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The molecular basis of human papillomavirus (HPV)-mediated cellular immortalization and malignant transformation has illustrated an indispensable role of viral E6/E7-oncoproteins. However, the impact of viral-oncoproteins on the metabolic phenotype of cancer cells remains ambiguous. We showed silencing of HPV18-encoded E6/E7-oncoprotein significantly reduced glucose consumption, lactate production, ATP level and viability. Silencing of HPV18-encoded E6/E7 in HeLa cells significantly down-regulated expression and activity of HK1, HK2, LDHA, and LDHB. Interestingly, there was an increased pyruvate kinase activity due to switch in expression from PKM2 isoform to PKM1. The switch in favor of alternatively spliced isoform PKM1, was regulated by viral-E6/E7-oncoprotein by inhibiting the c-Myc/hnRNP-axis. Further, the near absence of the PKM1 protein despite an adequate amount of PKM1 mRNA in HeLa cells was due to its proteasomal degradation. Our results suggests HPV18-encoded E6/E7 driven preferential expression of PKM2 is essential to support aerobic glycolysis and cell proliferation.The online version contains supplementary material available at 10.1007/s13337-022-00776-w.

We are amid the COVID-19 pandemic, the biggest public health event in a century that has caused illness in almost 180 million persons and loss of over 4 million lives worldwide, and disrupted the social and economic life of all societies. More such crises are predicted to occur in the coming decades, and there are massive pre-existing unaddressed public health needs in all countries. It is to be anticipated that in the post-COVID world there will be greater attention to the often neglected discipline of public health.

Against the backdrop of a decrepit healthcare system, where health for all is still a ‘distant’ dream, exposing stark gaps and shortfall of crucial inputs at various levels in catering to the health needs and requirements of a large population has compelled a review of the existing system and innovative course corrections. It has invoked a functional vision based on two parallel tracks; one, to rejuvenate and efficiently manage existing healthcare infrastructure and associated healthcare professionals at the village, tehsil and district level; and, the other of providing the modern facilities of diagnosis, prognosis, and preventive investigations with requisite interventions across all sections of the society. For more precise interventions to advance health the proposed two-pronged approach would ensure and safeguard the health, and inter alia reduce morbidity and mortality. Precision medicine in public health is predicated on OMICS technologies along with big data, machine learning and artificial intelligence for disease risk predictions and management in future. The capital intensive character of such technologies does raise concerns about their affordability, demanding an inter-institutional effort to generate simple and cost-effective high-throughput technology and tools for the diagnosis of diseases. The necessity to adopt modern medical biology technologies including Genomics has been amply evident in the COVID-19 pandemic through variant characterization of SARS-CoV2, and anticipated variations in the human host for differential susceptibilities and outcome of the disease.

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.

Preferential expression of the low-activity (dimeric) M2 isoform of pyruvate kinase (PK) over its constitutively active splice variant M1 isoform is considered critical for aerobic glycolysis in cancer cells. However, our results reported here indicate co-expression of PKM1 and PKM2 and their possible physical interaction in cancer cells. We show that knockdown of either PKM1 or PKM2 differentially affects net PK activity, viability, and cellular ATP levels of the lung carcinoma cell lines H1299 and A549. The stable knockdown of PK isoforms in A549 cells significantly reduced the cellular ATP level, whereas in H1299 cells the level of ATP was unaltered. Interestingly, the PKM1/2 knockdown in H1299 cells activated AMP-activated protein kinase (AMPK) signaling and stimulated mitochondrial biogenesis and autophagy to maintain energy homeostasis. In contrast, knocking down either of the PKM isoforms in A549 cells lacking LKB1, a serine/threonine protein kinase upstream of AMPK, failed to activate AMPK and sustain energy homeostasis and resulted in apoptosis. Moreover, in a similar genetic background of silenced PKM1 or PKM2, the knocking down of AMPKα1/2 catalytic subunit in H1299 cells induced apoptosis. Our findings help explain why previous targeting of PKM2 in cancer cells to control tumor growth has not met with the expected success. We suggest that this lack of success is because of AMPK-mediated energy metabolism rewiring, protecting cancer cell viability. On the basis of our observations, we propose an alternative therapeutic strategy of silencing either of the PKM isoforms along with AMPK in tumors.

We have previously observed concomitant events of mutations in mitochondrial and nuclear genes, along with elevated reactive oxygen species (ROS) and differential methylation within the promoters of nuclear genes in tumors and in vitro experiments of tumorigenesis. These observations have made it pertinent to replicate and understand the role of acquired mitochondrial condition in tuning a cell to accomplish a pro-cancerous state. Using a codon optimized vector system for exogenous over-expression and mitochondrial localization; we have characterized here the role of over-expressed wild type mtND5 and one of its non-synonymous somatic mutation, ND5:P265H. The ectopically over-expressed ND5:P265H in mitochondria resulted in a reduced Complex I activity, generation of higher ADP/ATP ratio, reactive oxygen species (ROS) and carbonylation of proteins as compared to mock-transfected cells. Cells over-expressing mtND5 variant produced both peroxide as well as super-oxide ROS; the generation of which was dependent on the functional status of P53; modulating epigenetically the expression of key apoptosis pathway genes. The pro-cancerous phenotypes, of anchorage dependent and independent growth; increased glucose uptake and lactate production, were selectively observed only in P53 non-functional cells over-expressing mutant ND5:P265H. We propose that somatic mutation in mtND5 resulting in down-regulated complex I enzyme activity, elevated ROS and up-regulation of a set of nuclear anti-apoptotic genes epigenetically in the P53 dysfunctional cellular background, has provided a unique understanding of the molecular mechanism of mitochondrial mutation; and the concomitant existence of somatically acquired mitochondrial and nuclear p53 mutations, in cancer progression and promotion.

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.

