Dynamic changes in extracellular vesicle-associated miRNAs elicited by ultrasound in inflammatory bowel disease patients.

Blood-based biomarkers that reliably indicate disease activity in the intestinal tract are an important unmet need in the management of patients with IBD. Extracellular vesicles (EVs) are cell-derived membranous microparticles, which reflect the cellular and functional state of their site of site of origin. As ultrasound waves may lead to molecular shifts of EV contents, we hypothesized that application of ultrasound waves on inflamed intestinal tissue in IBD may amplify the inflammation-specific molecular shifts in EVs like altered EV-miRNA expression, which in turn can be detected in the peripheral blood. 26 patients with IBD were included in the prospective clinical study. Serum samples were collected before and 30 min after diagnostic transabdominal ultrasound. Differential miRNA expression was analyzed by sequencing. Candidate inducible EV-miRNAs were functionally assessed in vitro by transfection of miRNA mimics and qPCR of predicted target genes. Serum EV-miRNA concentration at baseline correlated with disease severity, as determined by clinical activity scores and sonographic findings. Three miRNAs (miR-942-5p, mir-5588, mir-3195) were significantly induced by sonography. Among the significantly regulated EV-miRNAs, miR-942-5p was strongly induced in higher grade intestinal inflammation and correlated with clinical activity in Crohn's disease. Prediction of target regulation and transfection of miRNA mimics inferred a role of this EV-miRNA in regulating barrier function in inflammation. Induction of mir-5588 and mir-3195 did not correlate with inflammation grade. This proof-of-concept trial highlights the principle of induced molecular shifts in EVs from inflamed tissue through transabdominal ultrasound. These inducible EVs and their molecular cargo like miRNA could become novel biomarkers for intestinal inflammation in IBD.

Lymphocytic colitis can be transcriptionally divided into channelopathic and inflammatory lymphocytic colitis.

BACKGROUND: The pathobiology of the non-destructive inflammatory bowel disease (IBD) lymphocytic colitis (LC) is poorly understood. We aimed to define an LC-specific mucosal transcriptome to gain insight into LC pathology, identify unique genomic signatures, and uncover potentially druggable disease pathways. METHODS: We performed bulk RNA-sequencing of LC and collagenous colitis (CC) colonic mucosa from patients with active disease, and healthy controls (n = 4-10 per cohort). Differential gene expression was analyzed by gene-set enrichment and deconvolution analyses to identify pathologically relevant pathways and cells, respectively, altered in LC. Key findings were validated using reverse transcription quantitative PCR and/or immunohistochemistry. Finally, we compared our data with a previous cohort of ulcerative colitis and Crohn's disease patients (n = 4 per group) to distinguish non-destructive from classic IBD. RESULTS: LC can be subdivided into channelopathic LC, which is governed by organic acid and ion transport dysregulation, and inflammatory LC, which is driven by microbial immune responses. Inflammatory LC displays an innate and adaptive immunity that is limited compared to CC and classic IBD. Conversely, we noted a distinct induction of regulatory non-coding RNA species in inflammatory LC samples. Moreover, compared with CC, water channel and cell adhesion molecule gene expression decreased in channelopathic LC, whereas it was accentuated in inflammatory LC and associated with reduced intestinal epithelial cell proliferation. CONCLUSIONS: We conclude that LC can be subdivided into channelopathic LC and inflammatory LC that could be pathomechanistically distinct subtypes despite their shared clinical presentation. Inflammatory LC exhibits a dampened immune response compared to CC and classic IBDs. Our results point to regulatory micro-RNAs as a potential disease-specific feature that may be amenable to therapeutic intervention.

Prospects of microgreens as budding living functional food: Breeding and biofortification through OMICS and other approaches for nutritional security.

