plots were prepared using the R libraries, CummeRbund, and ggplot2, respectively. Comparisons of the early and late blood transcriptomes with the teneral conditions revealed a measurable genetic landscape with most of the same genes captured in each of the conditions, but the expression profiles differed noticeably between these conditions. For example, the most significant genetic response occurred during the 12 h interval after the blood meal, where we observed 8,414 genes with differential expression profiles between the two transcriptomes, nearly half of the genes shared between these two conditions. Similarly, 36 h after the course of the blood meal, the transcriptome still was responding significantly with 5,143 genes differentially expressed compared to teneral female midges. Comparisons between the early blood, late blood, teneral comparison had 3,476 overlapping genes with differential expression profiles suggesting the physiological response to blood feeding was drastically different than the response to sucrose feeding, where the comparisons revealed only 67 differentially-expressed genes.

Pursuing the idea that additional TF could be co-opted for coordinating gene expression that would contribute not only to epithelial-cell death but also to an adaptation of the organ in general and the epithelium in particular to varying hormonal conditions, notably complete androgen deprivation, and that these changes increase the susceptibility to progression to CRPC, we performed gene expression profiling using DNA microarrays to identify TF associated with the most-regulated genes after androgen deprivation by surgical castration. We included in the analyses a group of rats that received a high dose of 17b-estradiol and a group of rats that were castrated and treated with E2. Inspired by the study of Yeh et al., we attempted to identify regulatory networks among the genes obtained from microarray data, by examining the relatedness between the regulated genes and structural signatures in their promoters. In a first approach, we identified all genes showing differential expression in each experimental group when compared to the controls. The differentially expressed genes were arranged into enrichment terms, and the resulting regulatory gene networks constructed were used for the identification of candidate TF. In a second approach, we examined the 3,000 bp proximal promoter of the ten most differentially expressed genes for the presence of putative transcription-factor binding sites, and determined their relative abundance with respect to the corresponding promoter regions of two internal control genes. The filtered TF were then validated by qRT-PCR and localized in the gland by immunohistochemistry. The expression pattern and tissue location of these TF appear to be important for the fine-tuning of prostate adaptation to the androgen-deprived environment. We found MYBL2 to be concentrated in the epithelial cells and discretely in the cell nucleus. GATA2, on the other hand, was found in both epithelial and stromal cells, with clear nuclear localization in non-castrated animals. The number of cells showing nuclear localization was reduced in animals examined 3 days after castration. EVI1 was found in both epithelium and stroma, and showed nuclear localization in the epithelium after castration. ELK1 showed a restricted stromal localization in morphologically recognized smooth-muscle cells. NFYB was detected in the nucleus of epithelial cells in non-castrated animals. The nuclear localization was partly lost after castration. Stromal cells were also positive for NFYB. NFkB1 was found in both epithelium and stroma in noncastrated and castrated animals. The epithelium also expressed RelA and RelB, which, similarly to NFkB1, were not responsive to castration in terms of epithelial/stromal distribution. In contrast, REL was expressed in the epithelium after castration and was found in the cell nucleus. Since nuclear translocation of this occurs upon activation, this location is a good indication that REL is activated after castration.

We predicted that maternal stress would increase transcription of yolk-processing enzymes and the mitochondrial genes involved in oxidative metabolism. Second, stress influences brain gene expression therefore we predicted that genes associated with neural development might be differentially expressed between embryos of predator-exposed and control mothers. We also predicted that genes involved in eye development might be affected by maternal predator exposure because an earlier study in sticklebacks found that genes associated with eye development were differentially expressed in adults following exposure to predation risk. Third, previous studies have shown that exposing fish embryos to elevated cortisol alters the expression of genes encoding insulinlike growth factor, growth hormone, thyroid hormone receptors, and growth hormone receptors. Since stickleback mothers produce eggs with elevated cortisol after exposure to a predator we predicted that genes that are activated or repressed by cortisol or synthetic glucocorticoids would be differentially expressed between treatments. Three day post-fertilization stickleback embryos do not produce endogenous cortisol. Therefore we did not expect that the key gene products required for HPI axis function, such as corticotropinreleasing hormone, adrenocorticotropic hormone, and proopiomelanocortin to be expressed. Also, since glucocorticoids are known to affect the expression of immunityrelated genes, for example genes of the complement pathway, we predicted an effect of maternal exposure to predation risk on the expression of genes involved in immunity. Fourth, predation pressure is associated with a relatively ‘fast’ life history in many taxa, including sticklebacks therefore we predicted that genes related to accelerated growth and development would be upregulated in embryos as a result of maternal exposure to predation risk. Finally, given the broad epigenetic changes that occur across the genome during tissue differentiation, and previous studies showing that maternal care influences offspring DNA methylation patterns we also predicted that genes involved in epigenetic modifications to the genome, such as the DNA methyltransferases, would be influenced by maternal exposure to predation risk. This unbiased genome-wide expression survey identified some of the molecular mechanisms in embryos that respond to maternal experience. In general, maternal exposure to predation risk had an activating effect on offspring genome-wide expression and affected biological pathways involved in energy homeostasis, proliferation of cells, production of neurites and blood cells, differentiation of sensory neurons, and immunity. Many of these results are consistent with previous findings in sticklebacks and other animals that associated maternal stress with increased offspring growth and metabolism, altered immune function and behavior.

