De novo identification of viral pathogens from cell culture hologenomes

<p>Abstract</p> <p>Background</p> <p>Fast, specific identification and surveillance of pathogens is the cornerstone of any outbreak response system, especially in the case of emerging infectious diseases and viral epidemics. This process is generally tedious and time-consuming thus making it ineffective in traditional settings. The added complexity in these situations is the non-availability of pure isolates of pathogens as they are present as mixed genomes or hologenomes. Next-generation sequencing approaches offer an attractive solution in this scenario as it provides adequate depth of sequencing at fast and affordable costs, apart from making it possible to decipher complex interactions between genomes at a scale that was not possible before. The widespread application of next-generation sequencing in this field has been limited by the non-availability of an efficient computational pipeline to systematically analyze data to delineate pathogen genomes from mixed population of genomes or hologenomes.</p> <p>Findings</p> <p>We applied next-generation sequencing on a sample containing mixed population of genomes from an epidemic with appropriate processing and enrichment. The data was analyzed using an extensive computational pipeline involving mapping to reference genome sets and <it>de-novo </it>assembly. In depth analysis of the data generated revealed the presence of sequences corresponding to <it>Japanese encephalitis </it>virus. The genome of the virus was also independently <it>de-novo </it>assembled. The presence of the virus was in addition, verified using standard molecular biology techniques.</p> <p>Conclusions</p> <p>Our approach can accurately identify causative pathogens from cell culture hologenome samples containing mixed population of genomes and in principle can be applied to patient hologenome samples without any background information. This methodology could be widely applied to identify and isolate pathogen genomes and understand their genomic variability during outbreaks.</p

High Resolution Methylome Map of Rat Indicates Role of Intragenic DNA Methylation in Identification of Coding Region

DNA methylation is crucial for gene regulation and maintenance of genomic stability. Rat has been a key model system in understanding mammalian systemic physiology, however detailed rat methylome remains uncharacterized till date. Here, we present the first high resolution methylome of rat liver generated using Methylated DNA immunoprecipitation and high throughput sequencing (MeDIP-Seq) approach. We observed that within the DNA/RNA repeat elements, simple repeats harbor the highest degree of methylation. Promoter hypomethylation and exon hypermethylation were common features in both RefSeq genes and expressed genes (as evaluated by proteomic approach). We also found that although CpG islands were generally hypomethylated, about 6% of them were methylated and a large proportion (37%) of methylated islands fell within the exons. Notably, we obeserved significant differences in methylation of terminal exons (UTRs); methylation being more pronounced in coding/partially coding exons compared to the non-coding exons. Further, events like alternate exon splicing (cassette exon) and intron retentions were marked by DNA methylation and these regions are retained in the final transcript. Thus, we suggest that DNA methylation could play a crucial role in marking coding regions thereby regulating alternative splicing. Apart from generating the first high resolution methylome map of rat liver tissue, the present study provides several critical insights into methylome organization and extends our understanding of interplay between epigenome, gene expression and genome stability

Notes on herbal treatment practiced by the people of fringe villages of Manas National Park, India

155-160The Manas National Park spread across Baksa and Chirang districts in Assam where the Bhutan Hills rolls down towards the northern part. Altogether 62 fringe villages are located on the southern side of the Park. The present communication deals with the herbal recipes of 49 plant species belonging to 35 families administered against 30 diseases/ailments among the people of the fringe villages. The recipes along with mode of preparation and use, and dose presented in the paper are part of the empiric knowledge confined to the common people of the area.

Effects of Chronic Menthol at Alpha3Beta4 (α3β4)-Containing Nicotinic Acetylcholine Receptors

