Global hypomethylation and promoter methylation in small intestinal neuroendocrine tumors: An in vivo and in vitro study

Aberrant DNA methylation is a feature of human cancer affecting gene expression and tumor phenotype. Here, we quantified promoter methylation of candidate genes and global methylation in 44 small intestinal-neuroendocrine tumors (SI-NETs) from 33 patients by pyrosequencing. Findings were compared with gene expression, patient outcome and known tumor copy number alterations. Promoter methylation was observed for WIF1, RASSF1A, CTNNB1, CXCL14, NKX2–3, P16, LAMA1, and CDH1. By contrast APC, CDH3, HIC1, P14, SMAD2, and SMAD4 only had low levels of methylation. WIF1 methylation was significantly increased (P = 0.001) and WIF1 expression was reduced in SI-NETs vs. normal references (P = 0.003). WIF1, NKX2–3, and CXCL14 expression was reduced in metastases vs. primary tumors (P < 0.02). Low expression of RASSF1A and P16 were associated with poor overall survival (P = 0.045 and P = 0.011, respectively). Global methylation determined by pyrosequencing of LINE1 repeats was reduced in tumors vs. normal references, and was associated with loss in chromosome 18. The tumors fell into three clusters with enrichment of WIF1 methylation and LINE1 hypomethylation in Cluster I and RASSF1A and CTNNB1 methylation and loss in 16q in Cluster II. In Cluster III, these alterations were low-abundant and NKX2-3 methylation was low. Similar analyses in the SI-NET cell lines HC45 and CNDT2 showed methylation for CDH1 and WIF1 and/or P16, CXCL14, NKX2-3, LAMA1, and CTNNB1. Treatment with the demethylating agent 5-azacytidine reduced DNA methylation and increased expression of these genes in vitro. In conclusion, promoter methylation of tumor suppressor genes is associated with suppressed gene expression and DNA copy number alterations in SI-NETs, and may be restored in vitro.     

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.     

Expression proteomics of acute promyelocytic leukaemia cells treated with methotrexate

Methotrexate was first introduced as a cytotoxic agent that inhibits nucleotide biosynthesis in various cancer disorders; its molecular mechanism remains elusive. To understand the molecular mechanism by which methotrexate induces apoptosis, we analyzed the resulting intracellular protein changes in methotrexate-treated acute promyelocytic leukaemia (HL-60) cells by cysteine-labeled differential in-gel electrophoresis (CL-DIGE) combined with mass spectrometry. Initial CL-DIGE analysis revealed that 24 proteins were differentially expressed (p < 0.05) in the HL-60 cell proteome after treatment with 2.5 µM methotrexate for 72 h. We found that three structural α4, α5, α7 proteasome subunits, a non-catalytic β3 and two 26S regulatory proteasome subunits were down-regulated in methotrexate-treated HL-60 cells. Western blot analyses further showed that the inhibition of proteasome subunits is accompanied by suppression of NF-κB subunits and promotes the accumulation of ubiquitinated proteins. Furthermore, methotrexate activated unfolded protein response by inducing the expression of endoplasmic reticulum-resident proteins such as calreticulin, protein disulphide isomerase A3 and A4, and 78 kDa glucose regulated protein in a time-dependent manner. Altogether, our findings demonstrated that targeting NF-κB, structural and regulatory proteasome subunits with methotrexate may provide new insight into understanding methotrexate-induced apoptotic activities in HL-60 cells.     

Suppression by Red Light Irradiation of Corynespora Leaf Spot of Cucumber Caused by Corynespora cassiicola

The effect of red light irradiation on development of Corynespora leaf spot of cucumber plants (Cucumis sativus L. cv. Hokushin) caused by Corynespora cassiicola (Berk. & Court.) was investigated in greenhouses. In a greenhouse without red light (?Red), lesions enlarged, coalesced, and finally covered the entire leaves of cucumber. In a greenhouse with red light (+Red), however, lesion appearance was delayed relative to that under ?Red and its development was also significantly suppressed. Such difference in disease development was also observed in cucumbers grown under +Red and ?Red in the same greenhouse. Disease suppression under red light was also observed in glasshouse‐grown C. cassiicola‐inoculated cucumbers. Red light did not inhibit the infection behaviour of the pathogen. Our results indicated that the delay and suppression of Corynespora leaf spot of cucumber under +Red were due to induction of resistance in cucumber, and not to differences in environmental conditions or fungal population between the two greenhouses. Red light‐induced resistance might contribute to the development of new methods for controlling Corynespora leaf spot of greenhouse‐grown cucumber.     

