Selective cytotoxicity of Pancratistatin-related natural <Emphasis Type="Italic">Amaryllidaceae</Emphasis> alkaloids: evaluation of the activity of two new compounds

Background  

Pancratistatin (PST), a compound extracted from an Amaryllidaceae (AMD) family plant, has been shown to specifically induce apoptosis in cancer cells with no/minimal toxic effect on normal cells. A systematic synthetic approach has indicated that the minimum cytotoxic pharmacophore comprises the trans-fused b/c-ring system containing the 2, 3, 4-triol unit in the C-ring. To further explore the structure-activity relationship of this group of compounds we have investigated the anti-cancer efficacy and specificity of two PST-related natural compounds, AMD4 and AMD5. Both of these compounds lack the polyhydroxylated lycorane element of PST instead having a methoxy-substuituted crinane skeleton.     

Phylogenetic Study of Methanol Oxidizers from Chilika-Lake Sediments Using Genomic and Metagenomic Approaches

Group-wise diversity of sediment methylotrophs of Chilika lake (Lat. 19°28′–19°54′N; Long. 85°06′–85°35′E) Odisha, India at various identified sites was studied. Both the culturable and unculturable (metagenome) methylotrophs were investigated in the lake sediments employing both mxaF and 16S rRNA genes as markers. ARDRA profiling, 16S rRNA gene sequencing, PAGE profiling of HaeIII, EcoRI restricted mxaF gene and the mxaF gene sequences using culture-dependent approach revealed the relatedness of α-proteobacteria and Methylobacterium, Hyphomicrobium and Ancyclobacter sp. The total viable counts of the culturable aerobic methylotrophs were relatively higher in sediments near the sea mouth (S3; Panaspada), also demonstrated relatively high salinity (0.1 M NaCl) tolerance. Metagenomic DNA from the sediments, amplified using GC clamp mxaF primers and resolved through DGGE, revealed the diversity within the unculturable methylotrophic bacterium Methylobacterium organophilum, Ancyclobacter aquaticus, Burkholderiales and Hyphomicrobium sp. Culture-independent analyses revealed that up to 90 % of the methylotrophs were unculturable. The study enhances the general understandings of the metagenomic methylotrophs from such a special ecological niche.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0510-3) contains supplementary material, which is available to authorized users.     

CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity

CRISPR/Cas9 has emerged as a versatile genome-engineering tool that relies on a single guide RNA (sgRNA) and the Cas9 enzyme for genome editing. Simple, fast and economical methods to generate sgRNAs have made targeted mutagenesis routine in cultured cells, mice, zebrafish and other model systems. Pre-screening of sgRNAs for target efficacy is desirable both for successful mutagenesis and minimizing wasted animal husbandry on targets with poor activity. Here, we describe an easy, quick and cost-effective fluorescent polymerase chain reaction (PCR)-based method, CRISPR Somatic Tissue Activity Test (CRISPR-STAT), to determine target-specific efficiency of sgRNA. As a proof of principle, we validated our method using 28 sgRNAs with known and varied levels of germline transmission efficiency in zebrafish by analysis of their somatic activity in injected embryos. Our data revealed a strong positive correlation between the fluorescent PCR profiles of the injected embryos and the germline transmission efficiency. Furthermore, the assay was sensitive enough to evaluate multiplex gene targeting. This method is easy to implement by laboratories with access to a capillary sequencer. Although we validated the method using CRISPR/Cas9 and zebrafish, it can be applied to other model systems and other genome targeting nucleases.     

Antiproliferative and Apoptosis Inducing Effects of Non-Polar Fractions from Lawsonia inermis L. in Cervical (HeLa) Cancer Cells

Two non-polar fractions viz. hexane (Hex-LI) and chloroform fraction (CHCl₃-LI) of Lawsonia inermis were studied for their antiproliferative potential in various cancer cell lines viz. HeLa, MCF-7, A549 and C6 glioma cells. Both the fractions showed more than 60 % of growth inhibition in all the tested cell lines at highest tested concentration. In clonogenic assay, different concentrations of Hex-LI and CHCl₃-LI decreased the number and size of colonies as compared to control in HeLa cells. The apoptotic effects as nuclear condensation, fragmentation were visualized with Hoechst-33342 staining of HeLa cells using confocal microscope. Both fractions induced apoptotic cell death in human cervical carcinoma (HeLa) cells as evident from flow cytometric analysis carried out using Annexin V-FITC and propidium iodide dyes. CHCl₃-LI treated cells significantly induced apoptosis (25.43 %) in comparison to control. Results from Neutral Comet assay demonstrated that both fractions induced double stranded breaks (DSB’s) in HeLa cells. Our data indicated that Hex-LI and CHCl₃-LI treated cells showed significant increase of 32.2 and 18.56 % reactive oxygen species (ROS) levels in DCFH-DA assay respectively. Further, experimental studies to decipher exact pathway via which these fractions induce cell death are in progress.     

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.     

Dissecting electrostatic interactions in Bacillus circulans xylanase through NMR-monitored pH titrations

NMR-monitored pH titration curves of proteins provide a rich source of structural and electrostatic information. Although relatively straightforward to measure, interpreting pH-dependent chemical shift changes to obtain site-specific acid dissociation constants (pK (A) values) is challenging. In order to analyze the biphasic titrations exhibited by the side chain (13)C(γ) nuclei of the nucleophilic Glu78 and general acid/base Glu172 in Bacillus circulans xylanase, we have revisited the formalism for the ionization equilibria of two coupled acidic residues. In general, fitting NMR-monitored pH titration curves for such a system will only yield the two macroscopic pK (A) values that reflect the combined effects of both deprotonation reactions. However, through the use of mutations complemented with ionic strength-dependent measurements, we are able to extract the four microscopic pK (Ai) values governing the branched acid/base equilibria of Glu78 and Glu172 in BcX. These data, confirmed through theoretical calculations, help explain the pH-dependent mechanism of this model GH11 xylanase by demonstrating that the kinetically determined pK (A) values and hence catalytic roles of these two residues result from their electrostatic coupling.     

Microwave assisted synthesis and antimicrobial evaluation of new fused pyran derivatives bearing 2-morpholinoquinoline nucleus

A new series of fused pyran derivatives 5a-x bearing 2-morpholinoquinoline nucleus has been synthesized under microwave irradiation by a reaction of 2-morpholinoquinoline-3-carbaldehyde 2a-c, malononitrile 3 and compounds 4a-h in presence of NaOH as basic catalyst. All the compounds were screened against three Gram positive bacteria (Streptococcus pneumoniae, Clostridium tetani, Bacillus subtilis), three Gram negative bacteria (Salmonella typhi, Vibrio cholerae, Escherichia coli) and two fungi (Aspergillus fumigatus, Candida albicans) using broth microdilution MIC (Minimum Inhibitory Concentration) method. Of the compounds studied, compounds 5b, 5f, 5k, 5m, 5q, 5s and 5v have found to be most efficient members of the series.