Antitumor lectin Sclerotium rolfsii (SRL) induces apoptosis in human colon cancer cells by activation of multiple signaling pathways; A microarray analysis

Background: TF antigen specific Sclerotium rolfsii lectin (SRL) inhibits human colon epithelial cancer HT29 cell growth by induction of apoptosis through cell surface binding and has tumor suppressing effect in vivo as reported earlier. Here we report the purification, identification and characterization of SRL binding membrane proteins from HT29 cells. Methods and Findings: Membrane proteins from HT29 cells were isolated by phase separation and purified by affinity chromatography using SRL-Sepharose4B matrix. Affinity purified proteins were subjected to in-gel and in-solution trypsin digestion, analysed by ESI-Q-TOF LC-MS and spectrum mill software. Considering the specificity of SRL towards O-glycans, the presence of O-GalNAc sites in SRL interacting proteins were tested using NetOGlyc software. Western blotting was performed to validate the MS identified proteins. A major protein band around 25kDa following in-gel trypsin digestion was identified as Keratin 1 by MS. In-solution trypsin digestion followed by MS identified 25 SRL interacting proteins namely, keratins, heat shock proteins, tubulins, pyruvate kinase M1/M2, peroxiredoxin-1, ATP synthase subunit alpha, mitochondrial, retinal dehydrogenase 1, actin, annexin-A2, prohibitin, ADP/ATP translocase-2 and alpha enolase. NetOGlyc software analysis revealed 21 proteins positive for O-glycosylation sites including keratins alone containing 27 to 50 O-GalNAc sites. Keratin 1 identified and validated by western blotting as major SRL interacting protein showed 49 O-GalNAc sites. Conclusion: SRL binding membrane proteins from human colon epithelial cancer HT29 cells have been identified and characterized. Identified proteins contain O-GalNAc sites and are known to be involved in cell survival, apoptosis and tumorigenesis. The present study provides insights in studying the mechanism of SRL induced apoptosis and to explore lectin for its clinical implications. Key words: Sclerotium rolfsii lectin; HT29 cell membrane proteins; NetOGlyc version 4.0; Q-TOF-LC/MS; Spectrum Mill. Abbreviations: SRL: Sclerotium rolfsii lectin; LC/MS: Liquid chromatography/Mass spectrometry; ESI: Electro Spray Ionization; Q-TOF: Quadrupole- Time of Flight; PTM: Post Translational Modification; ACN: Acetonitrile; CBB: Coomassie Brilliant Blue; BSA: Bovine Serum Albumin

Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990–2017 : a systematic analysis for the Global Burden of Disease Study 2017

