Month: September 2020

Cre recombination could result in excision, inversion or translocation of the floxed genomic GDC-0449 regions depending on the locations and orientations of the loxP elements. By utilization of a specific promoter driving a spatially restricted Cre expression, conditional deletion of a gene or activation of transgene expression can be achieved in specific tissue in mice, thus offering the opportunity to study gene function with spatial control. A further refinement of the Cre/loxP technology is the development of inducible Cre transgene which permits temporal control of gene recombination, allowing investigation of gene functions in a particular developmental stage of the entire life-span of mice. The inducible Cre recombinase is consisted of mutated ligandbinding domain of the mouse estrogen or progesterone receptor and Cre recombinase. The mutated LBD fails to bind to estrogen or progesterone, but retains its ability in binding to synthetic ligands such as tamoxifen, 4-OHT and RU486. When ligand is absent, LBD-Cre is bound by HSP90 and retained in the cytoplasm. Upon ligand binding, LBD-Cre translocates into the nucleus and mediates genomic recombination. Therefore, Cre-mediated recombination is induced by the administration of synthetic ligand, allowing a temporal control of the recombination event. The tamoxifen inducible Cre recombinase protein is composed of Cre recombinase and two tamoxifen-binding domains of mutated mouse estrogen receptor a, one at each end of the Cre recombinase, ensuring efficient binding of MerCreMer to tamoxifen and 4- OHT, but at the same time retaining maximal Cre activity. Neural specific enolase is a glycolytic enzyme enolase abundantly but specifically expressed in terminally differentiated neurons and neuroendocrine cells. Transcript of mouse NSE was detectable from E12 onwards, and its expression was correlated with synaptogenesis. The 1.8 kb rat NSE promoter DNA fragment has been shown to drive expression of target genes in brain neurons of the transgenic mice. A NSE-Cre mouse line has been previously generated which exhibited spatially restricted Cre activity in neurons of the central nervous system. However, such mouse line did not allow temporal control of Cre recombination. In this study, we generated transgenic mouse line that expressed tamoxifen inducible Cre activity in neurons. Nutrient limitation, also known as caloric restriction or dietary restriction, can extend both the replicative and chronological lifespan of eukaryotes as diverse as yeast, nematodes, flies, rodents, and primates. These effects are mediated by the conserved nutrient sensing TOR, AKT/Sch9 and RAS/cAMP pathways, and reduced signaling by these pathways can delay aging even if nutrients are present. Yeast mutants with impaired nutrient sensing, such as tor1, sch9, or ras2 mutants, therefore have an extended lifespan, and rapamycin, an inhibitor of the TOR kinase, can extend the lifespan of mice. In the budding yeast Saccharomyces cerevisiae, the nutrient sensing pathways negatively regulate the activity and nuclear localization of the Rim15 protein kinase. Rim15 in turn is thought to activate the transcription factors Gis1, Msn2 and Msn4, which turn on genes that are needed for long term survival. Accordingly, rim15, gis1, msn2 and msn4 mutations are epistatic over tor1, sch9 and ras2 mutations, and reverse the lifespan-extending phenotypes of the latter.

To overcome the problem with generating antibodies against highly conserved antigens, mice with impaired immune tolerance have been exploited; however, concerns remain on this alternative approach due to the observations of multi-specificity and low-affinity on auto-antibodies developed from autoimmune mice. In order to generate antibodies against highly conserved ephrin-B2, we used phage display of single chain human antibody and screened them against ephrin-B2 expressed in yeast. From our previous work, we found that phage panning against antigens displayed in yeast is highly efficient in rapid enrichment of specific phage clones, obviating the need to produce soluble antigens as well as ensuring native conformation. With newly developed monoclonal antibody, we found that tumors of colon, breast, ovary, and lung upregulated ephrin-B2 compared to respective normal tissues. Antibody staining was also observed in the neovasculature within the tumor, corresponding to new vessel sprouts. Our antibody also exhibited properties such as its ability to cross-react with murine ephrin-B2, to inhibit EphB4 binding, and to be internalized into cells after binding to ephrin-B2. We anticipate that antibodies developed in this study will be useful in probing ephrin-B2 distribution in normal and disease processes, and in antagonizing the interaction between ephrin-B2 and EphB4 for scientific and therapeutic applications. Ephrin-B2 is preferentially expressed in arterial BU 4061T endothelium and smooth muscle cells, as well as neovasculature within the tumor. Expression of ephrin-B2 is modulated by VEGF, smooth muscle cell contact, and stress. Ephrin-B2 has also shown to be upregulated in many cancers, including colon, uterine, ovarian and esophageal cancers. Despite the importance of the role of ephrin-B2 in physiology and disease, up until now monoclonal antibody specific to ephrin-B2 has not been widely available. This may be attributed to the fact that human ephrin-B2 is highly homologous to those of other mammals including rodents, presenting a challenge to isolating high affinity antibody from immunization and hybridoma technique. Limited in vivo alternatives for making antibodies against highly conserved antigens, including using mice with impaired immune response, have been reported, yet the concerns remain on the multi-specificity and low-affinity of autoantibodies. Here we report the isolation of monoclonal antibody against ephrin-B2, which was selected from a phage library of human single chain antibody. Rather than panning phage clones against soluble antigens, we used yeast cells expressing antigens to pull down reactive phage clones, which was found to be highly efficient in rapidly enriching specific phage library. Given the library diversity and an enrichment factor of 102 –103 per each round of our screening strategy, we were able to observe specific phage clones reactive to ephrin-B2 as early as after two rounds of sorting. As antibody selection is based on monomeric interaction between antigen-antibody, after conversion into a dimer by fusion to IgG Fc, the affinity of ephrin-B2 antibody was comparable to those of high affinity monoclonal antibodies produced from hybridoma. Compared to many polyclonal antibodies generated from peptide fragments by immunization, EC8 selected against ephrin-B2 displayed in native conformation on yeast surface.

