Electronic Bond Structure of Carbon Nitride Thin Film Deposited by HiPIMS and dc Magnetron Plasma

We report the difference in electronic bond structure and surface chemical property of amorphous carbon nitride (a-CNx) film as a function of N/C which has been deposited by high power impulse magnetron sputtering (HiPIMS) and DC magnetron (dc-MS) plasma. The spectroscopic analyses suggest that the presence of oxide layer is the major cause for the surface charging in the core level peak. The core electron peaks (C1s, N1s and O1s) in x-ray photoelectron spectroscopy (XPS) show higher surface charging in HiPIMS compare to the film deposited by dc-MS plasma. The core peaks are moved gradually toward its virgin position as the etching duration is increased from 1 min to 80 mins. The films deposited by HiPIMS retain a higher N/C ratio following ion etching as compared to those deposited by DC magnetron sputtering suggesting denser films with a higher degree of cross-linking

Insulin like growth factor 2 (Igf2) and its receptor gene (Igf2r) showed opposite expression in diploid parthenogenetic embryos in Capra hircus

The present research work was proposed to compare the expression of development related genes (Igf2 and Igf2r) among diploid parthenogenetic (DIP), female sexed in vivo and female sexed in vitro produced embryonic cell colony. The good quality caprine oocytes were matured in presence of cytochalasin B (CCB) and activated by ethanol (7%) for 5 min followed by incubation with 2 mM 6-dimethyl amino purine (DMAP) for 4 h for DIP embryos production and embryo development was recorded. We have explored comparative expression profile of paternally (Igf2r) and maternally (Igf2) imprinted genes among DIP, female sexed IVF and female sexed in vivo derived embryos. Embryonic cell colony were developed from 8–16 and morula stage DIP, IVF and in vivo derived embryos and these colonies were used for studying the paternal and maternal imprinted genes. Overall, we observed higher expression of maternally expressed genes (Igf2r) in DIP compared to in vivo and IVF derived embryonic cell colony. The expression of paternal genes (Igf2) was low in DIP compared to in vivo and IVF derived embryonic cell colony

Necdin and neuronatin genes expression among diploid parthenogenetic, IVF and in vivo derived female sexed embryos during preimplantation development in goat

The present research work was undertaken to compare the expression of necdin (Ndn) and neuronatin (Nnat) genes among diploid parthenogenetic (DIP), female sexed in vivo and female sexed in vitro produced embryonic cell colony. The good quality caprine oocytes were matured in presence of cytochalasin B (CCB) and activated by ethanol (7%) for 5 min followed by incubation with 2mM 6-dimethyl amino purine (DMAP) for 4 hrs for DIP embryos production. Embryonic cell colony were developed from 8–16 and morula stage DIP, IVF and in vivo derived embryos and these colonies were used for studying the Ndn and Nnat gene expression. There was no expression of Ndn gene in both the stages of DIP embryos, while this gene was expressed almost similarly in IVF and in vivo derived 8–16 cell stages but down regulated significantly in morula of IVF compared to in vivo derived embryonic cell colony. The Nnat gene expression was absent in in vivo derived 8–16 cell and morula stage embryos, while it was expressed in both the stages of DIP and IVF embryos. Further, it was observed that the expression of this gene was significantly lower in DIP embryos of both the stages as compared to IVF one. There was no significant difference of this gene expression between 8–16 cell and morula of DIP but morula of IVF showed significantly higher expression than 8–16 cell stage

Long non-coding RNA lncMGC mediates the expression of TGF-β-induced genes in renal cells via nucleosome remodelers