Type 2 Diabetes Mellitus (T2DM), a multifactorial complex disorder, is emerging as a major cause of morbidity, mortality and socio-economic burden across the world. Despite huge efforts in understanding genetics of T2DM, only ∼10% of the genetic factors have been identified so far. Telomere attrition, a natural phenomenon has recently emerged in understanding the pathophysiology of T2DM. It has been indicated that Telomeres and associated pathways might be the critical components in the disease etiology, though the mechanism(s) involved are not clear. Recent Genome Wide (GWAS) and Candidate Gene Case-Control Association Studies have also indicated an association of Telomere and associated pathways related genes with T2DM. Single Nucleotide Polymorphisms (SNPs) in the telomere maintenance genes: TERT, TERC, TNKS, CSNK2A2, TEP1, ACD, TRF1 and TRF2, have shown strong association with telomere attrition in T2DM and its pathophysiology, in these studies. However, the assessment has been made within limited ethnicities (Caucasians, Han Chinese cohort and Punjabi Sikhs from South Asia), warranting the study of such associations in different ethnic groups. Here, we propose the possible mechanisms, in the light of existing knowledge, to understand the association of T2DM with telomeres and associated pathways.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with limited treatment modalities and poor prognosis. Metabolic reprogramming in cancer is considered a hallmark of therapeutic relevance. Here, we report disruption of metabolic reprogramming in TNBC cells by silibinin via modulation of EGFR-MYC-TXNIP signaling. Metabolic assays combined with LC-MS-based metabolomics revealed inhibition of glycolysis and other key biosynthetic pathways by silibinin, to induce metabolic catastrophe in TNBC cells. Silibinin-induced metabolic suppression resulted in decreased cell biomass, proliferation, and stem cell properties. Mechanistically, we identify EGFR-MYC-TXNIP as an important regulator of TNBC metabolism and mediator of inhibitory effects of silibinin. Highlighting the clinical relevance of our observations, the analysis of METABRIC dataset revealed deregulation of EGFR-MYC-TXNIP axis in TNBC and association of EGFRhigh -MYChigh -TXNIPlow signature with aggressive glycolytic metabolism and poor disease-specific and metastasis-free survival. Importantly, combination treatment of silibinin or 2-deoxyglucose (glycolysis inhibitor) with paclitaxel synergistically inhibited proliferation of TNBC cells. Together, our results highlight the importance of EGFR-MYC-TXNIP axis in regulating TNBC metabolism, demonstrate the anti-TNBC activity of silibinin, and argue in favor of targeting metabolic vulnerabilities of TNBC, at least in combination with mainstay chemotherapeutic drugs, to effectively treat TNBC patients.

The topological characteristics of biological networks enable us to identify the key nodes in terms of modularity. However, due to a large size of the biological networks with many hubs and functional modules across intertwined layers within the network, it often becomes difficult to accomplish the task of identifying potential key regulators. We use for the first time a generalized formalism of Hamiltonian Energy (HE) with a recursive approach. The concept, when applied to the Apoptosis Regulatory Gene Network (ARGN), helped us identify 11 Motif hubs (MHs), which influenced the network up to motif levels. The approach adopted allowed to classify MHs into 5 significant motif hubs (S-MHs) and 6 non-significant motif hubs (NS-MHs). The significant motif hubs had a higher HE value and were considered as high-active key regulators; while the non-significant motif hubs had a relatively lower HE value and were considered as low-active key regulators, in network control mechanism. Further, we compared the results of the HE analyses with the topological characterization, after subjecting to the three conditions independently: (i) removing all MHs, (ii) removing only S-MHs, and (iii) removing only NS-MHs from the ARGN. This procedure allowed us to cross-validate the role of 5 S-MHs, NFk-B1, BRCA1, CEBPB, AR, and POU2F1 as the potential key regulators. The changes in HE calculations further showed that the removal of 5 S-MHs could cause perturbation at all levels of the network, a feature not discernible by topological analysis alone.

Somatic mutations within mitochondrial DNA (mtDNA) encoded cytochrome c oxidase subunit I (MT-CO1 or MT-COI) are frequent in various cancer types. In addition, perturbation from orchestrated expression of mitochondrial DNA encoded genes is also associated with complex disorders, including cancer. Since codon bias and the mitochondrial translation system restricts functional characterization of over-expressed wild type or mutant mitochondrial DNA encoded genes, the codon optimization and artificial synthesis of entire MT-CO1 allowed us to over-express the wild type and one of its deleterious mutants into the mitochondria of the transfected cells. Ectopically expressed MT-CO1 was observed to efficiently express and localized to mitochondria but showed high level of aggregation under denaturing condition. Over-expression of wild type or mutant variant of MT-CO1 promoted anchorage dependent and independent proliferation potential in in-vitro experiments and introduced the cancer cell metabolic phenotype of high glucose uptake and lactate release. Reactive oxygen species generated in cells over-expressing MT-CO1 variants acted as key effectors mediating differential expression of apoptosis and DNA damage pathway related genes. High ROS generated also down-regulated the expression of global regulators of gene expression, DNMT3A and DNMT3B. The down-regulated expression of DNMTs co-related with differential methylation of the CpG islands in the promoter region of a select set of studied genes, in a manner to promote pro-cancerous phenotype. Apart from assigning the mechanistic role to the MT-CO1 variants and their perturbed expression in cancer development, the present study provides novel insights into the functional role of somatic mutations within MT-CO1 promoting cancer phenotype.

Warburg effect is an emerging hallmark of cancer cells with pyruvate kinase M2 (PKM2) as its key regulator. Curcumin is an extensively-studied anti-cancer compound, however, its role in affecting cancer metabolism remains poorly understood. Herein, we show that curcumin inhibits glucose uptake and lactate production (Warburg effect) in a variety of cancer cell lines by down-regulating PKM2 expression, via inhibition of mTOR-HIF1α axis. Stable PKM2 silencing revealed that PKM2 is required for Warburg effect and proliferation of cancer cells. PKM2 over-expression abrogated the effects of curcumin, demonstrating that inhibition of Warburg effect by curcumin is PKM2-mediated. High PKM2 expression correlated strongly with poor overall survival in cancer, suggesting the requirement of PKM2 in cancer progression. The study unravels novel PKM2-mediated inhibitory effect of curcumin on metabolic capacities of cancer cells. To the best of our knowledge, this is the first study linking curcumin with PKM2-driven cancer glycolysis, thus, providing new perspectives into the mechanism of its anticancer activity.

