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The mammalian transcriptome comprises a vast family of long noncoding (lnc)RNAs implicated in physiologic processes such as myogenesis, through which muscle forms during embryonic development and regenerates in the adult. However, the specific molecular mechanisms by which lncRNAs regulate human myogenesis are poorly understood. Here, we identified a novel muscle-specific lncRNA, lncFAM71E1-2:2 (lncFAM), which increased robustly during early human myogenesis. Overexpression of lncFAM promoted differentiation of human myoblasts into myotubes, while silencing lncFAM suppressed this process. As lncFAM resides in the nucleus, chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-MS) analysis was employed to identify the molecular mechanisms whereby it might promote myogenesis. Analysis of lncFAM-interacting proteins revealed that lncFAM recruited the RNA-binding protein HNRNPL to the promoter of MYBPC2, in turn increasing MYBPC2 mRNA transcription and enhancing production of the myogenic protein MYBPC2. These results highlight a mechanism whereby a novel ribonucleoprotein complex, lncFAM-HNRNPL, elevates MYBPC2 expression transcriptionally to promote myogenesis.

Cellular senescence is linked to chronic age-related diseases including atherosclerosis, diabetes, and neurodegeneration. Compared to proliferating cells, senescent cells express distinct subsets of proteins. In this study, we used cultured human diploid fibroblasts rendered senescent through replicative exhaustion or ionizing radiation to identify proteins differentially expressed during senescence. We identified acid ceramidase (ASAH1), a lysosomal enzyme that cleaves ceramide into sphingosine and fatty acid, as being highly elevated in senescent cells. This increase in ASAH1 levels in senescent cells was associated with a rise in the levels of ASAH1 mRNA and a robust increase in ASAH1 protein stability. Furthermore, silencing ASAH1 in pre-senescent fibroblasts decreased the levels of senescence proteins p16, p21, and p53, and reduced the activity of the senescence-associated β-galactosidase. Interestingly, depletion of ASAH1 in pre-senescent cells sensitized these cells to the senolytics Dasatinib and Quercetin (D+Q). Together, our study indicates that ASAH1 promotes senescence, protects senescent cells, and confers resistance against senolytic drugs. Given that inhibiting ASAH1 sensitizes cells towards senolysis, this enzyme represents an attractive therapeutic target in interventions aimed at eliminating senescent cells.

Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) modulate gene expression programs in physiology and disease. Here, we report a noncoding RNA regulatory network that modulates myoblast fusion into multinucleated myotubes, a process that occurs during muscle development and muscle regeneration after injury. In early stages of human myogenesis, the levels of lncRNA OIP5-AS1 increased, while the levels of miR-7 decreased. Moreover, OIP5-AS1 bound and induced miR-7 decay via target RNA-directed miRNA decay; accordingly, loss of OIP5-AS1 attenuated, while antagonizing miR-7 accelerated, myotube formation. We found that the OIP5-AS1-mediated miR-7 degradation promoted myoblast fusion, as it derepressed the miR-7 target MYMX mRNA, which encodes the fusogenic protein myomixer (MYMX). Remarkably, an oligonucleotide site blocker interfered with the OIP5-AS1-directed miR-7 degradation, allowing miR-7 to accumulate, lowering MYMX production and suppressing myotube formation. These results highlight a mechanism whereby lncRNA OIP5-AS1-mediated miR-7 decay promotes myotube formation by stimulating a myogenic fusion program.

Long noncoding (lnc)RNAs potently regulate gene expression programs in physiology and disease. Here, we describe a key function for lncRNA OIP5-AS1 in myogenesis, the process whereby myoblasts differentiate into myotubes during muscle development and muscle regeneration after injury. In human myoblasts, OIP5-AS1 levels increased robustly early in myogenesis, and its loss attenuated myogenic differentiation and potently reduced the levels of the myogenic transcription factor MEF2C. This effect relied upon the partial complementarity of OIP5-AS1 with MEF2C mRNA and the presence of HuR, an RNA-binding protein (RBP) with affinity for both transcripts. Remarkably, HuR binding to MEF2C mRNA, which stabilized MEF2C mRNA and increased MEF2C abundance, was lost after OIP5-AS1 silencing, suggesting that OIP5-AS1 might serve as a scaffold to enhance HuR binding to MEF2C mRNA, in turn increasing MEF2C production. These results highlight a mechanism whereby a lncRNA promotes myogenesis by enhancing the interaction of an RBP and a myogenic mRNA.

