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Stem cells are extraordinary cells with a unique ability of self-renewal and differentiation into various cell types such as muscle, nerve, bone, and blood cells. Historically, they have found significant applications in the biotech and pharma sectors. To grow and maintain stem cells artificially, researchers use basal media formulations supplemented with nutrients and growth factors, with Fetal bovine serum (FBS) as the key component of the culture medium. However, to maintain this supply every year, millions of pregnant cows are slaughtered for preparing FBS. The process of harvesting FBS also raises concerns about contamination with pathogens, animal proteins that may interfere with cellular behavior and ethical considerations regarding animal welfare. To overcome these limitations, here we report ClearX9-Stem™ - an affordable, sustainable, effective, and ethical replacement for an FBS-enriched stem cell culture medium. A specialized ClearX9-Stem™ cell culture medium formulation was designed to grow chicken embryonic fibroblast (SL-29) in the absence of FBS. Based on the results obtained, ClearX9-Stem™ is undergoing further refinement to meet the growing academic and industrial demand for serum-free culture media formulations. In the future, there is a need to customize and optimize ClearX9-Stem™ for the scalable growth of cells in bioreactors.HIGHLIGHTSClearX9-Stem™ provides good nutritional support for the growth of chicken embryonic fibroblast cells.ClearX9-Stem™ cell growth performance is comparable to the serum-enriched culture mediumClearX9-Stem™ maintains a healthy morphological profile of cells during divisionClearX9-Stem™ generates a stress-free environment within cellsClearX9-Stem™ does not require animal slaughter and reduces the environmental footprintClearX9-Stem™ has applications in the biotechnology, pharma, and cell-cultivated meat industries

Fetal Bovine Serum (FBS) is a nutrient-rich fluid that contains nutritional and macromolecular factors essential for cell growth. Every year millions of pregnant cows are slaughtered in search of FBS leading to huge environmental consequences. Here we report ClearX9™ - an affordable, sustainable, ethical, and effective replacement for FBS. ClearX9™ cell culture medium was used to grow HeLa (cervical cancer cells), HEK293T (embryonic kidney transformed cells) and Nthy Ori-3-1 (primary thyroid follicular transformed epithelial cells) and showed encouraging growth patterns and good cellular health. Compared with the FBS-enriched cell culture medium, ClearX9™ scored positive on all the parameters suggesting ClearX9™ as a credible alternative to FBS. In future, more work is required to establish the efficacy of ClearX9™ in toxicology testing, bio-manufacturing, regenerative medicine, and vaccine research.HIGHLIGHTSClearX9™ provides good nutritional support for the growth of animal cellsClearX9™ cell growth performance is comparable to the serum-enriched mediumClearX9™ maintains a healthy morphological profile of cells during divisionClearX9™ generates a stress-free environment within cellsClearX9™ does not require animal slaughter and reduces carbon footprintClearX9™ has applications in biotechnology and cell cultivated meat industry

We asked if transfer RNA (tRNA) ever got an opportunity of translating its own sequence during evolution, what would have been the function of such tRNA-encoded peptides (tREPs)? If not, could one artificially synthesize tREPs to study the corresponding functional outcomes? Here, we report a novel, first-in-the-class, chemically synthesized tREP-18 molecule originating from the

Background Despite several reports describing the dual role of miR-145 as an oncogene and a tumor suppressor in cancer, not much has been resolved and understood. Method In this study, the potential targets of miR-145 were identified bio-informatically using different target prediction tools. The identified target genes were validated in vitro by dual luciferase assay. Wound healing and soft agar colony assay assessed cell proliferation and migration. miR-145 expression level was measured quantitatively by RT-PCR at different stages of breast tumor. Western blot was used to verify the role of miR-145 in EMT transition using key marker proteins. Result Wound healing and soft agar colony assays, using miR-145 over-expressing stably transfected MCF7 cells, unraveled its role as a pro-proliferation candidate in cancerous cells. The association between miR-145 over-expression and differential methylation patterns in representative target genes (DR5, BCL2, TP53, RNF8, TIP60, CHK2, and DCR2) supported the inference drawn. These in vitro observations were validated in a representative set of nodal positive tumors of stage 3 and 4 depicting higher miR-145 expression as compared to early stages. Further, the role of miR-145 in epithelial–mesenchymal (EMT) transition found support through the observation of two key markers, Vimentin and ALDL, where a positive correlation with Vimentin protein and a negative correlation with ALDL mRNA expression were observed. Conclusion Our results demonstrate miR-145 as a pro-cancerous candidate, evident from the phenotypes of aggressive cellular proliferation, epithelial to mesenchymal transition, hypermethylation of CpG sites in DDR and apoptotic genes and upregulation of miR-145 in later stages of tumor tissues.

