Cas9 gRNA engineering for genome editing, activation and repression

We demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.National Human Genome Research Institute (U.S.) (P50 HG005550)United States. Department of Energy (DE-FG02-02ER63445)Wyss Institute for Biologically Inspired EngineeringUnited States. Army Research Office (DARPA W911NF-11-2-0054)National Science Foundation (U.S.)United States. National Institutes of Health (5R01CA155320-04)United States. National Institutes of Health (P50 GM098792)National Cancer Institute (U.S.) (5T32CA009216-34)Massachusetts Institute of Technology. Department of Biological EngineeringHarvard Medical School. Department of GeneticsDefense Threat Reduction Agency (DTRA) (HDTRA1-14-1-0006

Improved ability of biological and previous caries multimarkers to predict caries disease as revealed by multivariate PLS modelling

<p>Abstract</p> <p>Background</p> <p>Dental caries is a chronic disease with plaque bacteria, diet and saliva modifying disease activity. Here we have used the PLS method to evaluate a multiplicity of such biological variables (n = 88) for ability to predict caries in a cross-sectional (baseline caries) and prospective (2-year caries development) setting.</p> <p>Methods</p> <p>Multivariate PLS modelling was used to associate the many biological variables with caries recorded in thirty 14-year-old children by measuring the numbers of incipient and manifest caries lesions at all surfaces.</p> <p>Results</p> <p>A wide but shallow gliding scale of one fifth caries promoting or protecting, and four fifths non-influential, variables occurred. The influential markers behaved in the order of plaque bacteria > diet > saliva, with previously known plaque bacteria/diet markers and a set of new protective diet markers. A differential variable patterning appeared for new versus progressing lesions. The influential biological multimarkers (n = 18) predicted baseline caries better (ROC area 0.96) than five markers (0.92) and a single lactobacilli marker (0.7) with sensitivity/specificity of 1.87, 1.78 and 1.13 at 1/3 of the subjects diagnosed sick, respectively. Moreover, biological multimarkers (n = 18) explained 2-year caries increment slightly better than reported before but predicted it poorly (ROC area 0.76). By contrast, multimarkers based on previous caries predicted alone (ROC area 0.88), or together with biological multimarkers (0.94), increment well with a sensitivity/specificity of 1.74 at 1/3 of the subjects diagnosed sick.</p> <p>Conclusion</p> <p>Multimarkers behave better than single-to-five markers but future multimarker strategies will require systematic searches for improved saliva and plaque bacteria markers.</p

Prediction of Extracellular Proteases of the Human Pathogen Helicobacter pylori Reveals Proteolytic Activity of the Hp1018/19 Protein HtrA

Exported proteases of Helicobacter pylori (H. pylori) are potentially involved in pathogen-associated disorders leading to gastric inflammation and neoplasia. By comprehensive sequence screening of the H. pylori proteome for predicted secreted proteases, we retrieved several candidate genes. We detected caseinolytic activities of several such proteases, which are released independently from the H. pylori type IV secretion system encoded by the cag pathogenicity island (cagPAI). Among these, we found the predicted serine protease HtrA (Hp1019), which was previously identified in the bacterial secretome of H. pylori. Importantly, we further found that the H. pylori genes hp1018 and hp1019 represent a single gene likely coding for an exported protein. Here, we directly verified proteolytic activity of HtrA in vitro and identified the HtrA protease in zymograms by mass spectrometry. Overexpressed and purified HtrA exhibited pronounced proteolytic activity, which is inactivated after mutation of Ser205 to alanine in the predicted active center of HtrA. These data demonstrate that H. pylori secretes HtrA as an active protease, which might represent a novel candidate target for therapeutic intervention strategies

Diabetes-related molecular signatures in infrared spectra of human saliva

WOS: 000290261500001PubMed ID: 20630088Background: There is an ongoing need for improvements in non-invasive, point-of-care tools for the diagnosis and prognosis of diabetes mellitus. Ideally, such technologies would allow for community screening. Methods: In this study, we employed infrared spectroscopy as a novel diagnostic tool in the prediction of diabetic status by analyzing the molecular and sub-molecular spectral signatures of saliva collected from subjects with diabetes (n = 39) and healthy controls (n = 22). Results: Spectral analysis revealed differences in several major metabolic components - lipid, proteins, glucose, thiocyanate and carboxylate - that clearly demarcate healthy and diseased saliva. The overall accuracy for the diagnosis of diabetes based on infrared spectroscopy was 100% on the training set and 88.2% on the validation set. Therefore, we have established that infrared spectroscopy can be used to generate complex biochemical profiles in saliva and identify several potential diabetes-associated spectral features. Conclusions: Infrared spectroscopy may represent an appropriate tool with which to identify novel diseases mechanisms, risk factors for diabetic complications and markers of therapeutic efficacy. Further study into the potential utility of infrared spectroscopy as diagnostic and prognostic tool for diabetes is warranted