Cancer cells rewire metabolism to meet biosynthetic and energetic demands. The characteristic increase in glycolysis i.e. Warburg effect, now considered as a hallmark, supports cancer in various ways. To attain such metabolic reshuffle, cancer cells preferentially re-express the M2 isoform of pyruvate kinase (PKM2, M2-PK) and alter its quaternary structure to generate less-active PKM2 dimers. The relatively inactive dimers cause the accumulation of glycolytic intermediates that are redirected into anabolic pathways. In addition, dimeric PKM2 also benefits cancer cells through variety of non-glycolytic moonlight functions, such as gene transcription, protein kinase activity and redox balance. A large body of data has shown that several distinct post-translation modifications regulate PKM2 in a way that benefits cancer growth, e.g. formation of PKM2 dimers. This review discusses the recent advancements in our understanding of the variety of post-translation modifications (PTMs) and the benefits they impart to the sustenance of cancer. Understanding the PTMs in PKM2 is crucial to assess their therapeutic potential and to design novel anti-cancer strategies.

Whereas many previous studies have revealed that mitochondrial DNA (mtDNA) polymorphism T16189C is associated with the risk of cancer and Type 2 diabetes mellitus (T2DM), there are others that have disputed the same. As a result, clarity on the role of mitochondrial T16189C in these disorders is missing. The aim of this study is to evaluate the association of T16189C polymorphism with the risk of cancer and T2DM development by pooling all case-control studies available.Published studies till November 2017 were searched from PubMed, Google scholar, Google and EMBASE and isolated a total of 36 studies having 44,203 subjects (20,439 cases and 23,764 controls) based on strict inclusion and exclusion criteria. We used the statistical software "R" to calculate the Pooled Odds Ratios and 95% confidence intervals to evaluate the association of T16189C polymorphism with a possible risk towards cancer and T2DM development.From the meta-analysis, we obtained Pooled Odds Ratios using Random effect model for cancer (OR: 1.20, 95% CI: 0.96-1.49, P = 0.104) and for T2DM (OR: 1.22, 95% CI: 1.09-1.36, P = 0.0004). In the subgroup analysis with Random effect model, we found that both Asians and Caucasians were at a statistically significant risk (OR: 1.25, P 0.0001 and OR: 1.20, P 0.0001, respectively) for the development of T2DM, whereas, a statistically non-significant risk (OR: 1.28 P = 0.1965 and OR: 1.16, P = 0.1148) emerged for the development of cancer. There was no evidence of a significant publication bias (Egger's and Begg's test) in this meta-analysis. Further sensitivity analysis also demonstrated that our meta-analysis was relatively stable and credible.Individuals with 'C' allele at position 16,189 within the mitochondrial D-loop are seemingly at a higher risk of developing T2DM and cancer. However, before arriving at generalizations, it would be pertinent to conduct similar studies in different populations with larger numbers to corroborate these results, especially in cancer.

Jammu and Kashmir (J&K), the Northern most State of India, has been under-represented or altogether absent in most of the phylogenetic studies carried out in literature, despite its strategic location in the Himalayan region. Nonetheless, this region may have acted as a corridor to various migrations to and from mainland India, Eurasia or northeast Asia. The belief goes that most of the migrations post-late-Pleistocene were mainly male dominated, primarily associated with population invasions, where female migration may thus have been limited. To evaluate female-centered migration patterns in the region, we sequenced 83 complete mitochondrial genomes of unrelated individuals belonging to different ethnic groups from the state. We observed a high diversity in the studied maternal lineages, identifying 19 new maternal sub-haplogroups (HGs). High maternal diversity and our phylogenetic analyses suggest that the migrations post-Pleistocene were not strictly paternal, as described in the literature. These preliminary observations highlight the need to carry out an extensive study of the endogamous populations of the region to unravel many facts and find links in the peopling of India.

Resveratrol has been shown to exhibit its anti-cancer effect through a variety of mechanisms. Here, TIGAR (TP53-Induced Glycolysis and Apoptosis Regulator) was identified as an important target of resveratrol for exhibiting ROS-dependent-consequences on apoptosis and autophagy. Resveratrol treatment decreased TIGAR protein irrespective of cell line used. Down-regulated TIGAR protein triggered a drop in reduced-glutathione levels which resulted in sustained ROS, responsible for apoptosis and autophagy. Over-expression and silencing experiments demonstrated the importance of TIGAR in affecting the ROS-dependent anti-cancer effects of resveratrol. Resveratrol treated cells exhibited autophagy to escape apoptosis, however, chloroquine treatment along with resveratrol, blocked protective autophagy and facilitated apoptosis. Collectively, results unravel the effects of resveratrol on TIGAR in mediating its ROS dependent influence and suggest a better combination therapy of resveratrol and chloroquine for probable cancer treatment.

Metabolic changes that contribute to differentiation are not well understood. Overwhelming evidence shows the critical role of glycolytic enzyme pyruvate kinase (PK) in directing metabolism of proliferating cells. However, its role in metabolism of differentiating cells is unclear. Here we studied the role of PK in phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation in human leukemia K562 cells. We observed that PMA treatment decreased cancer-type anabolic metabolism but increased ATP production, along with up-regulated expression of two PK isoforms (PKM2 and PKR) in an ERK2-dependent manner. Interestingly, silencing of PK (PKM2 and PKR) inhibited PMA-induced megakaryocytic differentiation, as revealed by decreased expression of megakaryocytic differentiation marker CD61 and cell cycle behavior. Further, PMA-induced ATP production reduced greatly upon PK silencing, suggesting that PK is required for ATP synthesis. In addition to metabolic effects, PMA treatment also translocated PKM2, but not PKR, into nucleus. ERK1/2 knockdowns independently and together suggested the role of ERK2 in the up-regulation of both the isoforms of PK, proposing a role of ERK2-PK isoform axis in differentiation. Collectively, our findings unravel ERK2 guided PK-dependent metabolic changes during PMA induction, which are important in megakaryocytic differentiation.

Dysregulation or inhibition of apoptosis favors cancer and many other diseases. Understanding of the network interaction of the genes involved in apoptotic pathway, therefore, is essential, to look for targets of therapeutic intervention. Here we used the network theory methods, using experimentally validated 25 apoptosis regulatory proteins and identified important genes for apoptosis regulation, which demonstrated a hierarchical scale-free fractal protein-protein interaction network. TP53, BRCA1, UBIQ and CASP3 were recognized as a four key regulators. BRCA1 and UBIQ were also individually found to control highly clustered modules and play an important role in the stability of the overall network. The connection among the BRCA1, UBIQ and TP53 proteins was found to be important for regulation, which controlled their own respective communities and the overall network topology. The feedback loop regulation motif was identified among NPM1, BRCA1 and TP53, and these crucial motif topologies were also reflected in high frequency. The propagation of the perturbed signal from hubs was found to be active upto some distance, after which propagation started decreasing and TP53 was the most efficient signal propagator. From the functional enrichment analysis, most of the apoptosis regulatory genes associated with cardiovascular diseases and highly expressed in brain tissues were identified. Apart from TP53, BRCA1 was observed to regulate apoptosis by influencing motif, propagation of signals and module regulation, reflecting their biological significance. In future, biochemical investigation of the observed hub-interacting partners could provide further understanding about their role in the pathophysiology of cancer.