From the functional standpoint, broadly, three kinds of functional DNA sequences exist: one that encodes proteins, second that encodes only RNA (noncoding DNA), and third that does not transcribe at all (Noncoding DNA) historically people have paid attention to the protein-coding genes. For the last two-and-a-half decades, the noncoding RNA has taken center stage. However, the role of noncoding DNA (the dark matter of genome) is still largely unknown. We asked a simple question: How did nature decide to allocate protein-coding and RNA-coding jobs to a specific set of sequences? Did she sample all possibilities in the neighborhood of protein-coding genes, retaining good results (protein- and RNA-encoding genes), retiring not-so-relevant results (pseudogenes), and leaving some genome sequences untouched (nonexpressive genome) To answer these, we expressed some noncoding intergenic sequences from Escherichia coli into functional proteins. The success of this pilot experiment led us toward building a knowledge base that predicts the outcome of making user-defined genes from nonexpressive DNA sequences. In this chapter, the idea of lab-made genes shall be presented from the original concept to its present state where novel therapeutic molecules against cancer, infectious diseases, and neurodegenerative diseases have been found. We call this new platform “FLAGSHIP”—a novel drug discovery platform originating from the dark matter of the genome.

CRISPR-Cas9 has become a revolutionary, powerful and precise technology for genome editing in many organisms. It can be usd for precisely manipulating the cancer causing genes in order to treat it. CRISPR-Cas9 can aid to understand mechanism of tumorogenesis, drug target and potential solution for cell based therapy. Here, we describe current implementation of the CRISPR-Cas9 technology for accelerating the cancer research and its therapy. In this chapter, we further highlight the involvements of non coding region of genome in the progression of malignancy of cancer. We also provide an overview of challenges and advancement needed in CRISPR-Cas9 technology for cancer treatments.

Additional file of miR-24-2 controls H2AFX expression regardless of gene copy number alteration and induces apoptosis by targeting antiapoptotic gene BCL-2: a potential for therapeutic intervention

Additional file 1: Supplementary tables. Table S1. Bioinformatics prediction of microRNA targeting H2AFX transcript. Table S2. Normalization factor for each tissue sample calculated using geNorm software and the normalized expression values for H2AX. Table S3. Primer sequence for cloning the predicted miR-24-2 binding site in pGL3 vector. (DOC 80 KB)

Additional file 2: Supplementary figures. Figure S1. Expression analysis of microRNA (a) hsa-miR-145 and (b) hsa-miR-24-2 in sporadic breast cancer samples. Figure S2. TaqMan real-time confirmation of overexpression of miR-24-2 in MCF-7 cells after transfection with different concentration of precursor miR-24-2 oligonucleotides (10, 25 and 50 nmol/l). RNU 44 as an endogenous control shows amplification at same cycle threshold value. Figure S3. Bioinformatics analysis of miR-24-2 binding sites in transcripts of H2AFX, BCL-2 and MDM2 genes. Figure S4. Fluorescence-activated cell sorting (FACS) analysis of annexin V-stained MCF-7 cells treated with cisplatin (200 mmol/l). Figure S5. FACS analysis of annexin V-stained MCF-7 cells treated with H2O2 (25 mmol/l). Figure S6. Luciferase expression in HepG2 cells overexpressing miR-24-2 and transfected with pGL3 control vector or vector harboring the predicted miR-24-2 binding site present in 3'UTR of H2AFX/BCL-2 genes. Figure S7. Downregulation of miR-24-2 in MCF-7 cells increases the expression of (a) H2AFX and (b) BCL2 genes. Figure S8. Comparison of miR-24-2 overexpression at 48 hours and 72 hours posttransfection with pre-miR-24-2 (50 nmol/l). (DOC 962 KB)

The present study on the basis of a detailed bioinformatics analysis proposed a potential role of a miRNA, hsa-miR-19b-2-5p, in regulating the mitochondrial biogenesis. The miRNA has shown to be involved in important biological processes of cellular metabolic, cellular macromolecule biosynthetic processes and gene expression pathways. The miRNA, hsa-miR-19b-2-5p, was predicted to regulate the molecular function of nucleic acid, organic/heterocyclic compound, nucleic acid binding transcription factor activity. The pathway enrichment analysis suggested that this miRNA participated in several metabolic pathways which could be a key to the regulation of the mitochondrial gene expression and biogenesis. In addition, this miRNA targets a total of 112 mitochondria-related genes, establishing further the crucial role of the candidate miRNA in mitochondrial biology.