Nutrient deficiency has resulted in impaired growth and development of the population globally. Microgreens are considered immature greens (required light for photosynthesis and growing medium) and developed from the seeds of vegetables, legumes, herbs, and cereals. These are considered "living superfood/functional food" due to the presence of chlorophyll, beta carotene, lutein, and minerals like magnesium (Mg), Potassium (K), Phosphorus (P), and Calcium (Ca). Microgreens are rich at the nutritional level and contain several phytoactive compounds (carotenoids, phenols, glucosinolates, polysterols) that are helpful for human health on Earth and in space due to their anti-microbial, anti-inflammatory, antioxidant, and anti-carcinogenic properties. Microgreens can be used as plant-based nutritive vegetarian foods that will be fruitful as a nourishing constituent in the food industryfor garnish purposes, complement flavor, texture, and color to salads, soups, flat-breads, pizzas, and sandwiches (substitute to lettuce in tacos, sandwich, burger). Good handling practices may enhance microgreens'stability, storage, and shelf-life under appropriate conditions, including light, temperature, nutrients, humidity, and substrate. Moreover, the substrate may be a nutritive liquid solution (hydroponic system) or solid medium (coco peat, coconut fiber, coir dust and husks, sand, vermicompost, sugarcane filter cake, etc.) based on a variety of microgreens. However integrated multiomics approaches alongwith nutriomics and foodomics may be explored and utilized to identify and breed most potential microgreen genotypes, biofortify including increasing the nutritional content (macro-elements:K, Ca and Mg; oligo-elements: Fe and Zn and antioxidant activity) and microgreens related other traits viz., fast growth, good nutritional values, high germination percentage, and appropriate shelf-life through the implementation of integrated approaches includes genomics, transcriptomics, sequencing-based approaches, molecular breeding, machine learning, nanoparticles, and seed priming strategiesetc.

Deeper insights into long-term survival heterogeneity of pancreatic ductal adenocarcinoma (PDAC) patients using integrative individual- and group-level transcriptome network analyses.

Pancreatic ductal adenocarcinoma (PDAC) is categorized as the leading cause of cancer mortality worldwide. However, its predictive markers for long-term survival are not well known. It is interesting to delineate individual-specific perturbed genes when comparing long-term (LT) and short-term (ST) PDAC survivors and integrate individual- and group-based transcriptome profiling. Using a discovery cohort of 19 PDAC patients from CHU-Liège (Belgium), we first performed differential gene expression analysis comparing LT to ST survivor. Second, we adopted systems biology approaches to obtain clinically relevant gene modules. Third, we created individual-specific perturbation profiles. Furthermore, we used Degree-Aware disease gene prioritizing (DADA) method to develop PDAC disease modules; Network-based Integration of Multi-omics Data (NetICS) to integrate group-based and individual-specific perturbed genes in relation to PDAC LT survival. We identified 173 differentially expressed genes (DEGs) in ST and LT survivors and five modules (including 38 DEGs) showing associations to clinical traits. Validation of DEGs in the molecular lab suggested a role of REG4 and TSPAN8 in PDAC survival. Via NetICS and DADA, we identified various known oncogenes such as CUL1 and TGFB1. Our proposed analytic workflow shows the advantages of combining clinical and omics data as well as individual- and group-level transcriptome profiling.

Performance of model-based multifactor dimensionality reduction methods for epistasis detection by controlling population structure.

BACKGROUND: In genome-wide association studies the extent and impact of confounding due to population structure have been well recognized. Inadequate handling of such confounding is likely to lead to spurious associations, hampering replication, and the identification of causal variants. Several strategies have been developed for protecting associations against confounding, the most popular one is based on Principal Component Analysis. In contrast, the extent and impact of confounding due to population structure in gene-gene interaction association epistasis studies are much less investigated and understood. In particular, the role of nonlinear genetic population substructure in epistasis detection is largely under-investigated, especially outside a regression framework. METHODS: To identify causal variants in synergy, to improve interpretability and replicability of epistasis results, we introduce three strategies based on a model-based multifactor dimensionality reduction approach for structured populations, namely MBMDR-PC, MBMDR-PG, and MBMDR-GC. RESULTS: Simulation results comparing the performance of various approaches show that in the presence of population structure MBMDR-PC and MBMDR-PG consistently better control type I error rate at the nominal level than MBMDR-GC. Moreover, our proposed three methods of population structure correction outperform MDR-SP in terms of statistical power. CONCLUSION: We demonstrate through extensive simulation studies the effect of various degrees of genetic population structure and relatedness on epistasis detection and propose appropriate remedial measures based on linear and nonlinear sample genetic similarity.

PLANET-SNP pipeline: PLants based ANnotation and Establishment of True SNP pipeline.