Nutrient utilization is dramatically altered when cells receive signals to proliferate. Characteristic metabolic changes enable cells to meet the large biosynthetic demands associated with cell growth and division. Changes in rate-limiting glycolytic enzymes redirect metabolism to support growth and proliferation. Metabolic reprogramming in cancer is controlled largely by oncogenic activation of signal transduction pathways and transcription factors. Although less well understood, epigenetic mechanisms may seem to contribute to the regulation of metabolic gene expression in cancer. Reciprocally, accumulating evidence suggests that metabolic alterations may affect the epigenome. Understanding the relation between metabolism and epigenetics in cancer cells may open new avenues for anti-cancer strategies. In multi-cellular systems, molecular signals promoting cell growth and proliferation mediate the switch between catabolism and anabolism. Both normal proliferating and cancer cells must achieve high levels of macromolecular biosynthesis to provide the raw materials needed to produce new daughter cells. From a therapeutic view point, it is of great interest to determine metabolic differences that exist between normal proliferating cells and cancer cells. Cancer cells also exhibit significant alterations in the epigenome. Recent data indicate that cellular metabolism and epigenetic phenomenon are engaged in crosstalk [1, 2]. Considering current efforts to target both cancer metabolism and epigenetics, an understanding of the relationship between these two key features is of paramount importance [3, 4]. Here we discuss the role of cellular metabolism in regulation of the epigenome. Moreover, we discuss how epigenetic changes may contribute to establish cancer-specific metabolic features.

Background Genome-wide studies have identified both human leucocyte antigen (HLA) and non-HLA regions in association with leprosy. Involvement of novel functional loci within these regions has been proposed by us earlier. Methods We investigated the role of 23 single nucleotide polymorphisms (SNPs) in IL12B and IL12RB2 in a total of 2345 individuals from India, using MassArray platform, along with the copy number variations in IL23R , IL12RB2 and IL10 genes in a representative set of 257 individuals, using real-time PCR. Results SNP rs2853694 in IL12B gene (AA vs AC+CC, p=2.6E-04, OR=1.42 (1.17–1.70)) showed an association with leprosy. Pairwise interaction analysis followed by combined analysis of multiple SNPs identified that IL12B , TNF and BTNL2-DRA inter-genic SNPs provided a major risk towards leprosy (p=2.6E-08, OR=3.94 (2.43–6.38)), showing a further increase (p=3.6E-14) for combined risk genotype interactions. On the other hand, IL12B, BAT1 , NFKBIL1 and LTA SNPs together showed a disposition towards protection (p=0.000011, OR=0.32 (0.19–0.53)) with a further increase (p=6.38E-10) for combined protective genotype-interactions. Copy number variation analysis showed an increased copy number of the IL23R gene (PB=36.4%, controls=20.2%; p=0.026) associated with the pauci-bacillary form of leprosy, which correlated with a trend towards enhanced expression in memory T cells in a preliminary observation. Conclusions The observations made here highlight the importance of interaction between specific genetic backgrounds of immune response related genes in the outcome of Mycobacterium leprae infection.

An experimentally validated set of apoptosis-regulatory proteins was subjected to network analysis, depicting a scale-free hierarchical fractal network. The power-law distribution of the various topological properties of the network revealed the fractal nature of the network, a signature of self-organization of the network where the network maintained the democratic constitution of nodes at various levels and showed the absence of the centrality-lethality control system. Even though network breakdown under the absence of the centrality-lethality rule of hub removal did not happen, the change in the topological properties of the network could be observed. Depending on the amount of change observed in topological properties, we identified a few proteins (hubs) which could be functionally important in the combinatorial apoptosis gene regulatory network. The crosstalk of these important proteins within the network along with functional modules probably tries to maintain the structural features of the network. NFKB1 was found to be the most efficient signal transducer followed by SP1. In addition, hsa-let-7a controlled the modules independently, revealing its importance in the incoherent and coherent types of feed forward loop motif analysis. The sub-modules and sub-sub-modules predicted bi-fan and multi-layer-perceptron motifs, suggesting the role of multifunctional signals in regulating apoptosis. Finally, SP1, NFKB1 and hsa-let-7a were observed to regulate apoptosis by influencing motifs, signal transduction, and module regulation, which was validated through the removal of hubs, signifying their biological importance in association with cancers.

Type 2 diabetes (T2D) is a chronic metabolic disease which shows an exponential increase in all parts of the world. However, the disease is controllable by early detection and modified lifestyle. A series of factors have been associated with the pathogenesis of diabetes, and genes are considered to play a critical role. The individual risk of developing T2D is determined by an altered genetic background of the en-zymes involved in several metabolism-related biological mechanisms, including glucose homeostasis, insulin metab-olism, the glucose and ion transporters involved in glucose uptake, transcription factors, signaling intermediates of insulin signaling pathways, insulin production and secretion, pancreatic tissue development, and apoptosis. However, many candidate genes have shown heterogeneity of associations with the disease in different populations. A possible approach to resolving this complexity and under-standing genetic heterogeneity is to delineate the physiological phenotypes one by one as studying them in combination may cause discrepancies in association studies. A systems biology approach involving regulatory proteins, transcription factors, and microRNAs is one way to understand and identify key factors in complex diseases such as T2D. Our earlier studies have screened more than 100 single nucleotide polymorphisms (SNPs) belonging to more than 60 globally known T2D candidate genes in the Indian population. We observed that genes invariably involved in the activity of pancreatic β-cells provide susceptibility to type 2 diabetes (T2D). Encouraged by these results, we attempted to delineate in this review one of the commonest physiological phenotypes in T2D, namely impaired insulin secretion, as the cause of hyperglycemia. This review is also intended to explain the genetic basis of the pathophysiology of insulin secretion in the context of variations in the SIRT1 gene, a major switch that modulates insulin secretion, and a set of other genes such as HHEX, PGC-α, TCF7L2, UCP2, and ND3 which were found to be in association with T2D. The review aims to look at the genotypic and transcriptional regulatory relationships with the disease phenotype.