Background: The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2017 comparative risk assessment (CRA) is a comprehensive approach to risk factor quantification that offers a useful tool for synthesising evidence on risks and risk outcome associations. With each annual GBD study, we update the GBD CRA to incorporate improved methods, new risks and risk outcome pairs, and new data on risk exposure levels and risk outcome associations. Methods: We used the CRA framework developed for previous iterations of GBD to estimate levels and trends in exposure, attributable deaths, and attributable disability-adjusted life-years (DALYs), by age group, sex, year, and location for 84 behavioural, environmental and occupational, and metabolic risks or groups of risks from 1990 to 2017. This study included 476 risk outcome pairs that met the GBD study criteria for convincing or probable evidence of causation. We extracted relative risk and exposure estimates from 46 749 randomised controlled trials, cohort studies, household surveys, census data, satellite data, and other sources. We used statistical models to pool data, adjust for bias, and incorporate covariates. Using the counterfactual scenario of theoretical minimum risk exposure level (TMREL), we estimated the portion of deaths and DALYs that could be attributed to a given risk. We explored the relationship between development and risk exposure by modelling the relationship between the Socio-demographic Index (SDI) and risk-weighted exposure prevalence and estimated expected levels of exposure and risk-attributable burden by SDI. Finally, we explored temporal changes in risk-attributable DALYs by decomposing those changes into six main component drivers of change as follows: (1) population growth; (2) changes in population age structures; (3) changes in exposure to environmental and occupational risks; (4) changes in exposure to behavioural risks; (5) changes in exposure to metabolic risks; and (6) changes due to all other factors, approximated as the risk-deleted death and DALY rates, where the risk-deleted rate is the rate that would be observed had we reduced the exposure levels to the TMREL for all risk factors included in GBD 2017. Findings: In 2017,34.1 million (95% uncertainty interval [UI] 33.3-35.0) deaths and 121 billion (144-1.28) DALYs were attributable to GBD risk factors. Globally, 61.0% (59.6-62.4) of deaths and 48.3% (46.3-50.2) of DALYs were attributed to the GBD 2017 risk factors. When ranked by risk-attributable DALYs, high systolic blood pressure (SBP) was the leading risk factor, accounting for 10.4 million (9.39-11.5) deaths and 218 million (198-237) DALYs, followed by smoking (7.10 million [6.83-7.37] deaths and 182 million [173-193] DALYs), high fasting plasma glucose (6.53 million [5.23-8.23] deaths and 171 million [144-201] DALYs), high body-mass index (BMI; 4.72 million [2.99-6.70] deaths and 148 million [98.6-202] DALYs), and short gestation for birthweight (1.43 million [1.36-1.51] deaths and 139 million [131-147] DALYs). In total, risk-attributable DALYs declined by 4.9% (3.3-6.5) between 2007 and 2017. In the absence of demographic changes (ie, population growth and ageing), changes in risk exposure and risk-deleted DALYs would have led to a 23.5% decline in DALYs during that period. Conversely, in the absence of changes in risk exposure and risk-deleted DALYs, demographic changes would have led to an 18.6% increase in DALYs during that period. The ratios of observed risk exposure levels to exposure levels expected based on SDI (O/E ratios) increased globally for unsafe drinking water and household air pollution between 1990 and 2017. This result suggests that development is occurring more rapidly than are changes in the underlying risk structure in a population. Conversely, nearly universal declines in O/E ratios for smoking and alcohol use indicate that, for a given SDI, exposure to these risks is declining. In 2017, the leading Level 4 risk factor for age-standardised DALY rates was high SBP in four super-regions: central Europe, eastern Europe, and central Asia; north Africa and Middle East; south Asia; and southeast Asia, east Asia, and Oceania. The leading risk factor in the high-income super-region was smoking, in Latin America and Caribbean was high BMI, and in sub-Saharan Africa was unsafe sex. O/E ratios for unsafe sex in sub-Saharan Africa were notably high, and those for alcohol use in north Africa and the Middle East were notably low. Interpretation: By quantifying levels and trends in exposures to risk factors and the resulting disease burden, this assessment offers insight into where past policy and programme efforts might have been successful and highlights current priorities for public health action. Decreases in behavioural, environmental, and occupational risks have largely offset the effects of population growth and ageing, in relation to trends in absolute burden. Conversely, the combination of increasing metabolic risks and population ageing will probably continue to drive the increasing trends in non-communicable diseases at the global level, which presents both a public health challenge and opportunity. We see considerable spatiotemporal heterogeneity in levels of risk exposure and risk-attributable burden. Although levels of development underlie some of this heterogeneity, O/E ratios show risks for which countries are overperforming or underperforming relative to their level of development. As such, these ratios provide a benchmarking tool to help to focus local decision making. Our findings reinforce the importance of both risk exposure monitoring and epidemiological research to assess causal connections between risks and health outcomes, and they highlight the usefulness of the GBD study in synthesising data to draw comprehensive and robust conclusions that help to inform good policy and strategic health planning

Customisation of the Exome Data Analysis Pipeline Using a Combinatorial Approach

The advent of next generation sequencing (NGS) technologies have revolutionised the way biologists produce, analyse and interpret data. Although NGS platforms provide a cost-effective way to discover genome-wide variants from a single experiment, variants discovered by NGS need follow up validation due to the high error rates associated with various sequencing chemistries. Recently, whole exome sequencing has been proposed as an affordable option compared to whole genome runs but it still requires follow up validation of all the novel exomic variants. Customarily, a consensus approach is used to overcome the systematic errors inherent to the sequencing technology, alignment and post alignment variant detection algorithms. However, the aforementioned approach warrants the use of multiple sequencing chemistry, multiple alignment tools, multiple variant callers which may not be viable in terms of time and money for individual investigators with limited informatics know-how. Biologists often lack the requisite training to deal with the huge amount of data produced by NGS runs and face difficulty in choosing from the list of freely available analytical tools for NGS data analysis. Hence, there is a need to customise the NGS data analysis pipeline to preferentially retain true variants by minimising the incidence of false positives and make the choice of right analytical tools easier. To this end, we have sampled different freely available tools used at the alignment and post alignment stage suggesting the use of the most suitable combination determined by a simple framework of pre-existing metrics to create significant datasets

Dysregulated Cytokine Expression by CD4+ T cells from Post-Septic Mice Modulates both Th1 and Th2-Mediated Granulomatous Lung Inflammation