Acute prediction of SNPs (Single Nucleotide Polymorphisms) from high throughput sequencing data is a challenging problem, having potential to explore possible variation within plants species. For the extraction of profitable information from bulk of data, machine learning (ML) could lead to development of accurate model based on the learning of prior information. We performed state of art, in-depth learning on six different plant species. Comparative evaluation of five different algorithms showed that Random Forest substantially outperformed in selection of potential SNPs, with markedly improved prediction accuracy via 10-fold cross validation technique and integrated in system known as PLANET-SNP. We present the accurate method to extract the potential SNPs with user specific customizable parameters. It will facilitate the identification of efficient and functional SNPs in most easy and intuitive way. PLANET-SNP pipeline is very flexible in terms of data input and output formats. PLANET-SNP Pipeline is available at http://www.ncgd.nbri.res.in/PLANET-SNP-Pipeline.aspx.

Transcriptome analysis provides insight into prickle development and its link to defense and secondary metabolism in Solanum viarum Dunal.

Prickles are epidermal outgrowth found on the aerial surface of several terrestrial plants. Microscopic studies on prickles of S. viarum Dunal indicated a crucial role of glandular trichomes (GTs) in their development. A spontaneously obtained prickleless mutant showed normal epidermal GTs, but its downstream developmental process to prickle was perturbed. Thus, prickleless mutant offers an ideal opportunity to unveil molecular regulators working downstream to GTs in the prickle formation. Differential transcriptome analysis of epidermis of prickly and prickleless mutant revealed that expression of several defense regulators like ethylene, salicylic acid, PR-proteins, etc. were significantly down-regulated in prickleless mutant, provide an important link between defense and prickle development. It was also noteworthy that the expression of few essential development related TFs like MADS-box, R2R3-MYB, REM, DRL1, were also down-regulated in the stem, petioles, and leaves of prickleless mutant indicating their potential role in prickle development. Interestingly, the gene expression of terpenoid, steroid, flavonoid, glucosinolate, and lignin biosynthesis pathways were up-regulated in prickleless mutant. The biochemical and qRT-PCR analysis also confirmed metabolite elevation. These results indicated that the loss of prickle was compensated by elevated secondary metabolism in the prickleless mutant which played important role in the biotic and abiotic stress management.

In Silico identification of SNP diversity in cultivated and wild tomato species: insight from molecular simulations.

Single Nucleotide Polymorphisms (SNPs), an important source of genetic variations, are often used in crop improvement programme. The present study represented comprehensive In silico analysis of nucleotide polymorphisms in wild (Solanum habrochaites) and cultivated (Solanum lycopersicum) species of tomato to explore the consequence of substitutions both at sequence and structure level. A total of 8978 SNPs having Ts/Tv (Transition/Transversion) ratio 1.75 were identified from the Expressed Sequence Tag (EST) and Next Generation Sequence (NGS) data of both the species available in public databases. Out of these, 1838 SNPs were non-synonymous and distributed in 988 protein coding genes. Among these, 23 genes containing 96 SNPs were involved in traits markedly different between the two species. Furthermore, there were 28 deleterious SNPs distributed in 27 genes and a few of these genes were involved in plant pathogen interaction and plant hormone pathways. Molecular docking and simulations of several selected proteins showed the effect of SNPs in terms of compactness, conformation and interaction ability. Observed SNPs exhibited various types of motif binding effects due to nucleotide changes. SNPs that provide the evidence of differential motif binding and interaction behaviour could be effectively used for the crop improvement program.

PanCoreGen - Profiling, detecting, annotating protein-coding genes in microbial genomes.

A large amount of genomic data, especially from multiple isolates of a single species, has opened new vistas for microbial genomics analysis. Analyzing the pan-genome (i.e. the sum of genetic repertoire) of microbial species is crucial in understanding the dynamics of molecular evolution, where virulence evolution is of major interest. Here we present PanCoreGen - a standalone application for pan- and core-genomic profiling of microbial protein-coding genes. PanCoreGen overcomes key limitations of the existing pan-genomic analysis tools, and develops an integrated annotation-structure for a species-specific pan-genomic profile. It provides important new features for annotating draft genomes/contigs and detecting unidentified genes in annotated genomes. It also generates user-defined group-specific datasets within the pan-genome. Interestingly, analyzing an example-set of Salmonella genomes, we detect potential footprints of adaptive convergence of horizontally transferred genes in two human-restricted pathogenic serovars - Typhi and Paratyphi A. Overall, PanCoreGen represents a state-of-the-art tool for microbial phylogenomics and pathogenomics study.