BACKGROUND: Mycobacterium leprae is the etiologic pathogen that causes leprosy. The outcome of disease is dependent on the host genetic background. METHODS: We investigated the association of 51 single-nucelotide polymorphisms (SNPs) in anti-inflammatory cytokines (IL-10, TGFB1, IL-6, IL-4, and IL-13) and receptors (IL-10RA, IL-10RB, TGFBR1, TGFBR2, IL-6R, IL-4R, IL-5RA, IL-5RB, and IL-13RA1) with susceptibility to leprosy in a case-control study from New Delhi in northern India. This was followed by replication testing of associated SNPs in a geographically distinct and unrelated population from Orissa in eastern India. The functional potential of SNPs was established with in vitro reporter assays. RESULTS: Significant associations (P < .05) were observed for 8 polymorphisms (rs1800871, rs1800872, and rs1554286 of IL-10; rs3171425 and rs7281762 of IL-10RB; rs2228048 and rs744751 of TGFBR2; and rs1800797 of IL-6) with leprosy. This association was replicated for 4 SNPs (rs1554286 of IL-10, rs7281762 of IL-10RB, rs2228048 of TGFBR2, and rs1800797 of IL-6). The interaction study revealed a significantly greater association with leprosy risk than was obtained for any SNP individually. CONCLUSIONS: This study provides an interesting insight on the cumulative polygenic host component that regulates leprosy pathogenesis.

Type-specific detection of human papillomavirus (HPV) is indicated for better risk stratification and clinical management of women testing positive for HPV and for epidemiologic surveillance. MassARRAY spectrometry (MassARRAY; Sequenom) is a novel method for type-specific detection of 15 high-risk oncogenic HPV types: HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68, and -73. PreTect HPV-Proofer (Proofer; Norchip) is a type-specific assay that detects E6/E7 mRNA from five high-risk oncogenic HPV types: HPV-16, -18, -31, -33, and -45. The performance of these tests for type-specific identification of HPV was assessed with cervical specimens from 192 cases of cervical cancer in comparison with consensus MY09/MY11 PCR followed by nucleotide sequencing (consensus PCR). The overall HPV detection rates were 94.8% (95% confidence interval [CI], 91.7, 97.9), 83.3% (95% CI, 78.1, 88.5), and 86.5% (95% CI, 81.7, 91.3) for MassARRAY, Proofer, and consensus PCR, respectively. All tests were negative in six (3.1%) of the 192 cases. Considering only the specimens that contained at least one of the five types targeted by Proofer, the detection rates were 96.6%, 91.4%, and 86.9% for MassARRAY, Proofer, and consensus PCR, respectively. MassARRAY detected multiple infections in 14.1%, Proofer detected multiple infections in 3.6%, and consensus PCR failed to detect any multiple infections. The agreement was highest at 86.0% (kappa = 0.76) between MassARRAY and Proofer and lowest at 81.8% (kappa = 0.69) between Proofer and consensus PCR. In conclusion, MassARRAY is a highly sensitive and accurate method for type-specific detection of oncogenic HPV in cervical cancer, with Proofer showing impressive performance.

Iran is ethnically, linguistically and religiously diverse. However, little is known about the population genetics of Iranian religious communities.This study was performed in order to define the different paternal components of the Iranian gene pool.Fourteen Y chromosome bi-allelic markers were analysed in 130 male subjects from Assyrian, Armenian and Zoroastrian groups in comparison with 208 male subjects from three Iranian Muslim groups.Among the three Iranian Muslim groups, the Uromian people possessed a particularly close genetic relationship to the Armenian, whereas the Zoroastrian group was different from the Uromian, but had a close genetic relationship to the two other Muslim groups (Kermanian and Shirazian). The genetic results indicate a relationship between Armenian and Assyrian groups in Iran and a clear distinction of the former from the Zoroastrian group. However, Assyrians had elevated frequency (40%) of R*(xR1a) and low frequency (11%) of J.The results of this study may suggest that the Assyrian population either experienced Eurasian gene flow (possibly from Armenia) or that enforced relocations and expulsion of conquered people with different origin led to the integration of descendants with R haplogroup. This could also be due to genetic drift due to small population size and endogamy resulting from religious barriers.

Glycolysis, a central metabolic pathway, harbors evolutionary conserved enzymes that modulate and potentially shift the cellular metabolism on requirement. Pyruvate kinase, which catalyzes the last but rate-limiting step of glycolysis, is expressed in four isozymic forms, depending on the tissue requirement. M2 isoform (PKM2) is exclusively expressed in embryonic and adult dividing/tumor cells. This tetrameric allosterically regulated isoform is intrinsically designed to downregulate its activity by subunit dissociation (into dimer), which results in partial inhibition of glycolysis at the last step. This accumulates all upstream glycolytic intermediates as an anabolic feed for synthesis of lipids and nucleic acids, whereas reassociation of PKM2 into active tetramer replenishes the normal catabolism as a feedback after cell division. In addition, involvement of this enzyme in a variety of pathways, protein–protein interactions, and nuclear transport suggests its potential to perform multiple nonglycolytic functions with diverse implications, although multidimensional role of this protein is as yet not fully explored. This review aims to provide an overview of the involvement of PKM2 in various physiological pathways with possible functional implications.

