Marine bacterial isolates inhibit biofilm formation and disrupt mature biofilms of Pseudomonas aeruginosa PAO1

According to the Centers for Disease Control and Prevention, biofilms cause 65% of infections in developed countries. Pseudomonas aeruginosa biofilm cause life threatening infections in cystic fibrosis infection and they are 1,000 times more tolerant to antibiotic than the planktonic cells. As quorum sensing, hydrophobicity index and extracellular polysaccharide play a crucial role in biofilm formation, extracts from 46 marine bacterial isolates were screened against these factors in P. aeruginosa. Eleven extracts showed antibiofilm activity. Extracts of S6-01 (Bacillus indicus = MTCC 5559) and S6-15 (Bacillus pumilus = MTCC 5560) inhibited the formation of PAO1 biofilm up to 95% in their Biofilm Inhibitory Concentration(BIC) of 50 and 60 μg/ml and 85% and 64% in the subinhibitory concentrations (1/4 and 1/8 of the BIC, respectively). Furthermore, the mature biofilm was disrupted to 70–74% in their BIC. The antibiofilm compound from S6-15 was partially purified using solvent extraction followed by TLC and silica column and further characterized by IR analysis. Current study for the first time reveals the antibiofilm and antiquorum-sensing activity of B. pumilus, B. indicus, Bacillus arsenicus, Halobacillus trueperi, Ferrimonas balearica, and Marinobacter hydrocarbonoclasticus from marine habitat.     

Role of EGF receptor transactivation in phosphoinositide 3-kinase-dependent activation of MAP kinase by GPCRs

Many G protein coupled receptors (GPCRs) cause phosphorylation of MAP kinases through transactivation of the epidermal growth factor receptor (EGF-R), leading to increased cell survival and growth, motility, and migration. Phosphoinositide 3-kinase (PI3K) is one of the important cell survival signaling molecules activated by EGF-R stimulation. However, the extent to which EGF-R transactivation is essential for GPCR agonist-stimulated PI3K activation is not known. Here we examined the mechanism of PI3K activation that elicits GPCR-mediated ERK1/2 activation by pathways dependent and/or independent of EGF-R transactivation in specific cell types. Immortalized hypothalamic neurons (GT1-7 cells) express endogenous gonadotropin-releasing hormone receptors (GnRH-R) and their stimulation causes marked phosphorylation of ERK1/2 and Akt (Ser 473) through transactivation of the EGF-R and recruitment of PI3K. In C9 hepatocytes, agonist activation of AT1 angiotensin II (AT1-R), lysophosphatidic acid (LPA), and EGF receptors caused phosphorylation of Akt through activation of the EGF-R in a PI3K-dependent manner. However, ERK1/2 activation by these agonists in these cells was independent of PI3K activation. In contrast, agonist stimulation of HEK 293 cells stably expressing AT1-R caused ERK1/2 phosphorylation that was independent of EGF-R transactivation but required PI3K activation. LPA signaling in these cells showed partial and complete dependence on EGF-R and PI3K, respectively. These data indicate that GPCR-induced ERK1/2 phosphorylation is dependent or independent of PI3K in specific cell types, and that the involvement of PI3K during ERK1/2 activation is not dependent solely on agonist-induced transactivation of the EGF-R.     

Estimating overdispersion in sparse multinomial data

Multinomial data arise in many areas of the life sciences, such as mark-recapture studies and phylogenetics, and will often by overdispersed, with the variance being higher than predicted by a multinomial model. The quasi-likelihood approach to modeling this overdispersion involves the assumption that the variance is proportional to that specified by the multinomial model. As this approach does not require specification of the full distribution of the response variable, it can be more robust than fitting a Dirichlet-multinomial model or adding a random effect to the linear predictor. Estimation of the amount of overdispersion is often based on Pearson's statistic X² or the deviance D. For many types of study, such as mark-recapture, the data will be sparse. The estimator based on X² can then be highly variable, and that based on D can have a large negative bias. We derive a new estimator, which has a smaller asymptotic variance than that based on X², the difference being most marked for sparse data. We illustrate the numerical difference between the three estimators using a mark-recapture study of swifts and compare their performance via a simulation study. The new estimator has the lowest root mean squared error across a range of scenarios, especially when the data are very sparse.     