Development of a 63K SNP Array for Cotton and High-Density Mapping of Intraspecific and Interspecific Populations of Gossypium spp.

High-throughput genotyping arrays provide a standardized resource for plant breeding communities that are useful for a breadth of applications including high-density genetic mapping, genome-wide association studies (GWAS), genomic selection (GS), complex trait dissection, and studying patterns of genomic diversity among cultivars and wild accessions. We have developed the CottonSNP63K, an Illumina Infinium array containing assays for 45,104 putative intraspecific single nucleotide polymorphism (SNP) markers for use within the cultivated cotton species Gossypium hirsutum L. and 17,954 putative interspecific SNP markers for use with crosses of other cotton species with G. hirsutum. The SNPs on the array were developed from 13 different discovery sets that represent a diverse range of G. hirsutum germplasm and five other species: G. barbadense L., G. tomentosum Nuttal × Seemann, G. mustelinum Miers × Watt, G. armourianum Kearny, and G. longicalyx J.B. Hutchinson and Lee. The array was validated with 1,156 samples to generate cluster positions to facilitate automated analysis of 38,822 polymorphic markers. Two high-density genetic maps containing a total of 22,829 SNPs were generated for two F2 mapping populations, one intraspecific and one interspecific, and 3,533 SNP markers were co-occurring in both maps. The produced intraspecific genetic map is the first saturated map that associates into 26 linkage groups corresponding to the number of cotton chromosomes for a cross between two G. hirsutum lines. The linkage maps were shown to have high levels of collinearity to the JGI G. raimondii Ulbrich reference genome sequence. The CottonSNP63K array, cluster file and associated marker sequences constitute a major new resource for the global cotton research community.

Global nucleosome positioning regulates salicylic acid mediated transcription in Arabidopsis thaliana.

BACKGROUND: The nucleosome positioning regulates the gene expression and many other DNA-related processes in eukaryotes. Genome-wide mapping of nucleosome positions and correlation of genome-wide nucleosomal remodeling with the changes in the gene expression can help us understanding gene regulation on genome level. RESULTS: In the present study, we correlate the gene expression and the genomic nucleosomal remodeling in response to salicylic acid (SA) treatment in A. thaliana. We have mapped genome-wide nucleosomes by performing tiling microarray using 146 bp mononucleosomal template DNA. The average nucleosomal coverage is approximately 346 bp per nucleosome both under the control and the SA-treated conditions. The nucleosomal coverage is more in the coding region than in the 5' regulatory regions. We observe approximately 50% nucleosomal remodeling on SA treatment where significant nucleosomal depletion and nucleosomal enrichment around the transcription start site (TSS) occur in SA induced genes and SA repressed genes respectively in response to SA treatment. Especially in the case of the SA-induced group, the nucleosomal remodeling over the minimal promoter in response to SA is especially significant in the Non-expresser of PR1 (NPR1)-dependent genes. A detailed investigation of npr1-1 mutant confirms a distinct role of NPR1 in the nucleosome remodeling over the core promoter. We have also identified several motifs for various hormonal responses; including ABRE elements in the remodeled nucleosomal regions around the promoter region in the SA regulated genes. We have further identified that the W-box and TGACG/C motif, reported to play an important role in SA-mediated induction, are enriched in nucleosome free regions (NFRs) of the promoter region of the SA induced genes. CONCLUSIONS: This is the first study reporting genome-wide effects of SA treatment on the chromatin architecture of A. thaliana. It also reports significant role of NPR1 in genome-wide nucleosomal remodeling in response to SA.

Comparative Transcriptional Profiling of Contrasting Rice Genotypes Shows Expression Differences during Arsenic Stress.

