Sameer Srivastava

Dominantly Inherited Constitutional Epigenetic Silencing of MLH1 in a Cancer-Affected Family Is Linked to a Single Nucleotide Variant within the 5′UTR

Constitutional epimutations of tumor suppressor genes manifest as promoter methylation and transcriptional silencing of a single allele in normal somatic tissues, thereby predisposing to cancer. Constitutional MLH1 epimutations occur in individuals with young-onset cancer and demonstrate non-Mendelian inheritance through their reversal in the germline. We report a cancer-affected family showing dominant transmission of soma-wide highly mosaic MLH1 methylation and transcriptional repression linked to a particular genetic haplotype. The epimutation was erased in spermatozoa but reinstated in the somatic cells of the next generation. The affected haplotype harbored two single nucleotide substitutions in tandem; c.-27C > A located near the transcription initiation site and c.85G > T. The c.-27C > A variant significantly reduced transcriptional activity in reporter assays and is the probable cause of this epimutation.

Improved method of in vitro regeneration in Leucaena leucocephala - a leguminous pulpwood tree species

Leucaena leucocephala is a fast growing multipurpose legume tree used for forage, leaf manure, paper and pulp. Lignin in Leucaena pulp adversely influences the quality of paper produced. Developing transgenic Leucaena with altered lignin by genetic engineering demands an optimized regeneration system. The present study deals with optimization of regeneration system for L. leucocephala cv. K636. Multiple shoot induction from the cotyledonary nodes of L. leucocephala was studied in response to cytokinins, thidiazuron (TDZ) and N6-benzyladenine (BA) supplemented in half strength MS (½-MS) medium and also their effect on in vitro rooting of the regenerated shoots. Multiple shoots were induced from cotyledonary nodes at varied frequencies depending on the type and concentration of cytokinin used in the medium. TDZ was found to induce more number of shoots per explant than BA, with a maximum of 7 shoots at an optimum concentration of 0.23 µM. Further increase in TDZ concentration resulted in reduced shoot length and fasciation of the shoots. Liquid pulse treatment of the explants with TDZ did not improve the shoot production further but improved the subsequent rooting of the shoots that regenerated. Regenerated shoots successfully rooted on ½-MS medium supplemented with 0.54 µM α-naphthaleneacetic acid (NAA). Rooted shoots of Leucaena were transferred to coco-peat and hardened plantlets showed ≥ 90 % establishment in the green house.

Altered promoter nucleosome positioning is an early event in gene silencing

Gene silencing in cancer frequently involves hypermethylation and dense nucleosome occupancy across promoter regions. How a promoter transitions to this silent state is unclear. Using colorectal adenomas, we investigated nucleosome positioning, DNA methylation, and gene expression in the early stages of gene silencing. Genome-wide gene expression correlated with highly positioned nucleosomes upstream and downstream of a nucleosome-depleted transcription start site (TSS). Hypermethylated promoters displayed increased nucleosome occupancy, specifically at the TSS. We investigated 2 genes, CDH1 and CDKN2B, which were silenced in adenomas but lacked promoter hypermethylation. Instead, silencing correlated with loss of nucleosomes from the -2 position upstream of the TSS relative to normal mucosa. In contrast, permanent CDH1 silencing in carcinoma cells was characterized by promoter hypermethylation and dense nucleosome occupancy. Our findings suggest that silenced genes transition through an intermediary stage involving altered promoter nucleosome positioning, before permanent silencing by hypermethylation and dense nucleosome occupancy.

Biomedical Perspective of Electrochemical Nanobiosensor

Electrochemical biosensor holds great promise in the biomedical area due to its enhanced specificity, sensitivity, label-free nature and cost effectiveness for rapid point-of-care detection of diseases at bedside. In this review, we are focusing on the working principle of electrochemical biosensor and how it can be employed in detecting biomarkers of fatal diseases like cancer, AIDS, hepatitis and cardiovascular diseases. Recent advances in the development of implantable biosensors and exploration of nanomaterials in fabrication of electrodes with increasing the sensitivity of biosensor for quick and easy detection of biomolecules have been elucidated in detail. Electrochemical-based detection of heavy metal ions which cause harmful effect on human health has been discussed. Key challenges associated with the electrochemical sensor and its future perspectives are also addressed.