Structural insight into the inhibition of acetylcholinesterase by 2,3,4, 5-tetrahydro-1, 5-benzothiazepines

Benzothiazepines 1–3 inhibited acetylcholinesterase (AChE; EC 3.1.1.7) enzyme in a concentration-dependent fashion with IC₅₀ values of 1.0 ± 0.002, 1.2 ± 0.005 and 1.3 ± 0.001 μM, respectively. By using linear-regression equations, Lineweaver-Burk, Dixon plots and their secondary replots were constructed which indicated that compounds 1–3 are non-competitive inhibitors of AChE with K_i values of 0.8 ± 0.04, 1.1 ± 0.002, and 1.5 ± 0.001 μM, respectively. Molecular docking studies revealed that all the compounds are completely buried inside the aromatic gorge of AChE, extending deep into the gorge of AChE. A comparison of the docking results of compounds 1–3 displayed that these compounds generally adopt the same binding mode in the active site of AChE. The superposition of the docked structures demonstrated that the non-flexible benzothiazepine always penetrate into the aromatic gorge through the six-membered ring A, which allowed the ligands to interact simultaneously with more than one subsites of the active center of AChE. The higher AChE inhibitory potential of compounds 1–3 was found to be the cumulative effect of hydrophobic contacts and π-π interactions between the ligands and AChE. The relatively high affinity of benzothiazepine 1 with AChE was found to be due to additional hydrogen bond in benzothiazepine 1-AChE complex. The results indicated that substitution of halogen and methyl groups by hydrogen at aromatic ring of the benzothiazepine decreased the affinity of these molecules towards enzyme that may be due to the polar non-polar repulsions of these moieties with the amino acid residues in the active site of AChE. The observed binding modes of benzothiazepines 1–3 in the active site of AChE explain the affinities of benzothiazepines and provide a rational basis for the structure-based drug design of benzothiazepines with improved pharmacological properties.     

Exploring genome-wide DNA methylation profiles altered in hepatocellular carcinoma using Infinium HumanMethylation 450 BeadChips

Hepatocellular carcinoma (HCC) incidence has increased in the US and also has one of the fastest growing death rates of any cancer. The purpose of the current study was to discover novel genome-wide aberrant DNA methylation patterns in HCC tumors that are predominantly HCV-related. Infinium HumanMethylation 450K BeadChip arrays were used to examine genome-wide DNA methylation profiles in 66 pairs of HCC tumor and adjacent non-tumor tissues. After Bonferroni adjustment, a total of 130,512 CpG sites significantly differed in methylation level in tumor compared with non-tumor tissues, with 28,017 CpG sites hypermethylated and 102,495 hypomethylated in tumor tissues. Absolute tumor/non-tumor methylation differences ≥ 20% were found in 24.9% of the hypermethylated and 43.1% of the hypomethylated CpG sites; almost 10,000 CpG sites have ≥ 30% DNA methylation differences. Most (60.1%) significantly hypermethylated CpG sites are located in CpG islands, with 21.6% in CpG shores and 3.6% in shelves. In contrast, only a small proportion (8.2%) of significantly hypomethylated CpG sites are situated in islands, while most are found in open sea (60.2%), shore (17.3%) or shelf (14.3%) regions. A total of 2,568 significant CpG sites (2,441 hypermethylated and 127 hypomethylated) covering 589 genes are located within 684 differentially methylated regions defined as regions with at least two significant CpG sites displaying > 20% methylation differences in the same direction within 250-bp. The top 500 significant CpG sites can significantly distinguish HCC tumor from adjacent tissues with one misclassification. Within adjacent non-tumor tissues, we also identified 75 CpG sites significantly associated with gender, 228 with HCV infection, 17,207 with cirrhosis, and 56 with both HCV infection and cirrhosis after multiple comparisons adjustment. Aberrant DNA methylation profiles across the genome were identified in tumor tissues from US HCC cases that are predominantly related to HCV infection. These results demonstrate the significance of aberrant DNA methylation in HCC tumorigenesis.     