Some heteropentameric nicotinic acetylcholine receptors (nAChRs) are up-regulated by chronic nicotine. Menthol is present in ∼ 30% of tobacco cigarettes sold in the United States. Compared with non-mentholated cigarettes, menthol-containing cigarettes are associated with reduced smoking cessation. Chronic menthol favors the lower sensitivity (α4)3(β2)2, whereas chronic nicotine favors the higher sensitivity (α4)2(β2)3 stoichiometry. Following chronic nicotine treatment, total cell lysates displayed a shift in stoichiometry towards (α3)2(β4)3 from (α3)3(β4)2. α3β4 nAChRs are highly expressed in the medial habenula and interpeduncular nuclei, which are involved in reward processing, and possibly nicotine addiction and withdrawal. We studied effects following chronic treatment (at least 24 hours) of 500 nM menthol at α3β4 nAChR using Förster resonance energy transfer (FRET), total internal reflection fluorescence microscopy (TIRFM), and whole-cell patch-clamp electrophysiology of Neuro-2a cells transiently expressing fluorescently labeled subunits. FRET experiments indicated a shift in stoichiometry toward (α3)3(β4)2 from (α3)2(β4)3. TIRFM experiments revealed α3 subunit up-regulation in the endoplasmic reticulum and α3β4 nAChR reduction in the plasma membrane. Our FRET experiments, however, include contributions from intracellular α3β4 nAChR stoichiometry. In contrast, patch clamp experiments measuring Zn2+ inhibition of acetylcholine-evoked currents indicate exclusively the functional cell surface α3β4 nAChR stoichiometry. Neither chronic menthol, chronic nicotine, nor combined chronic menthol and nicotine detectably alters Zn2+ inhibition of acetylcholine, showing that neither alters functional plasma membrane α3β4 nAChR stoichiometry. Furthermore, chronic menthol treatment shifts by < 1.5-fold the EC50 of acetylcholine at α3β4 nAChRs. These findings are consistent with our fluorescence-based experiments showing a reduction in endoplasmic reticulum exit sites following chronic menthol, which consequently reduces α3β4 nAChR delivery to the plasma membrane. Therefore, despite their intracellular effects, neither menthol nor nicotine influences cell surface α3β4 nAChR stoichiometry. Support: DA037743, DA036061, DA40047

Effects of Chronic Menthol at Alpha3Beta4 (α3β4)-Containing Nicotinic Acetylcholine Receptors

Some heteropentameric nicotinic acetylcholine receptors (nAChRs) are up-regulated by chronic nicotine. Menthol is present in ∼ 30% of tobacco cigarettes sold in the United States. Compared with non-mentholated cigarettes, menthol-containing cigarettes are associated with reduced smoking cessation. Chronic menthol favors the lower sensitivity (α4)3(β2)2, whereas chronic nicotine favors the higher sensitivity (α4)2(β2)3 stoichiometry. Following chronic nicotine treatment, total cell lysates displayed a shift in stoichiometry towards (α3)2(β4)3 from (α3)3(β4)2. α3β4 nAChRs are highly expressed in the medial habenula and interpeduncular nuclei, which are involved in reward processing, and possibly nicotine addiction and withdrawal. We studied effects following chronic treatment (at least 24 hours) of 500 nM menthol at α3β4 nAChR using Förster resonance energy transfer (FRET), total internal reflection fluorescence microscopy (TIRFM), and whole-cell patch-clamp electrophysiology of Neuro-2a cells transiently expressing fluorescently labeled subunits. FRET experiments indicated a shift in stoichiometry toward (α3)3(β4)2 from (α3)2(β4)3. TIRFM experiments revealed α3 subunit up-regulation in the endoplasmic reticulum and α3β4 nAChR reduction in the plasma membrane. Our FRET experiments, however, include contributions from intracellular α3β4 nAChR stoichiometry. In contrast, patch clamp experiments measuring Zn2+ inhibition of acetylcholine-evoked currents indicate exclusively the functional cell surface α3β4 nAChR stoichiometry. Neither chronic menthol, chronic nicotine, nor combined chronic menthol and nicotine detectably alters Zn2+ inhibition of acetylcholine, showing that neither alters functional plasma membrane α3β4 nAChR stoichiometry. Furthermore, chronic menthol treatment shifts by < 1.5-fold the EC50 of acetylcholine at α3β4 nAChRs. These findings are consistent with our fluorescence-based experiments showing a reduction in endoplasmic reticulum exit sites following chronic menthol, which consequently reduces α3β4 nAChR delivery to the plasma membrane. Therefore, despite their intracellular effects, neither menthol nor nicotine influences cell surface α3β4 nAChR stoichiometry. Support: DA037743, DA036061, DA40047