Deepak P. Patil

m 6 A RNA methylation promotes XIST -mediated transcriptional repression

The long non-coding RNA X-inactive specific transcript (XIST) mediates the transcriptional silencing of genes on the X chromosome. Here we show that, in human cells, XIST is highly methylated with at least 78 N6-methyladenosine (m6A) residues—a reversible base modification of unknown function in long non-coding RNAs. We show that m6A formation in XIST, as well as in cellular mRNAs, is mediated by RNA-binding motif protein 15 (RBM15) and its paralogue RBM15B, which bind the m6A-methylation complex and recruit it to specific sites in RNA. This results in the methylation of adenosine nucleotides in adjacent m6A consensus motifs. Furthermore, we show that knockdown of RBM15 and RBM15B, or knockdown of methyltransferase like 3 (METTL3), an m6A methyltransferase, impairs XIST-mediated gene silencing. A systematic comparison of m6A-binding proteins shows that YTH domain containing 1 (YTHDC1) preferentially recognizes m6A residues on XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of m6A. These data reveal a pathway of m6A formation and recognition required for XIST-mediated transcriptional repression.

Reversible methylation of m6Am in the 5′ cap controls mRNA stability

Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5′ end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N6,2′-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5′ cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.

The miR-30 family microRNAs confer epithelial phenotype to human pancreatic cells

Epithelial-to-mesenchymal transition is a phenomenon necessary for embryonic development and also seen during certain pathological conditions. We show here for the first time that reduction in miR-30 family microRNAs, is responsible for mesenchymal transition of primary cultures of human pancreatic epithelial cells. We found that miR-30 family microRNAs target mesenchymal gene transcripts and maintain them in a translationally inactive state. Forced depletion using miR-30 family specific anti-miRs leads to mesenchymal transition while ectopic overexpression maintains the epithelial phenotype. We also show that miR-30 family microRNAs increase in abundance during differentiation of pancreatic islet-derived mesenchymal cells into hormone-producing islet-like cell aggregates. Our studies in human adult diseased pancreas also demonstrate that miR-30 family microRNAs are expressed at lower abundance in fibrotic lesions during pancreatitis. Together, our data confirm that miR-30 family microRNAs form a part of the regulatory signaling events involved in cellular response of pancreatic epithelial cells during mesenchymal transition.

Molecular analysis of gut microbiota in obesity among Indian individuals

Obesity is a consequence of a complex interplay between the host genome and the prevalent obesogenic factors among the modern communities. The role of gut microbiota in the pathogenesis of the disorder was recently discovered; however, 16S-rRNA-based surveys revealed compelling but community-specific data. Considering this, despite unique diets, dietary habits and an uprising trend in obesity, the Indian counterparts are poorly studied. Here, we report a comparative analysis and quantification of dominant gut microbiota of lean, normal, obese and surgically treated obese individuals of Indian origin. Representative gut microbial diversity was assessed by sequencing fecal 16S rRNA libraries for each group (n = 5) with a total of over 3000 sequences. We detected no evident trend in the distribution of the predominant bacterial phyla, Bacteroidetes and Firmicutes. At the genus level, the bacteria of genus Bacteroides were prominent among the obese individuals, which was further confirmed by qPCR (P < 0.05). In addition, a remarkably high archaeal density with elevated fecal SCFA levels was also noted in the obese group. On the contrary, the treated-obese individuals exhibited comparatively reduced Bacteroides and archaeal counts along with reduced fecal SCFAs. In conclusion, the study successfully identified a representative microbial diversity in the Indian subjects and demonstrated the prominence of certain bacterial groups in obese individuals; nevertheless, further studies are essential to understand their role in obesity.

Functional genomics of fuzzless-lintless mutant of Gossypium hirsutum L. cv. MCU5 reveal key genes and pathways involved in cotton fibre initiation and elongation