Growing evidence indicates that long intergenic noncoding RNAs play an important role in cancer progression by affecting gene regulation at the transcriptional and posttranscriptional levels. Recent studies have shown that long intergenic noncoding RNA functions as a competitive endogenous RNA, which can interact with and mitigate the function of microRNA. In this study, we investigated the molecular mechanism by which LINC00162 regulates cell proliferation and apoptotic cell death. By analyzing RNA sequencing data, LINC00162 was identified to be a target of heterogeneous nuclear ribonucleoprotein K (hnRNPK). HnRNPK positively regulated LINC00162 expression through p38 mitogen-activated protein kinase. Lowering the level of either hnRNPK or LINC00162 decreased proliferation and colony formation while it increased apoptotic cell death. Small RNA sequencing followed by the antisense oligonucleotide pulldown, revealed that LINC00162 interacts directly with miR-485-5p which exhibited tumor-suppressing effects by suppressing cell proliferation and colony formation, and increasing apoptotic cell death. Through the bioinformatic approaches, progestin and adipoQ receptor 4 (PAQR4) was selected as a common target of LINC00162 and miR-485-5p. miR-485-5p decreased the expression of PAQR4 by directly binding to the 3'-untranslated region of PAQR4 messenger RNA. Knockdown of hnRNPK and LINC00162 increased the level of functional miR-485-5p, indicating that LINC00162 may compete for miR-485-5p, thereby derepressing PAQR4 expression. Overexpression of either hnRNPK or LINC00162, or inhibition of miR-485-5p, protected cells against etoposide-induced apoptotic death. Our findings demonstrate that a regulatory paradigm implicating hnRNPK, LINC00162, miR-485-5p, and PAQR4 plays an important role in cell proliferation and apoptosis, and is a promising target for cancer therapeutics.

Glucose mediated insulin biosynthesis is tightly regulated and shared between insulin granule proteins such as its processing enzymes, prohormone convertases, PC1/3 and PC2. However, the molecular players involved in the co-ordinated translation remain elusive. The trans-acting factors like PABP (Poly A Binding Protein) and PDI (Protein Disulphide Isomerize) binds to a conserved sequence in the 5'UTR of insulin mRNA and regulates its translation. Here, we demonstrate that 5'UTR of PC1/3 and PC2 also associate with PDI and PABP. We show that a' and RRM 3-4 domains of PDI and PABP respectively, are necessary for RNA binding activity to the 5'UTRs of insulin and its processing enzymes.

Accumulating research suggests that the tumor immune microenvironment (TIME) plays an essential role in regulation of tumor growth and metastasis. The cellular and molecular nature of the TIME influences cancer progression and metastasis by altering the ratio of immune- suppressive versus cytotoxic responses in the vicinity of the tumor. Targeting or activating the TIME components show a promising therapeutic avenue to combat cancer. The success of immunotherapy is both astounding and unsatisfactory in the clinic. Advancements in RNA-based technology have improved understanding of the complexity and diversity of the TIME and its effects on therapy. TIME-related RNA or RNA regulators could be promising targets for anticancer immunotherapy. In this review, we discuss the available RNA-based cancer immunotherapies targeting the TIME. More importantly, we summarize the potential of various RNA-based therapeutics clinically available for cancer treatment. RNA-dependent targeting of the TIME, as monotherapy or combined with other evolving therapeutics, might be beneficial for cancer patients’ treatment in the near future.

