CpG island methylation in precursors of gastrointestinal malignancies

Gastrointestinal malignancies account for about 20% of all cancers worldwide. It is widely accepted that cancer evolves through several stepwise morphological stages such as the adenoma-carcinoma and hyperplastic polyp-serrated adenoma-carcinoma sequences in colorectal cancers, and the metaplasia-dysplasia-carcinoma sequences in esophageal and gastric cancers. The morphological progression is associated with the accumulation of multiple genetic and epigenetic events. It is now recognized that epigenetic silencing of gene expression by CpG island methylation is an important alternative mechanism of inactivating tumor suppressor genes. Inflammatory conditions of the gastrointestinal and pancreaticobiliary tracts and liver such as Barrett esophagus, Helicobacter pylori gastritis, inflammatory bowel disease and viral hepatitis, are associated with increased frequency of malignancies and CpG methylation. In addition, CpG methylation is present in aberrant crypt foci and pancreatic intraepithelial neoplasia that are considered putative precursors of colon and pancreatic carcinomas, respectively. Understanding of these early genetic and epigenetic changes allows for the discoveries of potential screening, monitoring and therapeutic strategies. Targeting of the epigenetic changes that occur before the development of frank malignancy offers a potential chemopreventive strategy.     

Human Enterovirus 109: a Novel Interspecies Recombinant Enterovirus Isolated from a Case of Acute Pediatric Respiratory Illness in Nicaragua