Background: MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) play key roles in diabetic kidney disease (DKD). The miR-379 megacluster of miRNAs and its host transcript lnc-megacluster (lncMGC) are regulated by transforming growth factor-β (TGF-β), increased in the glomeruli of diabetic mice, and promote features of early DKD. However, biochemical functions of lncMGC are unknown. Here, we identified lncMGC-interacting proteins by in vitro-transcribed lncMGC RNA pull down followed by mass spectrometry. We also created lncMGC-knockout (KO) mice by CRISPR-Cas9 editing and used primary mouse mesangial cells (MMCs) from the KO mice to examine the effects of lncMGC on the gene expression related to DKD, changes in promoter histone modifications, and chromatin remodeling.Methods:In vitro-transcribed lncMGC RNA was mixed with lysates from HK2 cells (human kidney cell line). lncMGC-interacting proteins were identified by mass spectrometry. Candidate proteins were confirmed by RNA immunoprecipitation followed by qPCR. Cas9 and guide RNAs were injected into mouse eggs to create lncMGC-KO mice. Wild-type (WT) and lncMGC-KO MMCs were treated with TGF-β, and RNA expression (by RNA-seq and qPCR) and histone modifications (by chromatin immunoprecipitation) and chromatin remodeling/open chromatin (by Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq) were examined.Results: Several nucleosome remodeling factors including SMARCA5 and SMARCC2 were identified as lncMGC-interacting proteins by mass spectrometry, and confirmed by RNA immunoprecipitation–qPCR. MMCs from lncMGC-KO mice showed no basal or TGF-β-induced expression of lncMGC. Enrichment of histone H3K27 acetylation and SMARCA5 at the lncMGC promoter was increased in TGF-β-treated WT MMCs but significantly reduced in lncMGC-KO MMCs. ATAC peaks at the lncMGC promoter region and many other DKD-related loci including Col4a3 and Col4a4 were significantly lower in lncMGC-KO MMCs compared to WT MMCs in the TGF-β-treated condition. Zinc finger (ZF), ARID, and SMAD motifs were enriched in ATAC peaks. ZF and ARID sites were also found in the lncMGC gene.Conclusion: lncMGC RNA interacts with several nucleosome remodeling factors to promote chromatin relaxation and enhance the expression of lncMGC itself and other genes including pro-fibrotic genes. The lncMGC/nucleosome remodeler complex promotes site-specific chromatin accessibility to enhance DKD-related genes in target kidney cells

A Thiazole Coumarin (TC) turn-on fluorescence probe for AT-base pair detection and multipurpose applications in different biological systems

Sequence-specific recognition of DNA by small turn-on fluorescence probes is a promising tool for bioimaging, bioanalytical and biomedical applications. Here, the authors report a novel cell-permeable and red fluorescent hemicyanine-based Thiazole Coumarin (TC) probe for DNA recognition, nuclear staining and cell cycle analysis. TC exhibited strong fluorescence enhancement in the presence of DNA containing AT-base pairs, but did not fluoresce with GC sequences, single-stranded DNA, RNA and proteins. The fluorescence staining of HeLa S3 and HEK 293 cells by TC followed by DNase and RNase digestion studies depicted the selective staining of DNA in the nucleus over the cytoplasmic region. Fluorescence-Activated Cell Sorting (FACS) analysis by flow cytometry demonstrated the potential application of TC in cell cycle analysis in HEK 293 cells. Metaphase chromosome and malaria parasite DNA imaging studies further confirmed the in vivo diagnostic and therapeutic applications of probe TC. Probe TC may find multiple applications in fluorescence spectroscopy, diagnostics, bioimaging and molecular and cell biology

Changes in expression of monocarboxylate transporters, heat shock proteins and meat quality of Large White Yorkshire and Ghungroo pigs during hot summer period

Objective Present study explores the effect of hot summer period on the glycolytic rate of early post-mortem meat quality of Ghungroo and Large White Yorkshire (LWY) pig and comparative adaptability to high temperature between above breeds by shifting the expression of stress related genes like mono-carboxylate transporters (MCTs) and heat shock proteins (HSPs). Methods Healthy pigs of two different breeds, viz., LYW and Ghungroo (20 from each) were maintained during hot summer period (May to June) with a mean temperature of about 38°C. The pigs were slaughtered and meat samples from the longissimus dorsi (LD) muscles were analyzed for pH, glycogen and lactate content and mRNA expression. Following 24 h of chilling, LD muscle was also taken from the carcasses to evaluate protein solubility and different meat quality measurements. Results LWY exhibited significantly (p<0.01) higher plasma cortisol and lactate dehydrogenase concentration than Ghungroo indicating their higher sensitivity to high temperature. LD muscle from LWY pigs revealed lower initial and ultimate pH values and higher drip loss compared to Ghungroo, indicating a faster rate of pH fall. LD muscle of Ghungroo had significantly lower lactate content at 45 min postmortem indicating normal postmortem glycolysis and much slower glycolytic rate at early postmortem. LD muscle of LWY showed rapid postmortem glycolysis, higher drip loss and higher degrees of protein denaturation. Ghungroo exhibited slightly better water holding capacity, lower cooking loss and higher protein solubility. All HSPs (HSP27, HSP70, and HSP90) and MCTs (MCT1, MCT2, and MCT4) in the LD muscle of pigs inclined to increase more in Ghungroo than LWY when exposed to high temperature. Conclusion Effect of high temperature on the variation of HSPs and MCTs may play a crucial role in thermal tolerance and adaptation to different climatic conditions, pH regulation, muscle acidification, drip loss, protein denaturation and also in postmortem meat quality development