In this work, we report the development of Drude polarizable force field parameters for the carboxylate and N-acetyl amine derivatives, extending the functionality of the existing Drude polarizable carbohydrate force field. The force field parameters have been developed in a hierarchical manner, reproducing the quantum mechanical gas-phase properties of small model compounds representing the key functional group in the carbohydrate derivatives, including optimization of the electrostatic and bonded parameters. The optimized parameters were then used to generate the models for carboxylate and N-acetyl amine carbohydrate derivatives. The transferred parameters were further tested and optimized to reproduce crystal geometries and J-coupling data from nuclear magnetic resonance experiments. The parameter development resulted in the incorporation of d-glucuronate, l-iduronate, N-acetyl-d-glucosamine (GlcNAc), and N-acetyl-d-galactosamine (GalNAc) sugars into the Drude polarizable force field. The parameters de...

The RNA-binding protein GRSF1 (G-rich RNA sequence-binding factor 1) critically maintains mitochondrial homeostasis. Accordingly, loss of GRSF1 impaired mitochondrial respiration and increased the levels of reactive oxygen species (ROS), triggering DNA damage, growth suppression, and a senescent phenotype characterized by elevated production and secretion of interleukin (IL)6. Here, we characterize the pathways that govern IL6 production in response to mitochondrial dysfunction in GRSF1-depleted cells. We report that loss of GRSF1 broadly altered protein expression programs, impairing the function of respiratory complexes I and IV. The rise in oxidative stress led to increased DNA damage and activation of mTOR, which in turn activated NF-κB to induce IL6 gene transcription and orchestrate a pro-inflammatory program. Collectively, our results indicate that GRSF1 helps preserve mitochondrial homeostasis, in turn preventing oxidative DNA damage and the activation of mTOR and NF-κB, and suppressing a transcriptional pro-inflammatory program leading to increased IL6 production.

Recent developments in high-throughput RNA sequencing methods coupled with innovative bioinformatic tools have uncovered thousands of circular (circ)RNAs. CircRNAs have emerged as a vast and novel class of regulatory RNAs with potential to modulate gene expression by acting as sponges for microRNAs (miRNAs) and RNA-binding proteins (RBPs). The biochemical enrichment of circRNAs by exoribonuclease treatment or by depletion of polyadenylated RNAs coupled with deep-sequencing is widely used for the systematic identification of circRNAs. Although these methods enrich circRNAs substantially, they do not eliminate efficiently non-polyadenylated and highly-structured RNAs. Here, we describe a method we termed RPAD, based on initial RNase R treatment followed by Polyadenylation and poly(A)(+) RNA Depletion. These joint interventions drastically depleted linear RNAs leading to isolation of highly pure circRNAs from total RNA pools. By facilitating the isolation of highly pure circRNAs, RPAD enables the elucidation of circRNA biogenesis, sequence, and function.

Antifreeze glycoproteins (AFGPs) are distinctively riveting class of bio-macromolecules, which endows the survival of organisms inhabiting polar and subpolar regions. These proteins are believed to hinder microscopic freezing by interacting with embryonic ice crystals and precluding their further growth. The underlying molecular mechanism by which AFGPs bind to ice has remained elusive due to insufficient structural characterization, with conflicting hypotheses on the possible binding mode of AFGPs - either via the hydrophobic peptide backbone or via the hydrophilic carbohydrate side chains - when interacting with ice. Chemical synthesis has allowed researchers to access synthetic variants of natural AFGPs. These studies revealed that AFGPs exhibit huge variations in their thermal hysteresis and ice shaping behavior with only slight structural variations, especially to the carbohydrate side chains. Four key structural motifs were identified as crucial to AFGP activity: the presence of a threonine γ-methyl group, an α-glycosidic carbohydrate-protein linkage, an acetylamide group (-NHCOCH3) at the C2 position of the carbohydrate linked to the protein, and the presence of carbohydrate hydroxyl groups. In this study, we use molecular dynamics (MD) simulations to probe the microscopic properties of water accompanying these structural variations of AFGPs. We find that these variations primarily influence the conformation space of AFGPs and also crucially control their hydration dynamics. Owing to the disordered nature of AFGPs we use Markov-state modeling to identify the conformational preferences of AFGPs. The simulations reveal the importance of steric bulk, intra-molecular carbohydrate-protein H-bonds and conformational preferences (α- vs. β-linkages) in controlling the spatial segregation of the hydrophilic and hydrophobic regions of AFGPs. We hypothesize that the hydrophobic component of AFGPs is crucial to their binding to ice, which determines the ice shaping ability of AFGPs. However, the hydrophilic carbohydrate hydroxyl groups and their ability to form water bridges control the subsequent hydration dynamics, which is key to the antifreeze properties. Investigating the tetrahedral order parameter of water molecules around the carbohydrates revealed competition between solute- and bulk-influenced solvent structures, with maximum restructuring being observed in the interfacial region 2.5-4.5 Å away from the AFGPs.