KIT Proto-oncogene Exon 8 Deletions at Codon 419 are Highly Frequent in Acute Myeloid Leukaemia with Inv(16) in Indian Population

The KIT gene is a receptor tyrosine kinase class III expressed by early hematopoietic progenitor cells and plays a significant role in hematopoietic stem cell proliferation, differentiation and survival which is considered to be a remarkable feature in the course of growth of acute myeloid leukaemia (AML). Owing to insufficient study of mutations in the KIT gene, the diagnosis and rate of recurrence of these mutations with divergent subtypes in AML cases in India is of concern. In order to find out the frequency of mutations of KIT gene exon 8 in 109 AML cases, we have performed polymerase chain reaction–single-strand conformation polymorphism (PCR–SSCP) followed by DNA sequencing and have identified 24 mutations in exon 8 in 13 cases, including deletions at codon 418 (n = 3), 419 (n = 11) and 420 (n = 5) as well as point mutations at codon 417 (n = 1) and 421 (n = 4). In eleven AML cases, exon 8 deletion and point mutations involved the loss at codon Asp419 immoderately conserved cross species placed in the receptor extracellular domain. Frequency elevation of the KIT proto-oncogene exon 8 deletion and point mutations in AML cases allude a crucial function for this region of the receptor extracellular domain. Thus, we report the incidence of acquired mutations in exon 8, with consistent loss at codon Asp419, in 10.09 % of AML cases in a selected Indian population.     

Distribution of Coliphages Against Four <Emphasis Type="Italic">E. Coli</Emphasis> Virotypes in Hospital Originated Sewage Sample and a Sewage Treatment Plant in Bangladesh

The distribution of coliphages infecting different Escherichia coli virotypes (EHEC, EIEC, EPEC, ETEC) and an avirulent strain (K-12) in sewage system of a hospital and a sewage treatment plant (STP) was investigated by culture-based agar overlay methods. Coliphages were found in all the samples except stool dumping site in the sewage system of the hospital and lagoon of the STP. Bacteriophage count (pfu/ml) infecting E. coli strains showed the following ascending pattern (EHEC < EIEC < EPEC < ETEC < E coli K-12) in all the collected samples except one. Phages capable of infecting avirulent E. coli K-12 strains were present in the highest number among all the examined locations. Phages specific for E. coli K-12 presented high diversity in plaque size on the bacterial lawn. Virulent E. coli specific coliphages rarely produced plaques with diameter of 1–2 mm or over. Conventional agar overlay method was found to be not satisfactory for phage community analysis from primary stool dumping site of the hospital, probably due to the presence of high concentration of antimicrobial substances. The gradual decrease seen in the five groups of coliphage quantity with the ongoing treatment process and then the absolute absence of coliphages in the outlet of the examined treatment plant is indicative of the usefulness of the treatment processes practiced there.     

Methylenetetrahydrofolate reductase C677T genetic polymorphisms and risk of leukaemia among the North Indian population

Background: Leukaemia is a heterogeneous disease in which haematopoietic progenitor cells acquire genetic lesions that lead to a block in differentiation, increased self-renewal, and unregulated proliferation. The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR), involved in folate metabolism, plays a crucial role in cells because folate availability is important for DNA integrity. The aim of this case–control study was to evaluate the association of the C677T MTHFR gene polymorphism with acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML) and chronic lymphocytic leukaemia (CLL). Materials and methods: A total of 275 leukaemia cases – including AML (n = 112), ALL (n = 81), CML (n = 43), CLL (n = 39) – and 251 age/sex-matched healthy control individuals participated in this study. MTHFR C677T polymorphisms in the cases and controls were evaluated by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Results: The average MTHFR 677CC, 677CT, 677TT genotype frequencies of total leukaemia cases were 68.73%, 19.64%, and 11.64% in cases, and 71.71%, 24.30%, and 3.98% in healthy controls, respectively. The average frequency of the MTHFR 677T allele was 21.45% among the cases compared to 16.13% among the controls. Conclusions: In the present case–control study we have observed a higher frequency of the MTHFR 677TT genotype in cases of leukaemia (AML, ALL, CML and CLL) as compared with controls; this might be due to ethnic and geographic variation. As per our findings, although the frequency of the MTHFR 677T allele is moderately high in AML, ALL and CLL, no statistically significant association was found; on the other hand statistically significant association was found in the context of CML cases.     