BackgroundFuzzless-lintless cotton mutants are considered to be the ideal material to understand the molecular mechanisms involved in fibre cell development. Although there are few reports on transcriptome and proteome analyses in cotton at fibre initiation and elongation stages, there is no comprehensive comparative transcriptome analysis of fibre-bearing and fuzzless-lintless cotton ovules covering fibre initiation to secondary cell wall (SCW) synthesis stages. In the present study, a comparative transcriptome analysis was carried out using G. hirsutum L. cv. MCU5 wild-type (WT) and it’s near isogenic fuzzless-lintless (fl) mutant at fibre initiation (0 dpa/days post anthesis), elongation (5, 10 and 15 dpa) and SCW synthesis (20 dpa) stages.ResultsScanning electron microscopy study revealed the delay in the initiation of fibre cells and lack of any further development after 2 dpa in the fl mutant. Transcriptome analysis showed major down regulation of transcripts (90%) at fibre initiation and early elongation (5 dpa) stages in the fl mutant. Majority of the down regulated transcripts at fibre initiation stage in the fl mutant represent calcium and phytohormone mediated signal transduction pathways, biosynthesis of auxin and ethylene and stress responsive transcription factors (TFs). Further, transcripts involved in carbohydrate and lipid metabolisms, mitochondrial electron transport system (mETS) and cell wall loosening and elongation were highly down-regulated at fibre elongation stage (5–15 dpa) in the fl mutant. In addition, cellulose synthases and sucrose synthase C were down-regulated at SCW biosynthesis stage (15–20 dpa). Interestingly, some of the transcripts (~50%) involved in phytohormone signalling and stress responsive transcription factors that were up-regulated at fibre initiation stage in the WT were found to be up-regulated at much later stage (15 dpa) in fl mutant.ConclusionsComparative transcriptome analysis of WT and its near isogenic fl mutant revealed key genes and pathways involved at various stages of fibre development. Our data implicated the significant role of mitochondria mediated energy metabolism during fibre elongation process. The delayed expression of genes involved in phytohormone signalling and stress responsive TFs in the fl mutant suggests the need for a coordinated expression of regulatory mechanisms in fibre cell initiation and differentiation.

Reading m6A in the Transcriptome: m6A-Binding Proteins

N6-Methyladenosine (m6A) is the most prevalent post-transcriptional modification of eukaryotic mRNA and long noncoding RNA. m6A mediates its effects primarily by recruiting proteins, including the multiprotein eukaryotic initiation factor 3 complex and a set of proteins that contain the YTH domain. Here we describe the mechanisms by which YTH domain-containing proteins bind m6A and influence the fate of m6A-containing RNA in mammalian cells. We discuss the diverse, and occasionally contradictory, functions ascribed to these proteins and the emerging concepts that are influencing our understanding of these proteins and their effects on the epitranscriptome.

Surface Engineering of Polycaprolactone by Biomacromolecules and their Blood Compatibility

Improving blood compatibility of biodegradable polymers is an area of intensive research in blood contacting devices. In this study, curdlan sulphate and heparin-modified poly (caprolactone) (PCL) hybrids were developed by physically entrapping these molecules on the PCL surface. This modification technique was performed by reversible gelation of the PCL surface region following exposure to a solvent and nonsolvent mixture. The presence of these biomacromolecules on the PCL surface was verified by atomic force microscopy (AFM) and scanning electron microscopy and energy dispersive X-ray analysis (SEM-EDAX) analysis, while wettability of the films was investigated by dynamic contact angle measurements. The blood compatibilities of the surface-modified films were examined using in vitro platelet and leukocyte adhesion and thrombus formation. Mouse RAW 264.7 macrophage cells were used to assess the cell adhesion and inflammatory response to the modified surface by quantifying mRNA expression levels of proinflammatory cytokines namely TNF-α and IL-1β using real-time polymerase chain reaction (RT-PCR). A lower platelet and leukocyte adhesion and activation was observed on the modified films incubated with whole human blood for 2 h. The thrombus formation on the PCL was significantly decreased upon immobilization of both curdlan sulphate (39%, *p<0.05) and heparin (28%, *p<0.01) when compared to bare PCL (80%). All of these results revealed that improved blood compatibility was obtained by surface entrapment of both curdlan sulphate (CURS) and heparin (HEP) onto PCL films. Both PCL-CURS and PCL-HEP films reduced RAW 264.7 macrophage cell adhesion (*p<0.05) with respect to the base unmodified PCL. The cellular inflammatory response was suppressed on the modified substrates. The mRNA expression levels of proinflammmatory cytokines (TNF-α and IL-1β) were upregulated on bare PCL, while it was significantly lower on PCL-CURS and PCL-HEP substrates (**p<0.001). Thus, this biomacromolecule entrapment process can be applied on PCL in order to achieve improved blood compatibility and reduced inflammatory host response for its future blood contacting applications.