It was isolated as a dosage suppressor of a snf1 mig1 srb8 triple mutant, but was later found to regulate gene expression after glucose depletion, when yeast cells shift their metabolism from fermentation of glucose to oxidation of ethanol. This transcriptional response is called the diauxic shift, and affects the expression of more than 2,000 genes in yeast. Induction of several genes at the diauxic shift is dependent on Gis1, which acts through a PDS motif, TAGGGAT, that is present in the promoters of these genes. Gis1 is also required for the induction of several mid-late and late genes during sporulation. Budding yeast also has a fourth related C2H2 zinc finger protein, Rph1, whose sequence is 34% similar to that of Gis1. The zinc fingers of Gis1 and Rph1 are almost identical, which suggests that they should bind to similar DNA motifs. Rph1 was cloned as a repressor of the PHR1 gene encoding photoreactivation lyase. Rph1 and Gis1 redundantly repress PHR1, and also the DPP1 gene encoding diacylglycerol pyrophosphate phosphatase. It is not clear what the roles of Gis1 and Rph1 as repressors of PHR1 and DPP1 have in common with each other, or with the role of Gis1 as an activator in the PDS response. However, several PDS motifs are present in the DPP1 promoter, and a STRE motif is found in PHR1, and there is evidence that the two proteins act through these motifs. Repression by Gis1 and Rph1 thus seems to be mediated at least in part by the same motifs as activation by Gis1. Furthermore, several studies have shown that Rph1 is able to bind the STRE motif. Gis1 and Rph1 also contain JmjN and JmjC domains, which were CP-358774 discovered during a study of Gis1. These domains are found in many eukaryotic proteins, and possess histone demethylase activity, with the catalytic site in the JmjC domain. Paradoxically, while Gis1 appears to have unique functions not shared by Rph1, only the latter has been shown to be an active histone demethylase. Rph1 demethylates di- and trimethylated lysine 36 on histone H3, and surprisingly also H3K9, which is not methylated in yeast. As for Gis1, there have been indications that it may demethylate H3K36me2 and H3K36me1, but there is also data suggesting that it is inactive. The Gis1 JmjC domain has a missense mutation in a key residue, which supports the notion that it is inactive. The JmjC domain is not required for transcriptional activation by Gis1. It is not clear which genes among the targets of Gis1, Msn2 and Msn4 that mediate the effect on aging. One proposed mechanism for life span extension is induction of oxidative stress response genes, such as SOD2. It has also been suggested that reduced nutrient signaling promotes cell cycle arrest, which protects the cells against replicative stress. Moreover, Gcn4- mediated depletion of ribosomal proteins has been implicated in life span extension. Recent work has further shown that the metabolism of glycerol and in particular acetate is important for aging. Yeast cells transiently form acetic acid as glucose is depleted, and acetic acid induced mortality has been proposed to be the primary mechanism of chronological aging. Conversely, glycerol protects yeast cells against stress, and the glycerol biosynthetic genes GPD1, GPD2 and RHR2 are upregulated in sch9, tor1 and ras2 mutants, resulting in higher glycerol levels.