MicroRNAs (miRNAs), are small non-coding RNAs of approximately 22 nucleotides in length, playing an important role in regulating gene expression post-transcriptionally. Understanding the effect of miRNA regulation in a pathway-specific manner unravels the approaches adopted to apprehend biological mechanisms, the information, which is scanty for researchers, not primed already for miR related research. Here, we describe a quick perspective in 5 steps with probable approaches and assays at every level to unravel the specific role of a microRNA, miR-145a-5p, as an example. This perspective as a guide would help in identifying novel targets for a microRNA, as shown for miR-145a-5p, which down-regulated the mRNA expression of ADD3 and BRCA2, using bioinformatic tools and experimental assays.

Regulation of metastasis continues to remain enigmatic despite our improved understanding of cancer. Identification of microRNAs associated with metastasis in the recent past has provided a new hope. Here, we show how microRNA-101 (miR-101) regulates two independent processes of cellular metastasis by targeting pro-metastatic upstream regulatory transcription factors, ZEB1 and ZEB2, and downstream effector-actin modulators, RHOA and RAC1, providing a single target for therapeutic intervention. Further, we depict how down-regulation of miR-101 by extracellular signal-regulated kinase-2 (ERK2) is vital for MAP kinase pathway induced cellular migration and mesenchymal transition. Importantly, EKR2 induced expression of ZEB1 seems essential for down-regulation of miR-101-1 and induction of EMT. Given the role of EMT in metastasis, we also observe a significant correlation between miR-101 expression and lymph node metastasis; and identify the ERK2-ZEB1-miR-101-1 pathway active in breast cancer tissues, with an apparent clinicopathological implication.

Additional file 1: Table S1. Percentage methylation values of 10 CpG positions: identified to be up regulated or down regulated in association with miR-145 expression under both in-vitro (Mock, miR-145) and in-vivo (Stage 1 + 2, Stage 3 + 4, Node 0, Node 1, Node 2, Node 3) and conditions.

MicroRNAs (miRNAs) are small non-coding RNA molecules of around 22 nucleotides in length. They are crucially involved in the post transcriptional regulation and thus play a significant role in the modulation of different diseases. Several studies have suggested that miRNA expression is dysregulated in various cancers through different mechanisms and the dysregulated miRNA in return affects different cancer hallmarks including cell proliferation, cell death suppression, metastasis and angiogenesis. Compilation of the available miRNA data can be a stimulator for proper understanding of the correlation between the miRNA expression and cancer progression. In this review, we have focussed on the role of miR-760 in the progression of different cancer. MicroRNA-760 (miR-760) has been found to be down regulated in various cancers, thus it can be utilized as a possible prognostic marker for cancer detection. Here, we have tried to fill a gap regarding the role of miR-760 in relation to cervical cancer also. Moreover, unravelling the role of miR-760 in different cancers will enlighten the researchers with proper understanding of biology of miR-760 in regulation of different cancers.

MicroRNAs the small (18–22 in length) noncoding RNA molecules are negative regulators of gene expression, modulating biological processes of cell differentiation, survival and death. The latter two phenomena are critical in tumour biology. We provide here the results of human genome wide target prediction of one such microRNA, hsa-miR-24-2, shown to target genes essential for initiating cellular stability and cell survival. The protein–protein interaction study showed important nodes which could affect cell cycle progression and differential oncogenesis. An analysis of hsa-miR-24-2 in sporadic breast tumours showed a negative correlation with metastasis and increasing nodes. The conclusion drawn of hsa-miR-24-2 targeting the genes of cell survival correlated with the methylation profile and resultant transcription factor binding site gain or loss in support of absence of cell survival. In order to accentuate the potential of hsa-miR-24-2 to reduce cellular viability under experimental conditions, in vitro studies in the presence and absence of anti-cancer drugs, such as docetaxel resulted in a significant decrease in cellular viability even at a 200-fold reduced dose of the drug in combination with hsa-miR-24-2.

mtDNA non-synonymous germ line variation (G10398A; p.A114T) has remained equivocal with least mechanistic understanding in showing an association with cancer. This has necessitated showing in-vitro how an over-expression within mitochondria of either of the variants produces higher intracellular ROS, resulting in differential anchorage dependent and independent growth. Both these features were observed to be relatively higher in ND3:114T variant. An elevated amount of intracellular carbonylated proteins and a reduced activity of a key glycolytic enzyme, Pyruvate kinase M2, along with high glucose uptake and lactate production were other pro-cancerous features observed. The retrograde signaling through surplus ROS was generated by post-ND3 over-expression regulated nuclear gene expression epigenetically, involving selectively the apoptotic-DDR-pathways. The feature of ND3 over-expression, inducing ROS mediated pro-cancerous features in the cells in in vitro, was replicated in a pilot study in a limited number of sporadic breast tumors, suggesting the importance of mitochondrial germ-line variant(s) in enabling the cells to acquire pro-cancerous features.