At the moment the most studied are the folding and unfolding processes of the apoform of this protein at different denaturant concentrations, various pH values and temperature. It was shown that the folding of this protein occurs via the formation of an intermediate state of the molten globule type and can be described using a twostage sequential scheme of reaction. It was demonstrated using the stopped-flow and quench-flow techniques that ureainduced apomyoglobin refolding goes via a kinetic intermediate that forms within 6 msec and is structurally similar to the equilibrium molten globule intermediate observed at pH 4.2. Subsequent kinetic studies suggested that this intermediate is onpathway. Quench-flow amide proton exchange combined with mass-spectrometry confirmed that apomyoglobin folds by a single pathway and that the intermediate is obligatory. In general, the formation of the molten globule state of apomyoglobin proceeds very rapidly during the burst-phase within several msec, which is in the range of the dead time of the instruments use. Therefore, in kinetic experiments it is almost impossible to detect the molten globule state of apomyoglobin. However, the rapidly formed molten globule state affects the parameters characteristic of the following stages of protein folding. The approach that allows taking into account the effect of the fast folding stage of protein folding on the rate of the slow folding stage of this protein is theoretically grounded. This approach permits one to obtain population values of the protein molten globule state versus the denaturant concentration, i.e. to study the stability of the molten globule state, and using the kinetic data of chevron plots it is possible to design the energy landscape of apomyoglobin. The effect of substitutions of different amino acid residues on the native state of apomyoglobin is studied quite well. Analogous experiments were performed with other proteins as well. The effects of substitutions of different amino acid residues on the stability and the rate of native state formation in various proteins have been studied systematically. Thus, for some proteins the so-called folding nucleus has been determined with the Q-analysis. The folding nucleus includes amino acid residues and protein parts the substitutions of which affect the folding/unfolding rate of the protein native state. In some cases, such studies involve several dozens of mutant proteins with single substitutions of amino acid residues. This permits revealing the localization of residues that determine the rate of formation and the stability of the protein native state. Nevertheless, there are virtually no investigations employing multiple substitutions of amino acid residues for analyzing intermediate states of proteins, although intermediate states of some proteins are functionally important and in many respects determine the properties of proteins.

Ataxia telangiectasia mutated protein , and ataxia telangiectasia and Rad3 related DNA-dependent protein kinase have also been implicated. Recently, some laboratories have suggested that the presence of serotonin in the serum is one of the key factors involved in the bystander effect, however this finding has been disputed. Most bystander effect studies have been performed using x-rays, gamma rays and alpha particles, however, little has been done concerning the effects of neutron radiation. Such information might be important for risk estimation in response to neutron exposure. No conclusive cytogenetic evidence exists to support or refute the existence of non-targeted effects in cellular responses to neutrons. A bystander effect following neutron exposure has been observed in Chinese hamster ovary cells, but no effect was seen in zebrafish irradiated in vivo. There are no available cytogenetic data concerning the bystander effect in human cells in response to neutrons. Here we used the cytokinesis-block micronucleus assay to address the question whether neutrons induce a bystander effect in normal human lymphoblastoid cell lines. The experiments described here provide no cytogenetic evidence that fast neutrons are capable of inducing a bystander effect through medium-borne factors. The neutron beam used in these experiments was contaminated with photons, necessitating parallel evaluations to determine whether there is a positive or an inhibitory effect of these photons on the results of the neutron experiments. Our observation of an absence of a bystander effect following doses of photons that are associated with exposure to neutrons confirmed that there is a lack of a bystander effect in response to neutrons, regardless of the presence of photons. Wang et al. have suggested that neutrons might suppress gamma ray-induced bystander signaling. They measured apoptosis and cell survival in zebrafish that received bystander signals from fish that were directly irradiated with neutrons. Since the doses of photons that contaminated these neutron exposures exceeded the minimum threshold for inducing a bystander effect, Wang et al. suggested that the gamma ray-induced bystander effect might have been suppressed by the neutron exposures. With our data it is difficult to determine whether neutrons have the ability to suppress any bystander effect produced by photons because a source of uncontaminated neutrons is not available. The results shown here suggest that contaminating photons are not a likely confounding factor that interfered with the ability to detect a neutron-induced bystander effect. Different responses to neutrons were observed in the two cell lines we used. GM15510 cells cultured in ICCM from neutron irradiated cells showed substantial variation in micronuclei frequencies but no consistent dose-related response. In contrast, GM15036 cells had micronuclei frequencies lower than the corresponding dose control value.