Recently, TRAIL function has been elucidated beyond its known classical role of mediating cellular homeostasis and immune surveillance against transformed cells. Here, we show how CC genotype of -716 TRAIL promoter SNP rendered risk for sporadic breast cancer as compared to the CT and TT genotypes (P (recessive model) = 0.018, OR = 1.4, 95% CI = 1.1-1.9; P (allele model) = 0.010, OR = 1.3, 95% CI = 1.1-1.7). The in silico prediction of the introduction of core Sp1/Sp3-binding motif suggested the functional significance of the SNP variation. This functional implication was validated by luciferase assay in HeLa (P = 0.026), MCF-7 (P = 0.022), HepG2 (P = 0.024), and HT1080 (P = 0.030) cells and also by real-time expression studies on tumor tissues (P = 0.01), revealing the transcriptionally repressed status of -716 T when compared to -716 C allele. The SNP-SNP interactions reflected an enhanced protective effect of CT and TT genotypes with the protective genetic backgrounds of TP53-BRCA2 (P = 0.002, OR = 0.2, 95% CI = 0.1-0.6), IFNG (P = 0.0000002, OR = 0.3, 95% CI = 0.2-0.4), and common variant Casp8 (P = 0.0003, OR = 0.5, 95% CI = 0.3-0.7). Interestingly, a comparison with clinical parameters showed overrepresented CT and TT genotypes in progressing (P = 0.041) and ER/PR negative tumors (P = 0.024/0.006). This was explained by increased apoptotic index, calculated as a ratio of selected pro-apoptotic and anti-apoptotic gene expression profiles, in CC genotyped tumors, favoring either intrinsic (P = 0.008,0.018) or extrinsic (P = 0.025,0.217) pathway depending upon the ER/PR status. Our study reveals for the first time that a promoter SNP of TRAIL functionally modulates the gene and consequently its role in breast cancer pathogenesis, cautioning to consider the -716 TRAIL SNP status in patients undergoing TRAIL therapy.

This study was designed to understand the mechanism and functional implication of the two heterozygous mutations (H391Y and K422R) of human pyruvate kinase M2 isozyme (PKM(2)) observed earlier in a Bloom syndrome background. The co-expression of homotetrameric wild type and mutant PKM(2) in the cellular milieu resulting in the interaction between the two at the monomer level was substantiated further by in vitro experiments. The cross-monomer interaction significantly altered the oligomeric state of PKM(2) by favoring dimerization and heterotetramerization. In silico study provided an added support in showing that hetero-oligomerization was energetically favorable. The hetero-oligomeric populations of PKM(2) showed altered activity and affinity, and their expression resulted in an increased growth rate of Escherichia coli as well as mammalian cells, along with an increased rate of polyploidy. These features are known to be essential to tumor progression. This study provides insight in understanding the modulated role of large oligomeric multifunctional proteins such as PKM(2) by affecting cellular behavior, which is an essential observation to understand tumor sustenance and progression and to design therapeutic intervention in future.

The present study was carried out in the Sahariya tribe of Central India, which reportedly have high prevalence of pulmonary tuberculosis. Total serum LDH and its tissue specific isoforms were estimated in TB patients and matched healthy controls to test the utility of LDH as diagnostic marker for tuberculosis. About 210 sputum positive cases and 328 age and sex matched sputum negative controls were recruited. The spectrophotometeric and densitometric analysis of each LDH isoform was carried out in both cases and controls. The mean values of serum LDH were estimated and compared for each class by t-test. The statistical comparisons were made between sputum negative controls and sputum positive cases by Mann-Whitney’s U test. The spectrophotometric estimation of serum LDH revealed significant (P=0.0016) increase in its level in cases (290 IU/L) as compared to controls (248 IU/L). The densitometric analysis of individual LDH isoforms in cases and controls demonstrated significant elevation in LDH1 (P>0.05), LDH2 (P>0.05) and LDH3 (P

In this study, we attempted to understand the mechanism of regulation of the activity and allosteric behavior of the pyruvate kinase M2 enzyme and two of its missense mutations, H391Y and K422R, found in cells from Bloom syndrome patients, prone to develop cancer. Results show that despite the presence of mutations in the intersubunit contact domain, the K422R and H391Y mutant proteins maintained their homotetrameric structure, similar to the wild-type protein, but showed a loss of activity of 75 and 20%, respectively. Interestingly, H391Y showed a 6-fold increase in affinity for its substrate phosphoenolpyruvate and behaved like a non-allosteric protein with compromised cooperative binding. However, the affinity for phosphoenolpyruvate was lost significantly in K422R. Unlike K422R, H391Y showed enhanced thermal stability, stability over a range of pH values, a lesser effect of the allosteric inhibitor Phe, and resistance toward structural alteration upon binding of the activator (fructose 1,6-bisphosphate) and inhibitor (Phe). Both mutants showed a slight shift in the pH optimum from 7.4 to 7.0. Although this study signifies the importance of conserved amino acid residues in long-range communications between the subunits of multimeric proteins, the altered behavior of mutants is suggestive of their probable role in tumor-promoting growth and metabolism in Bloom syndrome patients with defective pyruvate kinase M2.

To assess how the abnormal expression of DNA damage response (DDR) genes correlate with oncogenesis, we analyzed mRNA levels of ATM-CHK2-P53 axis in 65 sporadic breast tumors by real-time PCR followed by evaluation of P53 protein and its activation status in representative samples. Univariate analysis showed a significantly higher transcript level for ATM (P = 0.002), MDM2 (P = 0.015) and p21 (P = 0.013) in stage 1 tumors when compared against those of later stages. Although p53 transcript levels showed the characteristic increase in stage 1, a fourfold increase of p53 in N3 tumors than other nodal stages (P = 0.0007) significantly increased its expression in stage 3B. The accumulated p53 at stage 3B, confirmed also at the protein level (P = 0.012), was rendered nonfunctional by reduced P53 activation (p-P53Ser15; P = 0.00007) or increased rate of mutation, substantiated further by the corresponding failure of upregulation of downstream genes, MDM2 and p21. We conclude that the alteration of DDR expression facilitates tumor progression and its possible therapeutic implications need to be studied in future.

Mitochondrial DNA alterations in recent years have been suggested as modifier events, providing a possible proliferative advantage to the tumor cells. In order to provide further insight into the process of tumorigenesis, a study of whole mitochondria genome was conducted in 134 tissue samples obtained from 2 unrelated cancers (tumor and adjacent normal tissues from 36 breast cancer and 31 esophageal squamous cell carcinoma (ESCC) patients) with known p53 somatic mutation background. Fifteen of 36 (41.66%) breast and 12 of 31 (38.71%) ESCC tumors were found to contain at least 1 mtDNA somatic mutation, which correlated significantly with the concomitant presence of somatic mutation in DNA binding domain of the p53 gene (Breast cancer, p = 0.006; ESCC, p = 0.002). Interestingly, mutations in the non D-loop region of the mtDNA contributed significantly (Breast cancer, p = 0.004; ESCC, p = 0.032) in comparison to the hotspot-D-Loop-region. The concomitant presence of mutations in p53 and mtDNA were also predominant in breast cancer tumors with poor prognosis, that is, with the advanced stage, grade and the ER/PR negativity. Also, the observation made was apparently well explained in 10398A bearing N haplogroup genetic background with increased presence of novel and pathogenic germline mutation in mtDNA. Our study suggests that the concomitant presence of somatic alteration in mtDNA and the DNA binding domain of the p53 gene facilitates cell survival and tumorigenesis, requiring specialized therapeutic intervention because of a possible resistance to conventional chemotherapy.