Small molecules such as metabolites and drugs must pass through the membrane of the cell, a barrier primarily comprised of phospholipid bilayers and embedded proteins. To better understand the process of passive diffusion, knowledge of the ability of various functional groups to partition across bilayers and the associated energetics would be of utility. In the present study, the SILCS (Site Identification by Ligand Competitive Saturation) methodology has been applied to sample the distributions of a diverse set of chemical solutes representing the functional groups of small molecules across phospholipid bilayers composed of 0.9:0.1 POPC/Cholesterol, and a mixture of 0.52:0.18:0.3 DOPS:DOPC:cholesterol used in parallel artificial membrane permeability assay (PAMPA) experiments. A combination of oscillating chemical potential Grand Canonical Monte Carlo and Molecular Dynamics in the SILCS simulations was applied to achieve solute sampling through the bilayers and surrounding aqueous environment from which the distribution of solutes and the functional groups they represent were obtained. Results show differential distribution of aliphatic versus aromatic groups with the former having increased sampling in the center of the bilayers versus in the region of the glycerol linker for the latter. Variations in the distribution of different polar groups are evident, with large differences between negative acetate and positive methylammonium with accumulation of the polar-neutral and acetate solutes above the bilayer head groups. Conversion of the distributions to absolute free energies allows for a detailed understanding of energetics of functional groups in different regions of the bilayers and for calculation of absolute free energy profiles of multifunctional drug-like molecules across the bilayers from which partition coefficients and resistance factors suitable for insertion into the homogenous solubility diffusion equation for calculation of permeability were obtained. Comparisons of the calculated bilayer/solution partition coefficients with 1-octanol/water experimental data for both drug-like molecules and the solutes show overall good agreement, validating the calculated distributions and associated absolute free energy profiles.

Malignant characteristics of cancers, represented by rapid cell proliferation and high metastatic potential, are a major cause of high cancer-related mortality. As a multifunctional RNA-binding protein, heterogeneous nuclear ribonucleoprotein K (hnRNPK) is closely associated with cancer progression in various types of cancers. In this study, we sought to identify hnRNPK-regulated long intergenic non-coding RNAs (lincRNAs) that play a critical role in the regulation of cancer malignancy. We found that hnRNPK controlled malignant phenotypes including invasiveness, proliferation, and clonogenicity. RNA sequencing and functional studies revealed that LINC00263, a novel target of hnRNPK, is involved in the oncogenic functions of hnRNPK. Knockdown of LINC00263 mitigated the malignant capabilities. Conversely, increased malignant phenotypes were observed in LINC00263-overexpressing cells. Since LINC00263 was mainly localized in the cytosol and highly enriched in Argonaute 2-immunoprecipitation (Ago2-IP), we hypothesized that LINC00263 acts as a competitive endogenous RNA (ceRNA), and thus sought to identify LINC00263-associated microRNAs. Using small RNA sequencing followed by antisense oligonucleotide pull-down, miR-147a was selected for further study. We found that miR-147a negatively regulates LINC00263 via direct interaction, thus suppressing malignant capabilities. Moreover, knockdown of hnRNPK and LINC00263 upregulated miR-147a, indicating that LINC00263 serves as a ceRNA for miR-147a. By analyzing RNA sequencing data and miRNA target prediction, calpain 2 (CAPN2) was identified as a putative target of miR-147a. Ago2-IP and luciferase reporter assay revealed that miR-147a suppressed CAPN2 expression by directly binding to the 3′UTR of CAPN2 mRNA. In addition, we found that the weakened malignant capabilities following knockdown of hnRNPK or LINC00263 were restored by miR-147a inhibition or CAPN2 overexpression. Furthermore, our findings were validated in various other types of cancer cells including lung cancer, colorectal cancer, neuroblastoma, and melanoma. Collectively, we demonstrate that hnRNPK-regulated LINC00263 plays an important role in cancer malignancy by acting as a miR-147a decoy and thus upregulating CAPN2.