Association of MTHFR and PPARγ2 gene polymorphisms in relation to type 2 diabetes mellitus cases among north Indian population

Background

Type 2 diabetes mellitus is a multifactorial and polygenic disease, which is considered as a major life threatening problem all over the world. There has been a worldwide effort in the identification of susceptibility genes for type 2 diabetes mellitus and its complications. At present, adequate data is not available dealing with MTHFR (rs1801133) and PPARγ2 (rs1801282) gene polymorphisms and its association with type 2 diabetes mellitus cases among north Indian populations. Thus, we conceived the need for further studies to investigate MTHFR and PPARγ2 gene polymorphisms and their susceptibility to type 2 diabetes mellitus in north Indian population.

Materials and methods

In this study, a total 175 subjects including 87 type 2 diabetes mellitus cases and 88 controls were enrolled. MTHFR and PPARγ2 gene polymorphisms in the cases and controls were evaluated by polymerase chain reaction and restriction fragment length polymorphism (PCR–RFLP).

Results

The MTHFR gene CC, CT, TT genotype frequencies obtained were 40%, 43%, and 17% in type 2 diabetes mellitus cases and 56%, 29%, and 15% in healthy controls respectively. The OR for CC was 0.54 (95%CI 0.29–0.98, P = 0.041, χ2 = 4.18, power = 0.98), for CT 1.76 (95%CI 0.94–3.30, P = 0.07, χ2 = 3.2, power = 0.96), and for TT 1.2 (95%CI 0.53–2.70, P = 0.66, χ2 = 0.198, power = 0.76). The PPARγ2 gene GG CG, CC genotype frequencies obtained were 28%, 41%, and 31% in cases and 40%, 39%, and 21% in healthy controls respectively. OR for GG was 0.58 (95%CI 0.30–1.09, P = 0.08, χ2 = 2.9, power = 0.96), for CG 1.12 (95%CI 0.61–2.05, P = 0.71, χ2 = 0.137, power = 0.778), and for CC 1.63 (95%CI 0.82–3.23, P = 0.156, χ2 = 2.01, power = 0.92).

Conclusion

It might be recommended that MTHFR CC genotype seems to be a good marker for the early identification of population at risk of type 2 diabetes mellitus. While we have detected significant difference in allelic frequencies of PPARγ2 C (Proline) and G (Alanine), but at genotypic level significant difference was not detected in this case–control study. Further study with larger groups may be required to validate the study.     

Withanolide biosynthesis recruits both mevalonate and DOXP pathways of isoprenogenesis in Ashwagandha Withania somnifera L. (Dunal)

Withanolides are pharmaceutically important C(28)-phytochemicals produced in most prodigal amounts and diversified forms by Withania somnifera. Metabolic origin of withanolides from triterpenoid pathway intermediates implies that isoprenogenesis could significantly govern withanolide production. In plants, isoprenogenesis occurs via two routes: mevalonate (MVA) pathway in cytosol and non-mevalonate or DOXP/MEP pathway in plastids. We have investigated relative carbon contribution of MVA and DOXP pathways to withanolide biosynthesis in W. somnifera. The quantitative NMR-based biosynthetic study involved tracing of (13)C label from (13)C(1)-D-glucose to withaferin A in withanolide producing in vitro microshoot cultures of the plant. Enrichment of (13)C abundance at each carbon of withaferin A from (13)C(1)-glucose-fed cultures was monitored by normalization and integration of NMR signal intensities. The pattern of carbon position-specific (13)C enrichment of withaferin A was analyzed by a retro-biosynthetic approach using a squalene-intermediated metabolic model of withanolide (withaferin A) biosynthesis. The pattern suggested that both DOXP and MVA pathways of isoprenogenesis were significantly involved in withanolide biosynthesis with their relative contribution on the ratio of 25:75, respectively. The results have been discussed in a new conceptual line of biosynthetic load-driven model of relative recruitment of DOXP and MVA pathways for biosynthesis of isoprenoids. Key message The study elucidates significant contribution of DOXP pathway to withanolide biosynthesis. A new connotation of biosynthetic load-based role of DOXP/MVA recruitment in isoprenoid biosynthesis has been proposed.