Enteroviruses (Picornaviridae family) are a common cause of human illness worldwide and are associated with diverse clinical syndromes, including asymptomatic infection, respiratory illness, gastroenteritis, and meningitis. In this study, we report the identification and complete genome sequence of a novel enterovirus isolated from a case of acute respiratory illness in a Nicaraguan child. Unbiased deep sequencing of nucleic acids from a nose and throat swab sample enabled rapid recovery of the full-genome sequence. Phylogenetic analysis revealed that human enterovirus 109 (EV109) is most closely related to serotypes of human enterovirus species C (HEV-C) in all genomic regions except the 5′ untranslated region (5′ UTR). Bootstrap analysis indicates that the 5′ UTR of EV109 is likely the product of an interspecies recombination event between ancestral members of the HEV-A and HEV-C groups. Overall, the EV109 coding region shares 67 to 72% nucleotide sequence identity with its nearest relatives. EV109 isolates were detected in 5/310 (1.6%) of nose and throat swab samples collected from children in a pediatric cohort study of influenza-like illness in Managua, Nicaragua, between June 2007 and June 2008. Further experimentation is required to more fully characterize the pathogenic role, disease associations, and global distribution of EV109.The genus Enterovirus (EV) in the family Picornaviridae is a group of related viruses that are associated with a spectrum of disease, ranging from subclinical infections to acute respiratory and gastrointestinal illness to more severe manifestations, such as aseptic meningitis, encephalitis, and acute flaccid paralysis (16, 32). Enteroviruses are small, nonenveloped viruses that share a genomic organization. The RNA genome is a ∼7.5 kb single-stranded, positive-sense, polyadenylated molecule, with a single, long open reading frame flanked by 5′ and 3′ untranslated regions (UTRs). The 5′ UTR is ∼700 nucleotides in length and contains highly structured secondary elements with internal ribosomal entry site (IRES) function. The ∼2,200-amino-acid (aa) polyprotein is cotranslationally processed by viral proteases to yield structural (VP4, VP2, VP3, and VP1) and nonstructural (2A, 2B, 2C, 3A, 3B, 3C, and 3D) proteins (32). Current enterovirus classification is based on the high sequence divergence within the VP1 capsid region, which has been shown to correspond with serotype neutralization (27, 28). Human enterovirus (HEV) types are currently classified into four species, human enterovirus A (HEV-A), HEV-B, HEV-C (including poliovirus), and HEV-D, based on the four phylogenetic clusters observed in comparisons of the coding region sequences. An enterovirus is considered a new type within a species if it possesses <75% nucleotide identity and <85% amino acid identity with known members across the VP1 sequence (27, 30). Molecular identification methods play a crucial role in rapid, sensitive enterovirus diagnostics and have led to the recent discovery of several novel enteroviruses (29, 31, 40, 42, 44). Most approaches target a limited number of conserved regions in the 5′ UTR and VP4-VP2 junction or seek to ascertain serotype information by probing antigenic regions, such as VP1 (5).Picornavirus RNA-dependent RNA polymerases are highly error prone and lack proofreading ability, resulting in a misincorporation frequency of 1 per 10³ to 10⁴ nucleotides (48). The relative infidelity of these polymerases is believed to enable rapid adaptability under selective pressure. Large-impact evolutionary events, such as recombination within and between enterovirus serotypes, also contribute to their evolution and genetic diversity (3, 8, 26, 39) and may lead to changes in disease associations with human enterovirus infections. Human enteroviruses are classified into four species based on coding region sequence phylogeny, and intraspecies recombination events between enteroviruses that are closely related in the coding region are well documented (26, 38, 39). All known enterovirus 5′ UTR sequences, however, cluster into two groups containing either HEV-A and -B sequences or HEV-C and -D sequences. Recent findings have described enterovirus genomes with a coding region that clusters with one species and a 5′ UTR that clusters with a different species, suggesting possible interspecies recombination events (41, 44). Understanding the recombination-driven evolution of HEV-C viruses is of particular public health concern due to the viruses'' ability to recombine with vaccine poliovirus, resulting in circulating, highly neurovirulent vaccine-derived polioviruses (17, 21, 34). It is unclear whether recombination events between poliovirus and HEV-C viruses allow for the rapid acquisition of traits that increase pathogenic and circulation potential.The enterovirus pathogenicity spectrum is related to tissue tropism and is largely determined by cellular receptor usage. Most picornaviruses use receptors from the immunoglobulin superfamily of proteins, such as intracellular adhesion molecule-1 (ICAM-1) or coxsackievirus-adenovirus receptor (CAR) (36). A distinct subgroup of HEV-C viruses, which includes coxsackievirus (CAV) A1, A19, and A22 and enterovirus 104, has not yet been grown successfully in cell culture, and the receptor molecule for this subgroup is unknown (6). HEV-C viruses are believed to be the ancestral source of poliovirus, which resulted from a capsid mutation that caused a cellular receptor switch from ICAM-1 to CD155 (poliovirus receptor [PVR]) (17).In this study, we report the discovery and characterization of a novel human enterovirus type within species HEV-C, for which we propose the designation human enterovirus 109 (EV109). Sequence analysis reveals considerable nucleotide divergence in the 5′ UTR between EV109 and other HEV-C types, and scanning bootstrap analysis supports the hypothesis that EV109 is the product of an interspecies recombination event with an ancestral member of the HEV-A group. Viral capsid amino acid alignments and homology modeling reveal the predicted three-dimensional arrangement of divergent and conserved residues of EV109 compared with other related enteroviruses. We also report highly similar EV109 isolates within multiple cases of acute pediatric respiratory illness in Managua, Nicaragua.     

An Acid-loading Chloride Transport Pathway in the Intraerythrocytic Malaria Parasite,Plasmodium falciparum

The intraerythrocytic malaria parasite exerts tight control over its ionic composition. In this study, a combination of fluorescent ion indicators and ³⁶Cl⁻ flux measurements was used to investigate the transport of Cl⁻ and the Cl⁻-dependent transport of “H⁺-equivalents” in mature (trophozoite stage) parasites, isolated from their host erythrocytes. Removal of extracellular Cl⁻, resulting in an outward [Cl⁻] gradient, gave rise to a cytosolic alkalinization (i.e. a net efflux of H⁺-equivalents). This was reversed on restoration of extracellular Cl⁻. The flux of H⁺-equivalents was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and, when measured in ATP-depleted parasites, showed a pronounced dependence on the pH of the parasite cytosol; the flux was low at cytosolic pH values < 7.2 but increased steeply with cytosolic pH at values > 7.2. ³⁶Cl⁻ influx measurements revealed the presence of a Cl⁻ uptake mechanism with characteristics similar to those of the Cl⁻-dependent H⁺-equivalent flux. The intracellular concentration of Cl⁻ in the parasite was estimated to be ∼48 mm in situ. The data are consistent with the intraerythrocytic parasite having in its plasma membrane a 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid-sensitive transporter that, under physiological conditions, imports Cl⁻ together with H⁺-equivalents, resulting in an intracellular Cl⁻ concentration well above that which would occur if Cl⁻ ions were distributed passively in accordance with the parasite''s large, inwardly negative membrane potential.     