Aim: We proposed to investigate the combination effect of microRNA, nutraceuticals and drug (MND), in two pancreatic cancer cell lines to assess the therapeutic potential. Materials and methods: MIA PaCa-2 and PANC-1 cells transfected with miR-101 or miR-24-2 were treated with Betulinic acid or Thymoquinone and gemcitabine independently and in combination and assessed for the extent of synergism in both experimental and control conditions, considering significance at the p value of

Gemcitabine, an effective agent in treatment of cancer of pancreas, has undergone failures in many instances after multiple cycles of therapy due to emergence of drug resistance. Combination of dietary compounds with clinically validated drugs has emerged as an effective therapeutic approach to treat pancreatic tumors, refractory to gemcitabine therapy. In order to optimize a possible synergistic combination of Gemcitabine (GCB) with dietary molecules, Betuilnic acid (BA) and Thymoquinone (TQ), stand-alone IC50 dose of GCB, BA and TQ was calculated for pancreatic cancer cell lines. Fixed IC50 dose ratio of the dietary molecules in combination with reduced IC50 dose of GCB was tested on GCB resistant PANC-1 and sensitive MIA PaCa-2 cells for synergism, additive response and antagonism, using calcusyn. Combination index (CI) revealed that pre-treatment of BA and TQ along with GCB synergistically inhibited the cancer cell proliferation in in-vitro experiments. Pyruvate kinase (PK) M2 isoform, a promising target involved in cancer cell metabolism, showed down-regulation in presence of TQ or BA in combination with GCB. GCB with BA acted preferentially on tumor mitochondria and triggered mitochondrial permeability transition. Pre-exposure of the cell lines, MIA PaCa-2 and PANC-1, to TQ in combination with GCB induced apoptosis. Thus, the effectiveness of BA or TQ in combination with GCB to inhibit cell proliferation, induce apoptosis and down-regulate the expression of PKM2, reflects promise in pancreatic cancer treatment.

Moderately increased DNA damage due to the exogenous miR-101 (4 fold) over-expression in MCF7 cells was substantiated by an increase in the number of γ-H2AX foci, correlating with a simple-to-do Halo-assay. miR-101 induced mild/moderate DNA damage favoured senescence rather than apoptosis. An experimental support emanated from the induced mild/moderate DNA damage with 1 µM/5 µM etoposide in MCF7 cells, which resulted in an endogenous miR-101 over-expression (10/4 fold, respectively), followed by senescence. On the other hand, the severe DNA damage induced with 10 µM etoposide, resulted in a low (

Type 2 diabetes (T2D) is a syndrome of multiple metabolic disorders and is genetically heterogeneous. India comprises one of the largest global populations with highest number of reported type 2 diabetes cases. However, limited information about T2D associated loci is available for Indian populations. It is, therefore, pertinent to evaluate the previously associated candidates as well as identify novel genetic variations in Indian populations to understand the extent of genetic heterogeneity. We chose to do a cost effective high-throughput mass-array genotyping and studied the candidate gene variations associated with T2D in literature. In this case-control candidate genes association study, 91 SNPs from 55 candidate genes have been analyzed in three geographically independent population groups from India. We report the genetic variants in five candidate genes: TCF7L2, HHEX, ENPP1, IDE and FTO, are significantly associated (after Bonferroni correction, p