Yet, even DNA sequence-based identification of moulds has WY 14643 company several limitations. The DNA extraction yield may be relatively low because mould cells are hard to lyse. PCR amplification may fail due to the presence of PCR inhibitors in mould cultures. Moreover, although it may technically succeed, the molecular identification of moulds would require at least 5 to 7 days in the routine clinical laboratory setting. This delay negatively impacts the patients’ prognosis. Finally, only some clinical laboratories routinely use a molecular approach for microorganism identification. In 2007, only 17% of the US clinical laboratories performed molecular analysis. Therefore the identification of moulds remains problematic and misidentifications likely occur in the routine setting. A novel microorganism identification method has emerged in bacteriology that is based on MALDI-TOF mass spectrum analysis. This MALDI-TOF MS-based identification technique analyzes the protein content from treated or intact cells of microorganisms under the form of a spectrum that is considered as a protein fingerprint specific of a micro-organism. An unknown microorganism is identified by comparing its spectrum with the spectra in the reference library. MALDI-TOF MS-based identification is simple, fast, and accurate and has a high throughput for most bacteria. Numerous bacteriologists found that its identification accuracy outperformed that of conventional methods in the routine clinical laboratory setting. A few preliminary studies aimed to identify moulds using MALDI-TOF MS. However, each used only specific mould genera and culture conditions. Different extraction methods, types of matrix, and instruments were also used. This heterogeneity is particularly detrimental because mass spectra are influenced by culture conditions, extraction procedures, the type of matrix, and the spectrometer used. Our study therefore sought to elaborate a standardized procedure suitable for the MALDI-TOF MS-based identification of clinically relevant moulds in the routine laboratory setting. In the first step, the operating procedures for MALDI-TOF MS-based identification were optimized and validated on a large panel of clinically relevant moulds. In the second step, we evaluated the performances of this MALDI-TOF MS-based approach for the identification of mould clinical isolates prospectively collected from the routine activity of the Marseille teaching hospital laboratory. Moulds were considered regardless of their phylogeny and relation to any specific clinical situation. This is the first demonstration that a standardized MALDITOF procedure is capable to identify a large array of distinct mould species that are routinely isolated in the clinical laboratory setting. In this setting, MALDI-TOF MS-based identification has already revolutionized the identification of bacteria and yeasts. A growing number of clinical laboratories are now equipped with MALDI-TOF MS-based solutions for the MALDI-TOF MSbased identification of micro-organisms. Yet the lack of standardized procedure applicable to the routine identification of moulds isolated in the clinical laboratory routine remained the major gap in commercialized solutions to date.

We have previously reported characteristics of biological effects of RF SAR131675 1433953-83-3 exposure on auditory hair cells. Auditory hair cells could easily be exposed to mobile phone frequency and 1,763 MHz RF exposure, but this exposure did not induce cellular responses, including cell cycle distribution, DNA damage, stress response, or gene expression changes at 20 W/kg specific absorption rate in HEI-OCI auditory hair cells. RF ablation provides a controlled heating modality on microscopic tissue targets based on RF electrical current flow. Furthermore, a variety of models have been proposed to explain the mechanism of wound healing by electrical stimulation. Previous studies using high-voltage pulsed galvanic stimulation showed that the electrical stimulus induced cell migration and wound repair through increased protein and DNA synthesis. Maddin et al. demonstrated that a pulsed electrostatic field had positive biological effects on hair re-growth but the biophysical mechanism was not clear. The dermal papilla is a discrete population of specialized fibroblasts and plays a pivotal role in hair formation, growth, and cell cycling. To explore the effect of RF radiation in human hair follicle cells, we irradiated human dermal papilla cells to code division multiple access-type 1,763 MHz RF radiation and monitored alterations at molecular and cellular levels. In ex-vivo organ culture of hair follicles, we measured hair shaft elongation after RF exposure. Our results suggest that RF exposure could stimulate human hair growth in vitro. Various studies have been conducted to determine the biological effects of RF radiation, but it has not yet been determined if RF radiation poses a potential hazard. The biological effect of RF radiation remains controversial and the results from different studies may vary due to different experimental conditions and model systems. In an effort to find cell type-specific responses to RF radiation in our previous study, we tested the effect of 1,763 MHz RF radiation on Jurkat human T cells and HEI-OC1 mouse auditory hair cells using microarray analysis. However, we could not find significant alteration in gene expression and cell signaling. In this study, we examined the effect of RF exposure on primary cultured human HFs and hDPCs to find the increased hair growth of ex vivo cultured HFs through the expression of growth factors such as IGF-1. Hair growth is observable in the anagen stage before the regression phase and the resting phase of the hair follicle cycle. Growth factors are polypeptides that are involved in the regulation of hair morphogenesis, hair cell proliferation, and hair growth. Some reports have provided evidence that growth factors such as VEGF, IGF-1, and HGF have hair growth stimulatory activity, while TGF-b1 has inhibitory effects on hair growth. It has been reported that IGF-1, VEGF, fibroblast growth factor 5, and fibroblast growth factor 7 induce proliferation of cells in the matrix, dermal papilla, and dermal papillary vascular system and increase the amount of extracellular matrix in dermal papilla. These factors also maintain follicles in the anagen phase, whereas TGF-b1 evokes apoptosis of matrix cells and shifts the follicles from anagen to catagen.