Circular RNAs (circRNAs) comprise a vast class of covalently closed transcripts, generated primarily via backsplicing. Most circRNAs arise from full or partial exons, but they can also arise from introns, and from combinations of introns and exons. While high-throughput RNA-sequencing analysis has identified tens of thousands of circRNAs expressed in different tissues and growth conditions, the function of circRNAs has only been described for a handful of them. As most circRNAs appear not to encode peptides, their function is presumed to be linked to their interaction with a range of molecules, particularly other nucleic acids (notably microRNAs) and proteins. A major impediment to identifying circRNA-associated molecules is a lack of suitable methodologies capable of analyzing specifically circRNAs and not their linear RNA counter-parts with which they share most of their sequence. Here, we describe a flexible and robust method for identifying the proteins that associate with a given circRNA. The affinity pulldown assay is based on the use of a biotinylated antisense oligomer that recognizes the circRNA-specific junction sequence. Following pulldown using streptavidin beads, the proteins are eluted from the circRNP (circribonucleoprotein) complex and identified by mass spectroscopy; validation by Western blot analysis and other methods would then confirm the identity of the circRNA-associated proteins. We present a detailed step-by-step protocol, tips to optimize the analysis, troubleshooting suggestions, and assistance in interpreting the results. In sum, this protocol enables the discovery of proteins present in circRNPs, a critical effort toward elucidating circRNA function.

The CHARMM Drude-2013 polarizable force field (FF) was developed to include the explicit treatment of induced electronic polarizability, resulting in a more accurate description of the electrostatic interactions in molecular dynamics (MD) simulations. While the Drude-2013 protein FF has shown success in improving the folding properties of α-helical peptides and to reproduce experimental observables in simulations up to 1 μs, some limitations were noted regarding the stability of β-sheet structures in simulations longer than 100 ns as well as larger deviations from crystal structures in simulations of a number of proteins compared to the additive CHARMM36 protein FF. The origin of the instability has been identified and appears to be primarily due to overestimated atomic polarizabilities and induced dipole-dipole interactions on the Cβ, Cγ, and Cδ side chain atoms. To resolve this and other issues, a number of aspects of the model were revisited, resulting in Drude-2019 protein FF. Backbone parameters were optimized targeting the conformational properties of the (Ala)5 peptide in solution along with gas phase properties of the alanine dipeptide. Dipeptides that contain N-acetylated and N'-methylamidated termini, excluding Gly, Pro, and Ala, were used as models to optimize the atomic polarizabilities and Thole screening factors on selected Cβ, Cγ, and Cδ carbons by targeting quantum mechanical (QM) dipole moments and molecular polarizabilities. In addition, to obtain better conformational properties, side chain χ1 and χ2 dihedral parameters were optimized targeting QM data for the respective side chain dipeptide conformations as well as Protein Data Bank survey data based on the χ1, χ2 sampling from Hamiltonian replica-exchange MD simulations of (Ala)4-X-(Ala)4 in solution, where X is the amino acid of interest. Further improvements include optimizing nonbonded interactions between charged residues to reproduce QM interaction energies of the charged-protein model compounds and experimental osmotic pressures. Validation of the optimized Drude protein FF includes MD simulations of a collection of peptides and proteins including β-sheet structures, as well as transmembrane ion channels. Results showed that the updated Drude-2019 protein FF yields smaller overall root-mean-square differences of proteins as compared to the additive CHARMM36m and Drude-2013 FFs as well as similar or improved agreement with experimental NMR properties, allowing for long time scale simulation studies of proteins and more complex biomolecular systems in conjunction with the remainder of the Drude polarizable FF.