Leprosy is a chronic infectious disease caused by Mycobacterium Leprae, where the host genetic background plays an important role toward the disease pathogenesis. Various studies have identified a number of human genes in association with leprosy or its clinical forms. However, non-replication of results has hinted at the heterogeneity among associations between different population groups, which could be due to differently evolved LD structures and differential frequencies of SNPs within the studied regions of the genome. A need for systematic and saturated mapping of the associated regions with the disease is warranted to unravel the observed heterogeneity in different populations. Mapping of the PARK2 and PACRG gene regulatory region with 96 SNPs, with a resolution of 1 SNP per 1 Kb for PARK2 gene regulatory region in a North Indian population, showed an involvement of 11 SNPs in determining the susceptibility towards leprosy. The association was replicated in a geographically distinct and unrelated population from Orissa in eastern India. In vitro reporter assays revealed that the two significantly associated SNPs, located 63.8 kb upstream of PARK2 gene and represented in a single BIN of 8 SNPs, influenced the gene expression. A comparison of BINs between Indian and Vietnamese populations revealed differences in the BIN structures, explaining the heterogeneity and also the reason for non-replication of the associated genomic region in different populations., Author Summary Leprosy is a chronic granulomatous infection caused by the intracellular organism Mycobacterium leprae. The disease affects the skin and the peripheral nerves and can cause irreversible impairment of the nerve function with consequent chronic disabilities. The prevalence of leprosy has declined dramatically after the introduction of Multidrug therapy in the 1980s. However, the infection continues to survive as a major public health problem with more than 200,000 new cases reported globally every year, especially in China and India. The disease is governed by host genetic background, where several genes have been identified in association with leprosy or its clinical forms. The involvement of the PARK2 and PACRG genes with leprosy susceptibility in two distinct populations of the world, Vietnamese and Brazilian, and its non-replication in other populations suggests unravelling the reasons of heterogeneity between different population groups. The possibility of involvement of other variants and a differential LD structure for the PARK2 regulatory region in Indian populations as compared to Brazilian and Vietnamese provides an answer to the heterogeneity among associations observed previously in different population groups.

Introduction New levels of gene regulation with microRNA (miR) and gene copy number alterations (CNAs) have been identified as playing a role in various cancers. We have previously reported that sporadic breast cancer tissues exhibit significant alteration in H2AX gene copy number. However, how CNA affects gene expression and what is the role of miR, miR-24-2, known to regulate H2AX expression, in the background of the change in copy number, are not known. Further, many miRs, including miR-24-2, are implicated as playing a role in cell proliferation and apoptosis, but their specific target genes and the pathways contributing to them remain unexplored. Methods Changes in gene copy number and mRNA/miR expression were estimated using real-time polymerase chain reaction assays in two mammalian cell lines, MCF-7 and HeLa, and in a set of sporadic breast cancer tissues. In silico analysis was performed to find the putative target for miR-24-2. MCF-7 cells were transfected with precursor miR-24-2 oligonucleotides, and the gene expression levels of BRCA1, BRCA2, ATM, MDM2, TP53, CHEK2, CYT-C, BCL-2, H2AFX and P21 were examined using TaqMan gene expression assays. Apoptosis was measured by flow cytometric detection using annexin V dye. A luciferase assay was performed to confirm BCL-2 as a valid cellular target of miR-24-2. Results It was observed that H2AX gene expression was negatively correlated with miR-24-2 expression and not in accordance with the gene copy number status, both in cell lines and in sporadic breast tumor tissues. Further, the cells overexpressing miR-24-2 were observed to be hypersensitive to DNA damaging drugs, undergoing apoptotic cell death, suggesting the potentiating effect of mir-24-2-mediated apoptotic induction in human cancer cell lines treated with anticancer drugs. BCL-2 was identified as a novel cellular target of miR-24-2. Conclusions mir-24-2 is capable of inducing apoptosis by modulating different apoptotic pathways and targeting BCL-2, an antiapoptotic gene. The study suggests that miR-24-2 is more effective in controlling H2AX gene expression, regardless of the change in gene copy number. Further, the study indicates that combination therapy with miR-24-2 along with an anticancer drug such as cisplatin could provide a new avenue in cancer therapy for patients with tumors otherwise resistant to drugs.

Background Insulin is tightly associated with cancer progression; however, mechanistic insights into such observations are poorly understood. Recent studies show that metabolic transformation is critical to cancer cell proliferation. Here, we attempt to understand the role of insulin in promotion of cancer metabolism. To this end, the role of insulin in regulating glycolytic enzyme pyruvate kinase M2 (PKM2) was examined. Results We observed that insulin up-regulated PKM2 expression, through PI3K/mTOR mediated HIF1α induction, but significantly reduced PKM2 activity independent of this pathway. Drop in PKM2 activity was attributed to subunit dissociation leading to formation of low activity PKM2 oligomers, as assessed by density gradient centrifugation. However, tyrosine 105 phosphorylation of PKM2, known for inhibiting PKM2 activity, remained unaffected on insulin treatment. Interestingly, insulin-induced ROS was found responsible for PKM2 activity reduction. The observed changes in PKM2 status led to augmented cancer metabolism. Insulin-induced PKM2 up-regulation resulted in enhanced aerobic glycolysis as confirmed by PKM2 knockdown studies. Further, PKM2 activity reduction led to characteristic pooling of glycolytic intermediates and increased accumulation of NADPH; suggesting diversion of glucose flux towards macromolecular synthesis, necessary for cancer cell growth. Conclusion The study identifies new PKM2-mediated effects of insulin on cancer metabolism, thus, advancing the understanding of insulin’s role in cancer.