A novel approach to examining compositional heterogeneity of detergent-resistant lipid rafts

Lipid rafts play an important role in cell signalling, cell adhesion and other cellular functions. Compositional heterogeneity of lipid rafts provides one mechanism of how lipid rafts provide the spatial and temporal regulation of cell signalling and cell adhesion. The constitutive presence of some signalling receptors/molecules and accumulation of others in the lipid raft allows them to interact with each other and thereby facilitate relay of signals from the plasma membrane to the cell interior. Devising a method that can analyze these lipid microdomains for the presence of signalling receptors/molecules on an individual raft basis is required to address the issue of lipid raft heterogeneity. SDS-PAGE analysis, currently used for analyses of detergent-resistant lipid rafts, does not address this question. We have designed a cell-free assay that captures detergent-resistant lipid rafts with an antibody against a raft-resident molecule and detects the presence of another lipid raft molecule. Our results suggest that detergent-resistant lipid rafts, also known as detergent-resistant membranes, are heterogeneous populations on an immortalized mouse T-cell plasma membrane with respect to antigen receptor/signalling complex and other signalling/adhesion proteins. This cell-free assay provides a simple and quick way to examine the simultaneous presence of two proteins in the lipid rafts and has the potential to estimate trafficking of molecules in and out of the lipid microdomains during cell signalling on a single detergent-resistant lipid raft basis.     

GPCR-MPredictor: multi-level prediction of G protein-coupled receptors using genetic ensemble

G protein-coupled receptors (GPCRs) are transmembrane proteins, which transduce signals from extracellular ligands to intracellular G protein. Automatic classification of GPCRs can provide important information for the development of novel drugs in pharmaceutical industry. In this paper, we propose an evolutionary approach, GPCR-MPredictor, which combines individual classifiers for predicting GPCRs. GPCR-MPredictor is a web predictor that can efficiently predict GPCRs at five levels. The first level determines whether a protein sequence is a GPCR or a non-GPCR. If the predicted sequence is a GPCR, then it is further classified into family, subfamily, sub-subfamily, and subtype levels. In this work, our aim is to analyze the discriminative power of different feature extraction and classification strategies in case of GPCRs prediction and then to use an evolutionary ensemble approach for enhanced prediction performance. Features are extracted using amino acid composition, pseudo amino acid composition, and dipeptide composition of protein sequences. Different classification approaches, such as k-nearest neighbor (KNN), support vector machine (SVM), probabilistic neural networks (PNN), J48, Adaboost, and Naives Bayes, have been used to classify GPCRs. The proposed hierarchical GA-based ensemble classifier exploits the prediction results of SVM, KNN, PNN, and J48 at each level. The GA-based ensemble yields an accuracy of 99.75, 92.45, 87.80, 83.57, and 96.17% at the five levels, on the first dataset. We further perform predictions on a dataset consisting of 8,000 GPCRs at the family, subfamily, and sub-subfamily level, and on two other datasets of 365 and 167 GPCRs at the second and fourth levels, respectively. In comparison with the existing methods, the results demonstrate the effectiveness of our proposed GPCR-MPredictor in classifying GPCRs families. It is accessible at .     