Accumulation of arsenic (As) in rice (Oryza sativa L.) grain is a serious concern worldwide. Long-term exposure to As affects nutritional status in rice grain and is associated with higher rates of skin, bladder, and lung cancers, and heart disease. Genotypic variations in rice for As accumulation or tolerance are prevalent and are regulated by genetic and environmental factors. To understand molecular networks involved in As accumulation, genome-wide expression analysis was performed in roots of low- and high-As accumulating rice genotypes (LARGs and HARGs). Six rice genotypes with contrasting As accumulation potential and tolerance were used in this study. Genome-wide expression analysis suggested their differential response against As stress. This study suggests up- and downregulation of a number of unique genes involved in various pathways and biological processes in response to As stress in rice genotypes. A comparison of gene expression profiles, principal component analysis, and K-means clustering suggests that an independent pathway is operating during As stress tolerance or accumulation in contrasting genotypes. It was also observed that the differential behavior of aus genotype, Nayanmoni, from other LARGs might be due to its different genetic background. Cis-motif profiling of As-induced coexpressed genes in diverse rice genotypes led to the identification of unique cis-motifs present in differentially expressed genes. This study suggests that the genetic mechanism regulating the differential As accumulation in different genotypes may not be dependent on gene expression at the transcriptional level. However, many genes identified in this study can be analyzed and used for marker-trait associations related to As accumulation in diverse genotypes around the world.

A comparative antimicrobial analysis of various root canal irrigating solutions on endodontic pathogens: an in vitro study.

BACKGROUND AND OBJECTIVES: Evolution in understanding the poly-microbial environment of both endodontic infections and that of failed root canal treatments has been debatable over the years. The present study was designed to compare and analyze the effect of various root canal irrigation solutions on certain endodontic pathogens in vitro. MATERIALS AND METHODS: To analyze in vitro the zone of inhibition of the micro-organisms the following irrigating solutions were employed: a. Sodium hypochlorite (NaOCl) 5% b. Sodium hypochlorite (NaOCl) 3% c. Chlorhexidine 2% d. Chlorhexidine (CHX) 0.12% e. Doxycycline 0.01% f. Doxycycline 0.005% g. MTAD. An agar culture plate inoculated with four endodontic pathogens was used namely 1. Enterococcus faecalis (MTCC-439) 2. Candida albicans (MTCC-183) 3. Fusobacterium nucleatum (ATCC-25586) 4. Peptostreptococcus anaerobius (ATCC-27337) The inoculums were streaked out on the trypticase soy agar plate for discrete colonies with a wire loop using standard method. The inoculated plates carrying the antibiotic disks were incubated in an anaerobic jar in the following way-Anaerobic incubation-the following procedure was used to provide anaerobiosis with an increased concentration of carbon dioxide. By this method the zone of inhibition obtained by different irrigating solutions against different pathogens could be compared. RESULTS: MTAD showed maximum antibacterial activity. In case of C. albicans MTAD was less effective than 5% NaOCl, 3% NaOCl and 2% CHX, 0.12% CHX. However, it was more effective against E. faecalis, F. nuleatum and P. anaerobicus. In any case, antimicrobial activity is not the only prerequisite for an endodontic irrigant. E. faecalis strain used in this study showed resistance to doxycycline; also doxycycline was ineffective against C. albicans at 0.01% and 0.005% concentrations. CONCLUSION: It was found that MTAD was more antimicrobial than 5% NaOCl for some of the test micro-organisms; however the ability of MTAD to dissolve pulp tissue is not comparable to 5% NaOCl. In addition, 5 and 3% NaOCl showed signifcant antimicrobial activity against all test micro-organisms. The best option for a primary endodontic irrigant therefore is 5% NaOCl.

Evaluation of antifungal activity of white-colored mineral trioxide aggregate on different strains of Candida albicans in vitro.

AIM: The purpose of this study was to evaluate the antifungal action of various concentrations of white mineral trioxide aggregate (MTA) against seven different strains of Candida albicans using the tube dilution test. MATERIALS AND METHODS: Fresh mix of MTA was prepared at concentrations of 100, 50, 25, and 12.5 mg/ml and added to a broth tube containing Sabouraud's liquid medium. A total of 1287 broth tubes were prepared and divided into experimental and control groups. Stock cultures of seven strains of C. albicans were obtained. Fresh inoculate of the microorganism was prepared by growing overnight cultures. Aliquots of the test C. albicans were taken and added to the test tubes. All tubes were incubated at 37°C for 1-, 24-, 72-, and 168-h time periods. At each time period, the presence of C. albicans colonies was assessed. STATISTICAL ANALYSIS USED: Differences among the groups were statistically analyzed using Kruskal-Wallis and Mann-Whitney U tests. RESULTS: Results showed that one strain showed resistance even after 3 days at the lower MTA concentrations of 12.5 and 25 mg/ml. Growth reoccurred with three strains at MTA concentration of 12.5 mg/ml after 7 days. A significant difference was found between strain 3 and other strains at MTA concentrations of 12.5 and 25 mg/ml at the 3-days time period and between tubes containing 12.5 mg/ml and tubes containing higher concentrations of MTA at the 7-days time period. CONCLUSION: White MTA in concentrations of 100 and 50 mg/ml is effective in inhibiting the seven tested strains of C. albicans for periods up to 1 week.