In vivo modulation of foreign body response on polyurethane by surface entrapment technique

Implanted polymeric materials, such as medical devices, provoke the body to initiate an inflammatory reaction, known as the foreign body response (FBR), which causes several complications. In this study, polyurethane (Tecoflex®, PU) surface modified with the nonionic surfactant Tween80® (PU/T80) and the cell adhesive PLL-RGD peptide (PU/PLL-RGD) by a previously described entrapment technique were implanted in the peritoneal cavity of Wistar rats for 30 days. Implants were retrieved and examined for tissue reactivity and cellular adherence by various microscopic and analytical techniques. Surface-induced inflammatory response was assessed by real-time PCR based quantification of proinflammatory cytokine transcripts, namely, TNF-α and IL-1β, normalized to housekeeping gene GAPDH. Cellular adherence and their distribution profile were assessed by microscopic examination of H&E stained implant sections. It was observed that PU/PLL-RGD followed by the bare PU surface exhibited severe inflammatory and fibrotic response with an average mean thickness of 19 and 12 μm, respectively, in 30 days. In contrast, PU/T80 surface showed only a cellular monolayer of 2-3 μm in thickness, with a mild inflammatory response and no fibrotic encapsulation. The PU/PLL-RGD peptide-modified substrate promoted an enhanced rate of macrophage cell fusion to form foreign body giant cell (FBGCs), whereas FBGCs were rarely observed on Tween80®-modified substrate. The expression levels of proinflammatory cytokines (TNF-α and IL-1β) were upregulated on PU/PLL-RGD surface followed by bare PU, whereas the cytokine expressions were significantly suppressed on PU/T80 surface. Thus, our study highlights modulation of foreign body response on polyurethane surfaces through surface entrapment technique in the form of differential responses observed on PLL-RGD and Tween80® modified surfaces with the former effective in triggering tissue cell adhesion thereby fibrous encapsulation, while the later being mostly resistant to this phenomenon.

The m 6 A pathway facilitates sex determination in Drosophila

The conserved modification N6-methyladenosine (m6A) modulates mRNA processing and activity. Here, we establish the Drosophila system to study the m6A pathway. We first apply miCLIP to map m6A across embryogenesis, characterize its m6A ‘writer’ complex, validate its YTH ‘readers’ CG6422 and YT521-B, and generate mutants in five m6A factors. While m6A factors with additional roles in splicing are lethal, m6A-specific mutants are viable but present certain developmental and behavioural defects. Notably, m6A facilitates the master female determinant Sxl, since multiple m6A components enhance female lethality in Sxl sensitized backgrounds. The m6A pathway regulates Sxl processing directly, since miCLIP data reveal Sxl as a major intronic m6A target, and female-specific Sxl splicing is compromised in multiple m6A pathway mutants. YT521-B is a dominant m6A effector for Sxl regulation, and YT521-B overexpression can induce female-specific Sxl splicing. Overall, our transcriptomic and genetic toolkit reveals in vivo biologic function for the Drosophila m6A pathway.

The biocompatibility of sulfobetaine engineered poly (ethylene terephthalate) by surface entrapment technique.

Sulfobetaine-modified poly(ethylene terephthalate) (PET) systems were created by physically entrapping the zwitterionic species on the PET surface. The presence of the sulfobetiane molecules on these surfaces were verified by ATR-FTIR and SEM-EDAX analysis, while wettability of the films was investigated by water contact angle measurements. The blood compatibility of the modified films was evaluated by platelet adhesion in human platelet-rich plasma (PRP). The adhesion and inflammatory response of Mouse RAW 264.7 macrophage cells were studied. The surface induced cellular inflammatory response was determined by quantifying the expression levels of proinflammatory cytokines namely TNF-α and IL-1β by measuring their mRNA profiles in the cells using real time polymerase chain reaction normalized to the housekeeping gene GAPDH. L-929 fibroblast cells were used to assess the propensity of the materials to support the fibroblast cell adhesion. A lower platelet adhesion and activation were observed on the sulfobetaine-modified PET film incubated in PRP after 2h when compared to control. The modified film reduced cellular adhesion events ( p<0.05) with respect to the base material, which could be linked to the reduced protein adsorption observed on this surface. The cellular inflammatory response was suppressed on sulfobetaine-modified substrate. Expression levels of pro-inflammmatory cytokines (TNF-α and IL-1β) was found to be upregulated on bare PET, while it was significantly lower on modified PET ( p<0.001). Thus the sulfobetaine entrapment process can be applied on PET in order to achieve low biointeractions and reduced inflammatory host response for various biomedical and biotechnological applications.