Root colonization and growth promotion of sunflower (Helianthus annuus L.) by phosphate solubilizing Enterobacter sp. Fs-11

An Enterobacter sp. Fs-11 was isolated from sunflower rhizosphere, identified on the basis of 16S rRNA gene sequence analysis (GeneBank accession no. GQ179978) and studied for its root colonization and growth promotion ability in sunflower. Morphologically, it was rod shaped Gram-negative, motile bacterium, producing 4.5 μg mL(-1) indole acetic acid in tryptophan-supplemented medium. It utilized 27 out of 95 substrates in BIOLOG GN2 micro plate system. It was able to convert insoluble tri-calcium phosphate to soluble phosphorus up to 43.5 μg mL(-1) with decrease in pH of the medium up to 4.5 after 10 days incubation at 28 ± 2 °C in the Pikovskaya's broth. High performance liquid chromatography of cell free supernatant showed that Fs-11 produced malic acid and gluconic acid (2.43 and 16.64 μg mL(-1), respectively) in Pikovskaya's broth. Analysis of 900 bp fragment of pyrroloquinoline quinine pqqE gene sequence showed 98 % homology with that of E. cloacae pqqE gene. Confocal laser scanning microscope revealed strong colonization of fluorescently labeled Fs-11 with sunflower roots. Sunflower inoculation with Fs-11 and its rifampicin resistant derivative in sterile sand and natural soil showed that Fs-11 colonized sunflower roots up to 30 days after transplanting in both sterile sand as well as natural soil. Moreover, Fs-11 inoculation resulted in increased plant height, fresh weight, dry weight and total phosphorus contents as compared to un-inoculated plants. The data showed that Enterobacter sp. Fs-11 is an efficient phosphate solubilizing and plant growth promoting rhizobacterium and has great potential to be used as bio-inoculant for sunflower under phosphorus deficient conditions.     

Benzimidazole bearing oxadiazole and triazolo-thiadiazoles nucleus: Design and synthesis as anticancer agents

Two new series of benzimidazole bearing oxadiazole[1-(1H-benzo[d]imidazol-2-yl)-3-(5-substituted-1,3,4-oxadiazol-2-yl)propan-1-ones (4a-l)] and triazolo-thiadiazoles[1-(1H-benzo[d]imidazol-2-yl)-3-(6-(substituted)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)propan-1-one (7a-e)] have been synthesized successfully from4-(1H-benzo[d]imidazol-2-yl)-4-oxobutanehydrazide (3) with an aim to produce promising anticancer agents. In vitro anticancer activities of synthesized compounds were screened at the National Cancer Institute (NCI), USA, according to their applied protocol against full NCI 60 human cell lines panel; results showed good to remarkable anticancer activity. Among them, compound (4j, NCS: 761980) exhibited significant growth inhibition and further screened at 10-fold dilutions of five different concentrations (0.01, 0.1, 1, 10 and 100μM) with GI(50) values ranging from 0.49 to 48.0μM and found superior for the non-small cell lung cancer cell lines like HOP-92 (GI(50) 0.49, TGI 19.9,LC(50) >100 and Log(10)GI(50) -6.30, Log(10)TGI -4.70, Log(10)LC(50) >-4.00).     

Cineradiography of monkey lip-smacking reveals putative precursors of speech dynamics

A key feature of speech is its stereotypical 5 Hz rhythm. One theory posits that this rhythm evolved through the modification of rhythmic facial movements in ancestral primates. If the hypothesis has any validity, then a comparative approach may shed some light. We tested this idea by using cineradiography (X-ray movies) to characterize and quantify the internal dynamics of the macaque monkey vocal tract during lip-smacking (a rhythmic facial expression) versus chewing. Previous human studies showed that speech movements are faster than chewing movements, and the functional coordination between vocal tract structures is different between the two behaviors. If rhythmic speech evolved through a rhythmic ancestral facial movement, then one hypothesis is that monkey lip-smacking versus chewing should also exhibit these differences. We found that the lips, tongue, and hyoid move with a speech-like 5 Hz rhythm during lip-smacking, but not during chewing. Most importantly, the functional coordination between these structures was distinct for each behavior. These data provide empirical support for the idea that the human speech rhythm evolved from the rhythmic facial expressions of ancestral primates.