Conservation genomics reveals possible illegal trade routes and admixture across pangolin lineages in Southeast Asia

The use of genome-wide genetic markers is an emerging approach for informing evidence-based management decisions for highly threatened species. Pangolins are the most heavily trafficked mammals across illegal wildlife trade globally, but critically endangered Sunda pangolins (Manis javanica) have not been widely studied in insular Southeast Asia. We used?>?12,000 single nucleotide polymorphic markers (SNPs) to assign pangolin seizures from illegal trade of unknown origin to possible geographic sources via genetic clustering with pangolins of known origin. Our SNPs reveal three previously unrecognized genetic lineages of Sunda pangolins, possibly from Borneo, Java and Singapore/Sumatra. The seizure assignments suggest the majority of pangolins were traded from Borneo to Java. Using mitochondrial markers did not provide the same resolution of pangolin lineages, and to explore if admixture might explain these differences, we applied sophisticated tests of introgression using?>?2000 SNPs to investigate secondary gene flow between each of the three Sunda pangolin lineages. It is possible the admixture which we discovered is due to human-mediated movements of pangolins. Our findings impact a range of conservation actions, including tracing patterns of trade, repatriation of rescue animals, and conservation breeding. In order to conserve genetic diversity, we suggest that, pending further research, each pangolin lineage should as a precaution be protected and managed as an evolutionarily distinct conservation unit.     

Analysis of Comparative Sequence and Genomic Data to Verify Phylogenetic Relationship and Explore a New Subfamily of Bacterial Lipases

Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca²⁺-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter P_lac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65°C and retained ≥ 97% activity after incubation at 50°C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents.     

Agrobacterium rhizogenes-mediated transformation of Picrorhiza kurroa Royle ex Benth.: establishment and selection of superior hairy root clone

A protocol for induction and establishment of Agrobacterium rhizogenes-mediated hairy root cultures of Picrorhiza kurroa was developed through optimization of the explant type and the most suitable bacterial strain. The infection of leaf explants with the LBA9402 strain resulted in the emergence of hairy roots at 66.7% relative transformation frequency. Nine independent, opine and TL-positive hairy root clones were studied for their growth and specific glycoside (i.e., kutkoside and picroside I) productivities at different growth phases. Biosynthetic potentials for the commercially desirable active constituents have been expressed by all the tested hairy root clones, although distinct inter-clonal variations could be noted in terms of their quantity. The yield potentials of the 14-P clone, both in terms of biomass as well as individual glycoside contents (i.e., kutkoside and picroside I), superseded that of all other hairy root clones along with the non-transformed, in vitro-grown control roots of P. kurroa. The present communication reports the first successful establishment, maintenance, growth and selection of superior hairy root clone of Picrorhiza kurroa with desired phyto-molecule production potential, which can serve as an effective substitute to its roots and thereby prevent the indiscriminate up-rooting and exploitation of this commercially important, endangered medicinal plant species. CIMAP Publication No.: 2007-28J     

Construction and functionalization of pyranone ring fused with pyran moiety: Design and synthesis of novel pyrano[4,3-b]pyran-5(4H)-ones as potential inhibitors of sirtuins

Novel pyrano[4,3-b]pyran-5(4H)-one based small molecules were designed as potential inhibitors of sirtuins (i.e., yeast sir2, a homolog of human SIRT1). Elegant synthesis of these compounds was performed via a multi-step sequence consisting of MCR, Sandmeyer type iodination, Sonogashira type coupling followed by iodocyclization and then Pd-mediated various C–C bond forming reactions. The overall strategy involved the construction of a pyran ring followed by the fused pyranone moiety and subsequent functionalization at C-8 position of the resultant core pyrano[4,3-b]pyran-5(4H)-one framework. The crystal structure analysis of a representative iodolactonized product (6d) is presented. Some of the synthesized compounds showed promising inhibitory activities when tested against yeast sir2 in vitro. The compound 6g showed dose dependent inhibition (IC₅₀ = 78.05 μM) of yeast sir2 and good interactions with this protein in silico.