Previous epidemiological studies in humans and experimental studies in animals indicate that survivors of severe sepsis exhibit deficiencies in the activation and effector function of immune cells. In particular, CD4+ T lymphocytes can exhibit reduced proliferative capacity and improper cytokine responses following sepsis. To further investigate the cell-intrinsic defects of CD4+ T cells following sepsis, splenic CD4+ T cells from sham surgery and post-septic mice were transferred into lymphopenic mice. These recipient mice were then subjected to both TH1-(purified protein derivative) and TH2-(Schistosoma mansoni egg antigen) driven models of granulomatous lung inflammation. Post-septic CD4+ T cells mediated smaller TH1 and larger TH2 lung granulomas as compared to mice receiving CD4+ T cells from sham surgery donors. However, cytokine production by lymph node cells in antigen restimulation assays indicated increased pan-specific cytokine expression by post-septic CD4+ T cell recipient mice in both TH1 and TH2 granuloma models. These include increased production of TH2 cytokines in TH1 inflammation, and increased production of TH1 cytokines in TH2 inflammation. These results suggest that cell-intrinsic defects in CD4+ T cell effector function can have deleterious effects on inflammatory processes post-sepsis, due to a defect in the proper regulation of TH-specific cytokine expression

Improved Xenobiotic Metabolism and Reduced Susceptibility to Cancer in Gluten-Sensitive Macaques upon Introduction of a Gluten-Free Diet

A non-human primate (NHP) model of gluten sensitivity was employed to study the gene perturbations associated with dietary gluten changes in small intestinal tissues from gluten-sensitive rhesus macaques (Macaca mulatta).Stages of remission and relapse were accomplished in gluten-sensitive animals by administration of gluten-free (GFD) and gluten-containing (GD) diets, as described previously. Pin-head-sized biopsies, obtained non-invasively by pediatric endoscope from duodenum while on GFD or GD, were used for preparation of total RNA and gene profiling, using the commercial Rhesus Macaque Microarray (Agilent Technologies),targeting expression of over 20,000 genes.When compared with normal healthy control, gluten-sensitive macaques showed differential gene expressions induced by GD. While observed gene perturbations were classified into one of 12 overlapping categories--cancer, metabolism, digestive tract function, immune response, cell growth, signal transduction, autoimmunity, detoxification of xenobiotics, apoptosis, actin-collagen deposition, neuronal and unknown function--this study focused on cancer-related gene networks such as cytochrome P450 family (detoxification function) and actin-collagen-matrix metalloproteinases (MMP) genes.A loss of detoxification function paralleled with necessity to metabolize carcinogens was revealed in gluten-sensitive animals while on GD. An increase in cancer-promoting factors and a simultaneous decrease in cancer-preventing factors associated with altered expression of actin-collagen-MMP gene network were noted. In addition, gluten-sensitive macaques showed reduced number of differentially expressed genes including the cancer-associated ones upon withdrawal of dietary gluten. Taken together, these findings indicate potentially expanded utility of gluten-sensitive rhesus macaques in cancer research

A draft of the genome and four transcriptomes of a medicinal and pesticidal angiosperm Azadirachta indica

Abstract Background The Azadirachta indica (neem) tree is a source of a wide number of natural products, including the potent biopesticide azadirachtin. In spite of its widespread applications in agriculture and medicine, the molecular aspects of the biosynthesis of neem terpenoids remain largely unexplored. The current report describes the draft genome and four transcriptomes of A. indica and attempts to contextualise the sequence information in terms of its molecular phylogeny, transcript expression and terpenoid biosynthesis pathways. A. indica is the first member of the family Meliaceae to be sequenced using next generation sequencing approach. Results The genome and transcriptomes of A. indica were sequenced using multiple sequencing platforms and libraries. The A. indica genome is AT-rich, bears few repetitive DNA elements and comprises about 20,000 genes. The molecular phylogenetic analyses grouped A. indica together with Citrus sinensis from the Rutaceae family validating its conventional taxonomic classification. Comparative transcript expression analysis showed either exclusive or enhanced expression of known genes involved in neem terpenoid biosynthesis pathways compared to other sequenced angiosperms. Genome and transcriptome analyses in A. indica led to the identification of repeat elements, nucleotide composition and expression profiles of genes in various organs. Conclusions This study on A. indica genome and transcriptomes will provide a model for characterization of metabolic pathways involved in synthesis of bioactive compounds, comparative evolutionary studies among various Meliaceae family members and help annotate their genomes. A better understanding of molecular pathways involved in the azadirachtin synthesis in A. indica will pave ways for bulk production of environment friendly biopesticides. </jats:sec

Acromioclavicular joint dislocation: a comparative biomechanical study of the palmaris-longus tendon graft reconstruction with other augmentative methods in cadaveric models