Sniffing out pain: An in vivo intranasal study of analgesic efficacy.

BACKGROUND: Orofacial pain is a common encounter in dentistry (affecting 12% of the population) and is a primary reason for patients seeking emergency care. Dentists often prescribe oral analgesics, which have disadvantages of decreased absorption rates and delayed onset. Intranasal (IN) delivery takes advantage of a large surface area of mucosal tissue for rapid absorption. The purpose of this study was to evaluate the efficacy of IN ketorolac for endodontic pain using a randomized, double-blind, placebocontrolled parallel design study. MATERIALS & METHODS: Twenty patients presenting with moderate to severe endodontic pain were selected to receive IN treatment with placebo (n = 10) or ketorolac (n = 10) 30 minutes before endodontic treatment was started and immediately after the completion of endodontic treatment. Baseline pain levels were recorded before IN treatment. Pain levels were also recorded at 15 and 30 minutes after the initial IN dosing (before endodontic treatment); 30 minutes after completion of endodontic treatment; and 4, 8, and 12 hours after the initial IN spray. RESULTS: IN ketorolac alone or with endodontic treatment showed significantly better pain relief compared with IN placebo spray alone or with endodontic treatment at 30 minutes after the first or second intranasal dose and at 4 hours after the first intranasal dose. CONCLUSIONS: These results suggest that IN ketorolac may provide a novel and efficacious method for pain relief in endodontic pain patients. How to cite the article: Maroli S, Srinath HP, Goinka C, Yadav NS, Bhardwaj A, Varghese RK. Sniffing out pain: An in vivo intranasal study of analgesic efficacy. J Int Oral Health 2014;6(1):66-71.

Nanotechnology in dentistry: Present and future.

Nanotechnology is the manipulation of matter on the molecular and atomic levels. It has the potential to bring enormous changes into the fields of medicine and dentistry. A day may soon come when nanodentistry will succeed in maintaining near-perfect oral health through the aid of nanorobotics, nanomaterials and biotechnology. However, as with all developments, it may also pose a risk for misuse. Time, economical and technical resources, and human needs will determine the direction this revolutionizing development may take. This article reviews the current status and the potential clinical applications of nanotechnology, nanaomedicine and nanodentistry. How to cite the article: Bhardwaj A, Bhardwaj A, Misuriya A, Maroli S, Manjula S, Singh AK. Nanotechnology in dentistry: Present and future. J Int Oral Health 2013;6(1):121-6.

Large-scale resource development in Gossypium hirsutum L. by 454 sequencing of genic-enriched libraries from six diverse genotypes.

The sequence information has been proved to be an essential genomic resource in case of crop plants for their genetic improvement and better utilization by humans. To dissect the Gossypium hirsutum genome for large-scale development of genomic resources, we adopted hypomethylated restriction-based genomic enrichment strategy to sequence six diverse genotypes. Approximately 5.2-Gb data (more than 18.36 million reads) was generated which, after assembly, represents nearly 1.27-Gb genomic sequences. We predicted a total of 93,363 gene models (21,399 full length) and identified 35,923 gene models which were validated against already sequenced plant genomes. A total of 1,093 transcription factor-encoding genes, 3,135 promoter sequences and 78 miRNA (including 17 newly identified in Gossypium) were predicted. We identified significant no. of molecular markers including 47,093 novel simple sequence repeats and 66,364 novel single nucleotide polymorphisms. In addition, we developed NBRI-Comprehensive Cotton Genomics database, a web resource to provide access of cotton-related genomic resources developed at NBRI. This study contributes considerable amount of genomic resources and suggests a potential role of genic-enriched sequencing in genomic resource development for orphan crop plants.