S-, P- and D-wave resonances in positronium-sodium and positronium-potassium scattering

Scattering of positronium (Ps) by sodium and potassium atoms has been investigated employing a three-Ps-state coupled-channel model with Ps(1s,2s,2p) states using a time-reversal-symmetric regularized electron-exchange model potential fitted to reproduce accurate theoretical results for PsNa and PsK binding energies. We find a narrow S-wave singlet resonance at 4.58 eV of width 0.002 eV in the Ps-Na system and at 4.77 eV of width 0.003 eV in the Ps-K system. Singlet P-wave resonances in both systems are found at 5.07 eV of width 0.3 eV. Singlet D-wave structures are found at 5.3 eV in both systems. We also report results for elastic and Ps-excitation cross sections for Ps scattering by Na and K.Comment: 9 pages, 5 figures, Accepted in Journal of Physics

Rôle de la nucléoline dans le mécanisme moléculaire de la regulation de la transcription par la polymérase I et caractérisation de son acétylation

Nous montrons dans cette étude que dans les cellules déplétées pour la nucléoline, une plus faible accumulation de pré-ARNr est associée à une augmentation de marques d’hétérochromatine (H3K9me2) et une diminution de marques d’euchromatine (H4K12ac et H3K4me3) sur la chromatine des gènes ribosomiques. Des expériences de ChIP-seq montrent que la nucléoline est enrichie dans la région codante et promotrice de l’ADNr et est préférentiellement associée avec les gènes non méthylés des ARNr. La déplétion de la nucléoline entraîne une accumulation de l’ARN Pol I au début de l’ADNr et une diminution de UBF sur la région codante et promotrice. La nucléoline interfère avec la liaison de TTF-1 sur le promoteur-proximal T0, inhibant ainsi le recrutement de TIP5 du complexe NoRC, et établissant un état d’hétérochromatine répressive. Ces résultats révèlent l’importance de la nucléoline dans le maintien d’un état euchromatinien des ADNr et dans l’élongation de la transcription. Nous montrons aussi dans cette thèse que l’acétylation est une nouvelle modification post-traductionnelle de la nucléoline. Des études d’immunofluorescence utilisant l’anticorps anti nucléoline acétylée montrent que la nucléoline acétylée est exclue des nucléoles. De plus, par ChIP-seq nous n’avons jamais pu détecter d’association significative de la nucléoline acétylée sur la chromatine des ADNr. Aussi, nous n’avons détecté aucune activation de la transcription de Pol II sur des matrices de chromatine avec la nucléoline acétylée. Nous trouvons une distribution de la nucléoline acétylée majoritairement dans le nucléoplasme où elle co-localise parfaitement avec le facteur d’épissage SC35, et partiellement avec les structures marquées avec un anticorps dirigé contre Y12, mais ne co-localise pas avec des structures contenant la coïline, ce qui suggère que cette fraction de la nucléoline pourrait être impliquée dans la synthèse ou le métabolisme des pré-ARNm.Here we have shown that, in nucleolin depleted cells, lower accumulation of pre-rRNA is associated with the increase in heterochromatin marks (H3K9me2) and decrease of the euchromatin histone marks (H4K12Ac and H3K4me3) in rDNA chromatin. ChIP-seq experiments show that nucleolin is enriched in the coding and promoter region of the rDNA and is preferentially associated with the unmethylated rRNA genes. Nucleolin knockdown results in the accumulation of RNAPI at the beginning of the rDNA and a decrease of UBF in the coding and promoter regions. Nucleolin is able to interfere with the binding of TTF-1 on the promoter-proximal terminator T0 thus inhibiting the recruitment of the NoRC subunit TIP5 and HDAC1 and establishing a repressive heterochromatin state. These results reveal the importance of nucleolin in the maintenance of the euchromatin state of rDNA and transcription elongation.In this thesis we have also shown that acetylation is a novel post-translational modification of nucleolin. Immuno-fluorescence studies using anti-acetylated nucleolin antibody illustrated that acetylated nucleolin is excluded from nucleoli and interestingly, neither could we detect any significant binding of ac-nucleolin on rDNA chromatin by doing ChIP-Seq, nor did we detect any activation of Pol II transcription with ac-nucleolin from DNA and chromatin templates. Moreover, we found acetylated nucleolin had a predominant nucleoplasmic distribution where it associates with the splicing factor SC35 and partially with the structures labeled with Y12 antibody, but not with coilin containing structures

Absorption spectra of antimony pentachloride-aromatic hydrocarbon systems

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