Probing the active site of cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala.

Lack of three dimensional crystal structure of cinnamoyl CoA reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles. Mutagenesis of S136, Y170 and K174 showed complete loss of activity, indicating their pivotal roles in catalysis. Mutant S212G exhibited the catalytic efficiencies less than 10% of wild type, showing its indirect involvement in substrate binding or catalysis. R51G, D77G, F30V and I31N double mutants showed significant changes in Km values, specifying their roles in substrate binding. Finally, chemical modification and substrate protection studies corroborated the presence Ser, Tyr, Lys, Arg and carboxylate group at the active site of Ll-CCRH1.

Integrated Genetic, Epigenetic, and Transcriptional Profiling Identifies Molecular Pathways in the Development of Laterally Spreading Tumors

Laterally spreading tumors (LST) are colorectal adenomas that develop into extremely large lesions with predominantly slow progression to cancer, depending on lesion subtype. Comparing and contrasting the molecular profiles of LSTs and colorectal cancers offers an opportunity to delineate key molecular alterations that drive malignant transformation in the colorectum. In a discovery cohort of 11 LSTs and paired normal mucosa, we performed a comprehensive and unbiased screen of the genome, epigenome, and transcriptome followed by bioinformatics integration of these data and validation in an additional 84 large, benign colorectal lesions. Mutation rates in LSTs were comparable with microsatellite-stable colorectal cancers (2.4 vs. 2.6 mutations per megabase); however, copy number alterations were infrequent (averaging only 1.5 per LST). Frequent genetic, epigenetic, and transcriptional alterations were identified in genes not previously implicated in colorectal neoplasia (ANO5, MED12L, EPB41L4A, RGMB, SLITRK1, SLITRK5, NRXN1, ANK2). Alterations to pathways commonly mutated in colorectal cancers, namely, the p53, PI3K, and TGFβ pathways, were rare. Instead, LST-altered genes converged on axonal guidance, Wnt, and actin cytoskeleton signaling. These integrated omics data identify molecular features associated with noncancerous LSTs and highlight that mutation load, which is relatively high in LSTs, is a poor predictor of invasive potential. Implications: The novel genetic, epigenetic, and transcriptional changes associated with LST development reveal important insights into why some adenomas do not progress to cancer. The finding that LSTs exhibit a mutational load similar to colorectal carcinomas has implications for the validity of molecular biomarkers for assessing cancer risk. Mol Cancer Res; 14(12); 1217–28. ©2016 AACR.

Genetic and epigenetic markers in colorectal cancer screening: recent advances

ABSTRACT Introduction: Colorectal cancer (CRC) is a heterogenous disease which develops from benign intraepithelial lesions known as adenomas to malignant carcinomas. Acquired alterations in Wnt signaling, TGFβ, MAPK pathway genes and clonal propagation of altered cells are responsible for this transformation. Detection of adenomas or early stage cancer in asymptomatic patients and better prognostic and predictive markers is important for improving the clinical management of CRC. Area covered: In this review, the authors have evaluated the potential of genetic and epigenetic alterations as markers for early detection, prognosis and therapeutic predictive potential in the context of CRC. We have discussed molecular heterogeneity present in CRC and its correlation to prognosis and response to therapy. Expert commentary: Molecular marker based CRC screening methods still fail to gain trust of clinicians. Invasive screening methods, molecular heterogeneity, chemoresistance and low quality test samples are some key challenges which need to be addressed in the present context. New sequencing technologies and integrated omics data analysis of individual or population cohort results in GWAS. MPE studies following a GWAS could be future line of research to establish accurate correlations between CRC and its risk factors. This strategy would identify most reliable biomarkers for CRC screening and management.