In this report a family is described where cerebellar ataxia occurred along with total albinism. A possibility of syntenic condition of the two genes responsible for the traits could explain the occurrence of the two conditions. The presence of two traits singly in the sibs reflects a recombinant event and suggests that the linkage is not absolute. Chromosomal study did not show any structural or numerical anomalies except in 1/50 metaphase plates scored in the proband (with cerebellar ataxia) where in chromosome No. 14 proximal intense band (21) seemed to be shifted, inv. (14) (q13q23?). The affected sibs with ataxia and albinism or ataxia alone depicted relatively a lower sister chromatid exchange (SCE) rate than their parents and age matched controls.

In addition to being a structural component of chromatin, histone H2AX also has an important role in preserving genetic integrity. The histone H2AFX gene maps to the chromosome region 11q23.2 approximately 11q23.3 that is deleted in most human cancers. Mouse model studies also have clearly shown its involvement in tumorigenesis in a dosage-dependent manner. Therefore, in this study, DNA from 65 paired sporadic breast cancer tissues was systematically screened for gene mutations and changes in gene copy numbers. Although whole H2AFX gene scans showed an absence of mutation in the studied samples, the H2AFX gene copy number was altered in 37% of tumor samples. Furthermore, a twofold reduction in gene copy number in the MCF7 cell line strongly suggests the involvement of H2AFX alteration in breast carcinogenesis. Analysis of clinicopathologic association revealed a convincing correlation with positive estrogen/progesterone receptor status. To our knowledge, this is the first report of a change in H2AFX gene copy number in human cancer.

Pyruvate phosphate dikinase (PPDK) is the key enzyme essential for the glycolytic pathway in most common and perilous parasite Entamoeba histolytica. Inhibiting the function of this enzyme could control the wide spread of intestinal infections caused by Entamoeba histolytica in humans. With this objective, we modeled the three dimensional structure of the PPDK protein. We used templates with 51% identity and 67% similarity to employ homology-modeling approach. Stereo chemical quality of protein structure was validated by protein structure validation program PROCHECK and VERIFY3D. Experimental proof available in literature along with the in silico studies indicated Lys21, Arg91, Asp323, Glu325 and Gln337 to be the probable active sites in the target protein. Virtual screening was carried out using the genetic docking algorithm GOLD and a consensus scoring function X-Score to substantiate the prediction. The small molecule libraries (ChemDivision database, Diversity dataset, Kinase inhibitor database) were used for screening process. Along with the high scoring results, the interaction studies provided promising ligands for future experimental screening to inhibit the function of PPDK in Entamoeba histolytica. Further, the phylogeny study was carried out to assess the possibility of using the proposed ligands as inhibitors in related pathogens.

Pyruvate kinase M2, an important metabolic enzyme, promotes aerobic glycolysis (Warburg effect) to facilitate cancer cell proliferation. Unravelling the status of this important glycolytic pathway enzyme under sub-lethal doses of etoposide, a commonly used anti-proliferative genotoxic drug to induce mild/moderate DNA damage in HeLa cells as a model system and discern its effect on: PKM2 expression, phosphorylation, dimer: tetramer ratio, activity and associated effects, was pertinent. Protein expression and phosphorylation of PKM2 from HeLa cells was estimated using Western blotting. Same protein lysate was also used to estimate total pyruvate kinase activity and the total dimer: tetramer content evaluated using glycerol gradient ultra-centrifugation. Intracellular PEP was estimated manually using standard curve; while NADPH was assessed by NADPH estimation kit. Unpaired t test and two-way-ANOVA was used for statistical analysis. A relative decrease in PKM2 expression and a subsequent dose and time dependent increase in Y105-phosphorylation were observed. A concomitant increase in PKM2 dimer content and Y105-phosphorylation responsible for reduced PKM2 activity promoted PEP accumulation and NADPH production, representing increased metabolic flux into PPP, a feature that favours cancer cells. It was apparent that the sub-lethal doses of etoposide induced inadequate damage to DNA in cancer cells in culture promoted pro-survival conditions due to Y105-phosphorylation of PKM2, its stable dimerization and inactivation, a unique association not known earlier, indicating what might happen in tumour revivals or recurrences.

Genotypic status of the signal peptide region of transforming growth factor beta1 (TGF-beta1) showed a significant difference in C/C-genotype frequency at +29 position (codon 10) between a range of viral hepatitis patients and controls (P = 0.009, OR = 3.15, CI = 1.29-7.678), contributed by those who were infected with hepatitis B virus (HBV) alone or HBV + hepatitis delta virus (HDV) (P = 0.003, OR = 5.0, CI = 1.78-13.97).

Transforming growth factor beta (TGF-beta) is an example for a large and still-growing family of growth factors. TGF-beta1 is known to act both as a tumour suppressor and as a stimulator of tumour progression. This study examines the relationship amongst putative enhancer, promoter, 5'-untranslated-region (UTR) and exon-1 polymorphisms of the TGF-beta1 gene (region I from -1881 to -1613; region II from -1410 to -1123, and region III from -55 to +176, as per human genome organisation (HUGO) nomenclature) in 26 breast cancer patients and 97 healthy control subjects. The germline and somatic status of the four known polymorphisms was ascertained, and a significant difference was observed for the germline C/T and T/T genotype distribution between patients and controls in comparison to C/C genotypes at position -1349 (chi2 = 6.193; P = 0.009). In addition to the somatic variations observed for some of the regions studied, in 10/26 (38%) sporadic breast cancer cases, a novel somatic mutation in codon 47 of exon 1 (GenBank accession number AY059373) was also detected in tumour samples. The risk of cancer was found to be significant (OR = 4.525) for the -1349 C/T and T/T genotype background, suggesting that this genetic background may act as a risk factor for sporadic breast cancer.