By interacting with proteins and nucleic acids, the vast family of mammalian circRNAs is proposed to influence many biological processes. Here, RNA sequencing analysis of circRNAs differentially expressed during myogenesis revealed that circSamd4 expression increased robustly in mouse C2C12 myoblasts differentiating into myotubes. Moreover, silencing circSamd4, which is conserved between human and mouse, delayed myogenesis and lowered the expression of myogenic markers in cultured myoblasts from both species. Affinity pulldown followed by mass spectrometry revealed that circSamd4 associated with PURA and PURB, two repressors of myogenesis that inhibit transcription of the myosin heavy chain (MHC) protein family. Supporting the hypothesis that circSamd4 might complex with PUR proteins and thereby prevent their interaction with DNA, silencing circSamd4 enhanced the association of PUR proteins with the Mhc promoter, while overexpressing circSamd4 interfered with the binding of PUR proteins to the Mhc promoter. These effects were abrogated when using a mutant circSamd4 lacking the PUR binding site. Our results indicate that the association of PUR proteins with circSamd4 enhances myogenesis by contributing to the derepression of MHC transcription.

Eukaryotic cells express a myriad of circular RNAs (circRNAs), many of them displaying tissue-specific expression patterns. They arise from linear precursor RNAs in which 5' and 3' ends become covalently ligated. Given these features, biochemical and computational approaches traditionally used to study linear RNA must be adapted for analysis of circular RNAs. Such circRNA-specific methodologies are allowing the systematic identification of circRNAs and the analysis of their biological functions. Here, we review the resources and molecular methods currently utilized to quantify circRNAs, visualize their distribution, identify interacting partners, and elucidate their function. We discuss the challenges of analyzing circRNAs and propose alternative approaches for studying this unique class of transcripts. This article is characterized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Methods > RNA Analyses in vitro and In Silico RNA Methods > RNA Analyses in Cells.

Insulin maintains glucose homeostasis by stimulating glucose uptake from extracellular environment to adipose and muscle tissue through glucose transporter (GLUT4). Insulin resistance plays a significant role in pathologies associated with type2 diabetes. It has been previously shown that hyperinsulinemia can lead to insulin resistance. In these studies very high levels of insulin was used to achieve insulin resistance. We hypothesized that one of the causes of type 2 diabetes could be insulin synthesis in the absence of glucose stimulation. We used CHO cell line, stably expressing Myc-GLUT4-GFP along with human insulin receptor to study the effect of hyperinsulinemia in the presence of low glucose (6.5 mM) or high glucose (20 mM). The insulin responsiveness of these cells was assessed by FRAP, FACS and subcellular fractionation. The results suggest that exposure of cells to insulin in low glucose conditions made these cells insulin resistant within 10 passages, while the same level of insulin in the presence of high glucose did not result in insulin resistance. These results clearly suggest that hyperinsulinemia combined with hypoglycaemia may lead to insulin resistance and may be one of the causes for the typ2 diabetes.