Genetic Engineering of Phenylpropanoid Pathway in Leucaena leucocephala

The phenylpropanoid pathway is responsible for the biosynthesis of a variety of products that include lignin, flavonoids and hydroxycinnamic acid conjugates. Many intermediates and end products of this pathway play important role in plants as phytoalexins, antiherbivory compounds, antioxidants, ultra-violet (UV) protectants, pigments and aroma compounds. Lignin has far reaching impacts on agriculture, industry and the environment, making phenylpropanoid metabolism a globally important part of plant biochemistry. The mechanical support provided by lignin prevents lodging, a problem in many agronomically important plants, it also provides a hydrophobic surface, essential for longitudinal water transport, and provides a barrier against pathogens. Finally the many functions of lignin and related products in resistance to biotic and abiotic stresses make the phenylpropanoid pathway vital to the health and survival of plants. Besides its critical role in normal plant health and development, high lignin levels are problematic in the agro-industrial exploitation of various plant species. Lignin is considered an undesirable component in paper manufacture, and has a negative impact on forage crop digestibility. Leucaena leucocephala is one of the most versatile, fast growing commercially important trees for paper and pulp industry in India, contributing 1/4th of the total raw material. Lignin composition, quantity and distribution are known to affect the agro-industrial utilization of plant biomass. High quantity and low Syringyl (S) to Guaiacyl (G) lignin ratio plays a detrimental role in economy and ecology of paper production. Every unit increase in S/G ratio decreases the cost of paper production by two and half times. Moreover chemical processing of pulp for lignin removal releases chlorinated organic compounds in effluent, which are hazardous and a serious threat to the environment. Hence, there is currently intense interest in modifying the content and/or composition of the cell wall structural polymer (lignin) as a means of improving the efficiency of the paper pulping process for forest trees. To engineer plants with agronomically useful lignin related traits, we need to devise strategies that can flexibly and predictably yield reductions in lignin content and/or changes in lignin monomer composition. Our studies have concentrated on attempts to alter the levels of enzymes involved in early as well as late phenylpropanoid pathway, mainly by downregulation or upregulation of the phenylpropanoid pathway genes in transgenic L. leucocephala and tobacco plants. Besides, we are also working on some R2R3 type MYB transcription factors supposed to play important role in lignin biosynthesis and some other genes which are not directly involved in phenylpropanoid pathway, but are important for carrying out polymerization of monolignols (peroxidases) and defense mechanism of plants (β glucosidase: family 1 Glycosyl hydrolase). Major phenylpropanoid pathway genes (C4H, 4-CL, CCoAOMT, CCR, Cald5H and CAD) were isolated using PCR based approach. Their 5’ and 3’ UTR determined by rapid amplification of cDNA race (RACE). We could isolate multiple isoforms of most of the genes in this way well supported by Southern hybridization experiment. All the genes were expressed in E. coli and/or yeast with/without the signal sequence. The over-expressed proteins were purified using suitable methods and were used for raising polyclonal antibodies against them. The most un-conserved region of each gene was cloned in antisense orientation in suitable binary vector and L. leucocephala and tobacco explants were transformed using the antisense construct to down-regulate the targeted gene. In order to increase the S/G ratio of lignin monomers sense construct of the target gene was used. We have also done RNAi based downregulation of Cinnamate 4-Hydroxylase (C4H), a key enzyme of phenylpropanoid pathway and a member of cytochrome P450 family in tobacco. Spatio-temporal expression of each gene was studied in L. leucocephala in different tissues at different periods of their growth. Kinetics study of some of the enzymes has been carried out in our lab. We are also trying to establish the structure function correlation of some enzymes. We have also isolated promoters of some phenylpropanoid pathway genes and have identified R2-R3 type MYB binding domain(s) in them. Two MYB genes have been isolated from L. leucocephala and heterologously expressed in Escherichia coli. Their DNA binding efficiency and role in regulating phenylpropanoid pathway remain to be seen. All the isolated genes shared 70-80% homology with the already reported sequences from other species at nucleotide level and more than 80% identity at amino acid level. Different isoforms of different genes had varying degree of identity between them ranging from 80% to more than 95%. We could easily locate the ribosome binding site in the 5’ UTR and the polyadenylation signal in the 3’ UTR in all the genes. Proteins were expressed in both prokaryotic and eukaryotic system. Some of the genes were difficult to express in BL21 (DE3), primarily because of the translational incompatibility of some of the codons in E. coli and may be partly because of the signal sequence present in most of the proteins. All the genes were found to be actively expressing in lignifying tissues and roots in comparison to leaves albeit a time dependent regular expression pattern could not be drawn in case of every gene. We used three methods of genetic transformation for transferring our gene constructs to L. leucocephala embryo viz: Agrobacterium mediatd, gene gun mediated and gene gun followed by cocultivation with Agrobacterium. The transformation and regeneration efficiency varied with each protocol. The transgenic plants invariably showed stunted vigour and slow growth irrespective of the nature of the gene downregulated or upregulated. Initial screening of the transformants was done on MS medium containing appropriate antibiotic and later confirmation was done using PCR with hptII/nptII, gus specific and CaMV35S promoter specific primers. At least one tobacco plant downregulated for C4H showed rudimentary root system and curled leaves with brown tip. Transformed Leucaena and tobacco plants had reduced lignin content with varying degree in case of every downregulated gene. Histochemical staining of transverse root and stem tissue sections showed reduced lignification as evidenced by immunocytolocalization patterns of the candidate protein under study. Promoters of few lignin biosynthetic pathway genes having R2R3 type MYB-binding signal sequences (AC elements) have been cloned. Two R2R3 type MYB transcription factors from L. leucocephala that have been expressed and purified from E. coli will be used for gel retardation studies with the promoter sequences. Also in vitro synthesized oligonucleotides having the highly conserved MYB-binding motifs will be designed to determine the most probable binding sites of the two MYB proteins. Sense construct for the two MYB genes have been transformed in tobacco and antisense construct have been transformed in Leucaena plants to study the after effects of gene manipulation. In short, we have isolated and characterized several genes belonging to phenylpropanoid pathway and have expressed them in different systems. Transgenics for down-regulation or upregulation of those genes have shown very interesting results.