We investigated 21 affected individuals in two unrelated monilethrix families of Indian origin and identified point mutation (g.4624G>A) in the HTM motif (exon-7) of the KRTHB6 gene in all the affected members leading to E413K change in this basic keratin. The HTM motif of KRTHB1, however, showed previously unreported two allelic variants, one with three novel variations (SNPs) in cis: g.4421insT (intronic); g.4461T>C (exonic); g.4485A>G (exonic) and second with only intronic variation (SNP) (g.4421insT). Interestingly, the two distinct phenotypes of: localized severe hair defect with beaded appearance confined to the scalp of all the affected members of Family 1 and of generalized unbeaded hair defect of moderate severity in Family 2, segregated in the two families, respectively, correlating with the two separate genotypes for the functionally critical HTM region of KRTHB1 gene in the background of E413K mutation in the KRTHB6 gene. Presence of E413K mutation in the HTM of KRTHB6 gene was not observed in the background of the allelic variant with three SNPs in KRTHB1 gene in homozygous condition in all the affected members of Family 1, affected with a localized but severe form of the disease. However, the same (E413K) mutation existed in the KRTHB6 gene in the background of the allelic variant with three SNPs in the KRTHB1 gene in homozygous condition, consistently in all the affected members of Family 2, where all its affected members showed the segregation of a milder form of the disease. Presence of both E413K mutation in the KRTHB6 and the variations in the KRTHB1 genes were not observed together in randomly selected 150 unaffected controls outside the two affected families. This is also the first report of HTM mutation of KRTHB6 gene in monilethrix cases of Indian origin and the first report of SNPs in the KRTHB1 gene in literature to our knowledge.

Summary Two new single nucleotide mutations were observed within the promoter region of human interleukin-6 gene (IL-6) in the tumour sample of a patient with sporadic breast cancer, which was a somatic change. Both mutations, one at −125 (C > G) and the other at position −173 (G > T) from the translation start site, were transversions observed at new positions, not reported earlier. In addition to these two novel mutations in this patient, a known somatic polymorphism was also observed at position −174 (G > C) (from the transcription initiation site, redesignated as −236 from the translational initiation site as per the HUGO nomenclature). Further, a preliminary comparative analysis of the studied promoter region by the ‘ConsInspector 3.0’ program, where the mutated sequence (AF362378) was compared with the sequence existing in the database (Y00081), depicted the presence of the variations in putative binding sites for transcription factors such as glucocorticoid response element (GRE) and nuclear factor kappa-B (NFκ-B), which could lead to differential expression of this gene.

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.

Tyrosine phosphorylation (p-Y105) of pyruvate kinase (PK) M2, in recent years, has been suggested to facilitate Warburg effect and tumor cell growth. However, a comparison of the structural dynamics of the un-phosphorylated, the active, and the phosphorylated-at-Y105, the inactive-states, is not clear. We studied molecular dynamics of the two states to unravel these features, where phosphorylated PKM2 showed a rapid global conformation change in the initial stages of the simulation. The overall simulation identified that the phosphorylation event results in more buried and less flexible PKM2 conformation, as compared to the un-phosphorylated form, resulting in an open and closed conformation of the active site in un-phosphorylated and phosphorylated forms, respectively, due to the movement of B domain. This conformational shift in Y105-phosphorylated-PKM2 (p-Y105-PKM2) with closed active site, responsible for inhibition of PKM2 activity, was an outcome of the bending residues (117-118, 218-219, 296-297, and 301-308) within the loop connecting A and B domains and the presence of helix-loop-helix motif in A domain. The un-phosphorylated PKM2 formed a helix bend (H4) due to less fluctuation of the residue S-100; where the other end of the helix (H4) was connected to the substrate binding pocket. Further, simulation analysis showed that phosphorylation did not affect the FBP binding predominantly. We propose that p-Y105 inhibits the activity of PKM2 without influencing FBP binding directly and not allowing the open binding conformation by influencing G128, S100, G506 and gamma turn, G126 and S127 residues. Phosphorylated PKM2 was also identified to gain the transcriptional factor function which was not the case with un-phosphorylated form. These structurally important residues in PKM2 could have a bearing on cancer metabolism, since PKM2 has been implicated in the promotion of cancer in the recent past.

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 (

The DNA mismatch repair (MMR) pathway plays a prominent role in the correction of errors made during DNA replication and genetic recombination and in the repair of small deletions and loops in DNA. Mismatched nucleotides can occur by replication errors, damage to nucleotide precursors, damage to DNA, or during heteroduplex formation between two homologous DNA molecules in the process of genetic recombination. Defects in MMR can precipitate instability in simple sequence repeats (SSRs), also referred to as microsatellite instability (MSI), which appears to be important in certain types of cancers, both spontaneous and hereditary. Variations in the highly polymorphic alleles of specific microsatellite repeats can be identified using PCR with primers derived from the unique flanking sequences. These PCR products are analyzed on denaturing polyacrylamide gels to resolve differences in allele sizes of2 bp. Although (CA)n repeats are the most abundant class among dinucleotide SSRs, trinucleotide and tetranucleotide repeats are also frequent. These polymorphic repeats have the advantage of producing band patterns that are easy to analyze and can be used as an indication of a possible MMR defect in a cell. The presumed association between such allelic variation and an MMR defect should be confirmed by molecular analysis of the structure and/or expression of MMR genes.

Single-strand conformation polymorphism (SSCP) for screening mutations/single-nucleotide polymorphisms (SNPs) is a simple, cost-effective technique, saving an expensive exercise of sequencing each and every polymerase chain reaction product and assisting in choosing only the amplicons of interest with expected mutations. The principle of detection of small changes in DNA sequences is based on changes in single-strand DNA conformations. The changes in electrophoretic mobility that SSCP detects are sequence dependent. The limitations faced in SSCP range from routine polyacrylamide gel electrophoresis (PAGE) problems to the problems of resolving mutant DNA bands. Both these problems can be solved by controlling PAGE conditions and by varying physical and environmental conditions such as pH, temperature, voltage, gel type and percentage, addition of additives or denaturants, and others. Despite much upgrading of the technology for mutation detection, SSCP remains the method of choice to analyze mutations and SNPs in order to understand genomic variations, both spontaneous and induced, and the genetic basis of diseases.