Cell-penetrating peptides (CPPs) facilitate the transport of pharmacologically active molecules, such as plasmid DNA, short interfering RNA, nanoparticles, and small peptides. The accurate identification of new and unique CPPs is the initial step to gain insight into CPP activity. Experiments can provide detailed insight into the cell-penetration property of CPPs. However, the synthesis and identification of CPPs through wet-lab experiments is both resource- and time-expensive. Therefore, the development of an efficient prediction tool is essential for the identification of unique CPP prior to experiments. To this end, we developed a kernel extreme learning machine (KELM) based CPP prediction model called KELM-CPPpred. The main data set used in this study consists of 408 CPPs and an equal number of non-CPPs. The input features, used to train the proposed prediction model, include amino acid composition, dipeptide amino acid composition, pseudo amino acid composition, and the motif-based hybrid features. We further used an independent data set to validate the proposed model. In addition, we have also tested the prediction accuracy of KELM-CPPpred models with the existing artificial neural network (ANN), random forest (RF), and support vector machine (SVM) approaches on respective benchmark data sets used in the previous studies. Empirical tests showed that KELM-CPPpred outperformed existing prediction approaches based on SVM, RF, and ANN. We developed a web interface named KELM-CPPpred, which is freely available at http://sairam.people.iitgn.ac.in/KELM-CPPpred.html.

The senescence-associated secretory phenotype (SASP) is a major trait of senescent cells, but the molecular regulators of SASP factor secretion are poorly understood. Mass spectrometry analysis revealed that secretory carrier membrane protein 4 (SCAMP4) levels were strikingly elevated on the surface of senescent cells compared with proliferating cells. Interestingly, silencing SCAMP4 in senescent fibroblasts reduced the secretion of SASP factors, including interleukin 6 (IL6), IL8, growth differentiation factor 15 (GDF-15), C-X-C motif chemokine ligand 1 (CXCL1), and IL7, while, conversely, SCAMP4 overexpression in proliferating fibroblasts increased SASP factor secretion. Our results indicate that SCAMP4 accumulates on the surface of senescent cells, promotes SASP factor secretion, and critically enhances the SASP phenotype.

Human malignant melanoma is highly aggressive and metastatic in nature and exhibits phenotypic plasticity. Melanoma has multiple phenotypically distinct subpopulations which involved in tumor progression and markedly resistant to conventional therapy. Some of the melanoma subpopulation exhibits stem cells feature and defined as cancer stem cells (CSCs) or tumor initiating cells (TICs). Identification and characterization of CSCs in melanoma may have therapeutic implication for combating melanoma progression. In recent study, we have identified that CD133+ subpopulation of melanoma potentially involved in tumor initiation, metastasis, epithelial to mesenchymal (EMT) and angiogenesis. Genetic and pharmacological screening revealed that functional properties of CD133+ melanoma cells are regulated by Notch1-MAPK signaling axis. Andrographolide (herbal product) abrogates Notch1-MAPK pathway in CD133+ cells that leads to attenuation of melanoma progression, metastasis and angiogenesis.

Aim: To determine the prevalence of diabetes and to explore various modifiable and nonmodifiable factors associated with diabetes in Ahmednagar city. Method: The data was then collected using convenient sampling method. The subjects were selected from VPMH hospital, various camps and door to door survey. The subjects were assessed based on the assessment form including Demographic data, Socio-economic data, Family history, Medical history, Surgical history, Personal history, B.M.I., Waist Hip ratio, Diabetes history, Drug history and human activity profile questionnaire. Result: 38.6% of adult diagnosed with diabetes in Ahmednagar city population the prevalence came out to be 46.6% among males and 30.5% in females. The mean age for male and female are 57.3 years and 57.2 years respectively. About 47.8% diabetic samples belongs to upper middle class and 4.3% belongs to lower class. 58.6% have a positive family history and 41.3% have a negative family history. 50% have no medical history and 30.4% have positive history of HTN and 3.5% have positive history of CVD and HTN & CVD. Conclusion: The mean age for diabetes in male is 57.3% and for female is 57.2%. The mean duration of diabetes in males is 13.1 year in males and 9.8 year in females. This is mostly the first study to include use of sugar in beverage by diabetic population i.e. 56.5% out total diabetic population