<p>Abstract</p> <p>Background</p> <p>Acromioclavicular injuries are common in sports medicine. Surgical intervention is generally advocated for chronic instability of Rockwood grade III and more severe injuries. Various methods of coracoclavicular ligament reconstruction and augmentation have been described. The objective of this study is to compare the biomechanical properties of a novel palmaris-longus tendon reconstruction with those of the native AC+CC ligaments, the modified Weaver-Dunn reconstruction, the ACJ capsuloligamentous complex repair, screw and clavicle hook plate augmentation.</p> <p>Hypothesis</p> <p>There is no difference, biomechanically, amongst the various reconstruction and augmentative methods.</p> <p>Study Design</p> <p>Controlled laboratory cadaveric study.</p> <p>Methods</p> <p>54 cadaveric native (acromioclavicular and coracoclavicular) ligaments were tested using the Instron machine. Superior loading was performed in the 6 groups: 1) in the intact states, 2) after modified Weaver-Dunn reconstruction (WD), 3) after modified Weaver-Dunn reconstruction with acromioclavicular joint capsuloligamentous repair (WD.ACJ), 4) after modified Weaver-Dunn reconstruction with clavicular hook plate augmentation (WD.CP) or 5) after modified Weaver-Dunn reconstruction with coracoclavicular screw augmentation (WD.BS) and 6) after modified Weaver-Dunn reconstruction with mersilene tape-palmaris-longus tendon graft reconstruction (WD. PLmt). Posterior-anterior (horizontal) loading was similarly performed in all groups, except groups 4 and 5. The respective failure loads, stiffnesses, displacements at failure and modes of failure were recorded. Data analysis was carried out using a one-way ANOVA, with Student's unpaired t-test for unpaired data (S-PLUS statistical package 2005).</p> <p>Results</p> <p>Native ligaments were the strongest and stiffest when compared to other modes of reconstruction and augmentation except coracoclavicular screw, in both posterior-anterior and superior directions (p < 0.005).</p> <p>WD.ACJ provided additional posterior-anterior (P = 0. 039) but not superior (p = 0.250) stability when compared to WD alone.</p> <p>WD+PLmt, in loads and stiffness at failure superiorly, was similar to WD+CP (p = 0.066). WD+PLmt, in loads and stiffness at failure postero-anteriorly, was similar to WD+ACJ (p = 0.084).</p> <p>Superiorly, WD+CP had similar strength as WD+BS (p = 0.057), but it was less stiff (p < 0.005).</p> <p>Conclusions and Clinical Relevance</p> <p>Modified Weaver-Dunn procedure must always be supplemented with acromioclavicular capsuloligamentous repair to increase posterior-anterior stability. Palmaris-Longus tendon graft provides both additional superior and posterior-anterior stability when used for acromioclavicular capsuloligamentous reconstruction. It is a good alternative to clavicle hook plate in acromioclavicular dislocation.</p

High Density Microarray Analysis Reveals New Insights into Genetic Footprints of Listeria monocytogenes Strains Involved in Listeriosis Outbreaks

Listeria monocytogenes, a foodborne bacterial pathogen, causes invasive and febrile gastroenteritis forms of listeriosis in humans. Both invasive and febrile gastroenteritis listeriosis is caused mostly by serotypes 1/2a, 1/2b and 4b strains. The outbreak strains of serotype 1/2a and 4b could be further classified into several epidemic clones but the genetic bases for the diverse pathophysiology have been unsuccessful. DNA microarray provides an important tool to scan the entire genome for genetic signatures that may distinguish the L. monocytogenes strains belonging to different outbreaks. We have designed a pan-genomic microarray chip (Listeria GeneChip) containing sequences from 24 L. monocytogenes strains. The chip was designed to identify the presence/absence of genomic sequences, analyze transcription profiles and identify SNPs. Analysis of the genomic profiles of 38 outbreak strains representing 1/2a, 1/2b and 4b serotypes, revealed that the strains formed distinct genetic clusters adhering to their serotypes and epidemic clone types. Although serologically 1/2a and 1/b strains share common antigenic markers microarray analysis revealed that 1/2a strains are further apart from the closely related 1/2b and 4b strains. Within any given serotype and epidemic clone type the febrile gastroenteritis and invasive strains can be further distinguished based on several genetic markers including large numbers of phage genome, and intergenic sequences. Our results showed that the microarray-based data can be an important tool in characterization of L. monocytogenes strains involved in both invasive and gastroenteritis outbreaks. The results for the first time showed that the serotypes and epidemic clones are based on extensive pan-genomic variability and the 1/2b and 4bstrains are more closely related to each other than the 1/2a strains. The data also supported the hypothesis that the strains causing these two diverse outbreaks are genotypically different and this finding might be important in understanding the pathophysiology of this organism