Anticancer Activity of Herbal Medicine: Mechanism of Action

Cancer is an alarming disease and quite lethal in nature in developed and developing nations. Many new therapeutic agents and therapies are available in the market but have some severe side effects on human beings’ organs. These therapeutic agents are quite costly and not easily available in some of the developing nations. Various scientific reports have shown that chemoprevention through naturally derived herbal and dietary phytochemicals is an innovative therapeutic tool against different cancer types. These herbal phytochemicals have shown their potential anticancer activity in both in vitro and in vivo studies. Further, many of them have been successfully proved for their chemopreventive property by inducing apoptosis equivalent to certain other chemical drugs without causing any side effects. The combinational role of herbal and dietary phytochemicals has proved to be very effective against cancer prevention. The present chapter summarised the effectiveness of herbal and dietary phytochemicals for chemoprevention and also highlighted their combinational role on various kinds of cancer.

H2O2 sensing through electrochemically deposited thionine coated ITO thin film

Progression and initiation of different diseases including pulmonary diseases, alzheimers and tumors are linked with the oxidative stress, an important cause of cell damage. Different antioxidant enzymes are involved in detoxifying reactive oxygen species including hydrogen peroxide (H2O2) that is generated in response to various stimuli and has important role in cell activation & bio-signaling processes. Herein, we developed hydrogen peroxide electrochemical sensor based on horseradish peroxidase (HRP) entrapped polymerized thionine (PTH) film. Electrochemical deposition of thionine (dye) on indium tin oxide (ITO) surface was carried out through chornoamperometry followed by cyclic voltammetry. Deposited thionine thin film obtained was checked for its stability at different scan rates. The PTH-modified electrodes showed linear dependence of peak current with scan rate within the range of 20 to 100 mV s-1. Thionine used as electron transfer mediator between heme site of HRP and electrode. Cyclic voltammetry showed increase in the reduction peak current due to electrocatalytic reduction of H2O2. The sensor detection limit range from 10-1 _ 102 µM and limit of detection was 0.1µM. The proposed sensor has good storage response, cost effective, high sensitivity and wide linear range that could be used for the fabrication of other enzyme based biosensors.