Carbohydrates are known to closely modulate their surrounding solvent structures and influence solvation dynamics. Spectroscopic investigations studying far-IR regions (below 1000 cm(-1)) have observed spectral shifts in the libration band (around 600 cm(-1)) of water in the presence of monosaccharides and polysaccharides. In this paper, we use molecular dynamics simulations to gain atomistic insight into carbohydrate-water interactions and to specifically highlight the differences between additive (nonpolarizable) and polarizable simulations. A total of six monosaccharide systems, α and β anomers of glucose, galactose, and mannose, were studied using additive and polarizable Chemistry at HARvard Macromolecular Mechanics (CHARMM) carbohydrate force fields. Solvents were modeled using three additive water models TIP3P, TIP4P, and TIP5P in additive simulations and polarizable water model SWM4 in polarizable simulations. The presence of carbohydrate has a significant effect on the microscopic water structure, with the effects being pronounced for proximal water molecules. Notably, disruption of the tetrahedral arrangement of proximal water molecules was observed due to the formation of strong carbohydrate-water hydrogen bonds in both additive and polarizable simulations. However, the inclusion of polarization resulted in significant water-bridge occupancies, improved ordered water structures (tetrahedral order parameter), and longer carbohydrate-water H-bond correlations as compared to those for additive simulations. Additionally, polarizable simulations also allowed the calculation of power spectra from the dipole-dipole autocorrelation function, which corresponds to the IR spectra. From the power spectra, we could identify spectral signatures differentiating the proximal and bulk water structures, which could not be captured from additive simulations.

Understanding the regulation of insulin biosynthesis is important as it plays a central role in glucose metabolism. The mouse insulin gene2 (Ins2) has two splice variants; long (Ins2L) and short (Ins2S), that differ only in their 5'UTR sequence and Ins2S is the major transcript which translate more efficiently as compared to Ins2L. Here, we show that cellular factors bind preferentially to the Ins2L 5'UTR, and that PABP and HuD can bind to Ins2 splice variants and regulate its translation. In vitro binding assay with insulin 5'UTR and different HuD isoforms indicate that the 'N' terminal region of HuD is important for RNA binding and insulin translation repression. Using reporter assay we showed that specifically full-length HuD A isoform represses translation of reporter containing insulin 5'UTR. We further show that PABP and HuD interact with each other in RNA-dependent manner and this interaction is affected by glucose and PDI (5'UTR associated translation activator). These results suggest that PABP interacts with HuD in basal glucose conditions making translation inhibitory complex, however upon glucose stimulation this association is affected and PABP is acted upon by PDI resulting in stimulation of insulin translation. Together, our findings snapshot the mechanism of post-transcriptional regulation of insulin biosynthesis.

Leptin and Insulin mediated signalling have been known to play a significant role in energy utilization and body weight. Obese mice with leptin mutations were known to have several impairments but the contribution of micro RNA to the manifestation of these impairments in the Ob/ Ob mice (lacking functional leptin) were not explored in detail. miRNA play an important role in regulating obesity was indicated by the obese phenotype of the mice lacking Dicer in the PMOC neuron [1]. Leptin is an essential neurotrophic factor in early development and also reduced Leptin during early development results in obesity, thus it can be hypothesized that some of the effects of the leptin deprivation could be mediated by miRNAs. Recently Crepin and co-workers have studied differential expression of micro RNAs in the hypothalamus of the Ob/Ob mice compared to control mice [2]. In the initial screen using Taqman Low Density assay (TLDA) array they identified eleven miRNA’s that are differentially expressed and three of these miRNA (miR-200a, miR-200b and miR-429) were further analysed by quantitative PCR to confirm their over expression in Ob/Ob mice. Further, the authors have demonstrated that leptin treatment in ob/ob mice reduces the expression of miR-200a, miR-200b and miR-429 when compared to the vehicle control. miR200a was also shown to be upregulated in the db/db mice (lacking functional leptin receptors). IRS2 and Leptin receptor (ObRb) mRNA were identified as functional target of miR-200a. Using luciferase reporter assay they, have demonstrated that miR-200a targets the 3’UTR region of insulin receptor substrate 2 (IRS2) and leptin receptor (ObRb) in CHO cells. Interestingly, Commentary