Explorative visual analytics on interval-based genomic data and their metadata

Background: With the wide-spreading of public repositories of NGS processed data, the availability of user-friendly and effective tools for data exploration, analysis and visualization is becoming very relevant. These tools enable interactive analytics, an exploratory approach for the seamless "sense-making" of data through on-the-fly integration of analysis and visualization phases, suggested not only for evaluating processing results, but also for designing and adapting NGS data analysis pipelines. Results: This paper presents abstractions for supporting the early analysis of NGS processed data and their implementation in an associated tool, named GenoMetric Space Explorer (GeMSE). This tool serves the needs of the GenoMetric Query Language, an innovative cloud-based system for computing complex queries over heterogeneous processed data. It can also be used starting from any text files in standard BED, BroadPeak, NarrowPeak, GTF, or general tab-delimited format, containing numerical features of genomic regions; metadata can be provided as text files in tab-delimited attribute-value format. GeMSE allows interactive analytics, consisting of on-the-fly cycling among steps of data exploration, analysis and visualization that help biologists and bioinformaticians in making sense of heterogeneous genomic datasets. By means of an explorative interaction support, users can trace past activities and quickly recover their results, seamlessly going backward and forward in the analysis steps and comparative visualizations of heatmaps. Conclusions: GeMSE effective application and practical usefulness is demonstrated through significant use cases of biological interest. GeMSE is available at http://www.bioinformatics.deib.polimi.it/GeMSE/ , and its source code is available at https://github.com/Genometric/GeMSEunder GPLv3 open-source license

Parameters for accurate genome alignment

<p>Abstract</p> <p>Background</p> <p>Genome sequence alignments form the basis of much research. Genome alignment depends on various mundane but critical choices, such as how to mask repeats and which score parameters to use. Surprisingly, there has been no large-scale assessment of these choices using real genomic data. Moreover, rigorous procedures to control the rate of spurious alignment have not been employed.</p> <p>Results</p> <p>We have assessed 495 combinations of score parameters for alignment of animal, plant, and fungal genomes. As our gold-standard of accuracy, we used genome alignments implied by multiple alignments of proteins and of structural RNAs. We found the HOXD scoring schemes underlying alignments in the UCSC genome database to be far from optimal, and suggest better parameters. Higher values of the X-drop parameter are not always better. E-values accurately indicate the rate of spurious alignment, but only if tandem repeats are masked in a non-standard way. Finally, we show that γ-centroid (probabilistic) alignment can find highly reliable subsets of aligned bases.</p> <p>Conclusions</p> <p>These results enable more accurate genome alignment, with reliability measures for local alignments and for individual aligned bases. This study was made possible by our new software, LAST, which can align vertebrate genomes in a few hours <url>http://last.cbrc.jp/</url>.</p

Activating Transcription Factor 4 Confers a Multidrug Resistance Phenotype to Gastric Cancer Cells through Transactivation of SIRT1 Expression

BACKGROUND: Multidrug resistance (MDR) in gastric cancer remains a major challenge to clinical treatment. Activating transcription factor 4 (ATF4) is a stress response gene involved in homeostasis and cellular protection. However, the expression and function of ATF4 in gastric cancer MDR remains unknown. In this study, we investigate whether ATF4 play a role in gastric cancer MDR and its potential mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that ATF4 overexpression confered the MDR phenotype to gastric cancer cells, while knockdown of ATF4 in the MDR variants induced re-sensitization. In this study we also showed that the NAD(+)-dependent histone deacetylase SIRT1 was required for ATF4-induced MDR effect in gastric cancer cells. We demonstrated that ATF4 facilitated MDR in gastric cancer cells through direct binding to the SIRT1 promoter, resulting in SIRT1 up-regulation. Significantly, inhibition of SIRT1 by small interfering RNA (siRNA) or a specific inhibitor (EX-527) reintroduced therapeutic sensitivity. Also, an increased Bcl-2/Bax ratio and MDR1 expression level were found in ATF4-overexpressing cells. CONCLUSIONS/SIGNIFICANCE: We showed that ATF4 had a key role in the regulation of MDR in gastric cancer cells in response to chemotherapy and these findings suggest that targeting ATF4 could relieve therapeutic resistance in gastric cancer

Proteomic and Phospho-Proteomic Profile of Human Platelets in Basal, Resting State: Insights into Integrin Signaling

During atherogenesis and vascular inflammation quiescent platelets are activated to increase the surface expression and ligand affinity of the integrin αIIbβ3 via inside-out signaling. Diverse signals such as thrombin, ADP and epinephrine transduce signals through their respective GPCRs to activate protein kinases that ultimately lead to the phosphorylation of the cytoplasmic tail of the integrin αIIbβ3 and augment its function. The signaling pathways that transmit signals from the GPCR to the cytosolic domain of the integrin are not well defined. In an effort to better understand these pathways, we employed a combination of proteomic profiling and computational analyses of isolated human platelets. We analyzed ten independent human samples and identified a total of 1507 unique proteins in platelets. This is the most comprehensive platelet proteome assembled to date and includes 190 membrane-associated and 262 phosphorylated proteins, which were identified via independent proteomic and phospho-proteomic profiling. We used this proteomic dataset to create a platelet protein-protein interaction (PPI) network and applied novel contextual information about the phosphorylation step to introduce limited directionality in the PPI graph. This newly developed contextual PPI network computationally recapitulated an integrin signaling pathway. Most importantly, our approach not only provided insights into the mechanism of integrin αIIbβ3 activation in resting platelets but also provides an improved model for analysis and discovery of PPI dynamics and signaling pathways in the future

An Evolutionarily Conserved Arginine Is Essential for Tre1 G Protein-Coupled Receptor Function During Germ Cell Migration in Drosophila melanogaster

BACKGROUND: G protein-coupled receptors (GPCRs) play central roles in mediating cellular responses to environmental signals leading to changes in cell physiology and behaviors, including cell migration. Numerous clinical pathologies including metastasis, an invasive form of cell migration, have been linked to abnormal GPCR signaling. While the structures of some GPCRs have been defined, the in vivo roles of conserved amino acid residues and their relationships to receptor function are not fully understood. Trapped in endoderm 1 (Tre1) is an orphan receptor of the rhodopsin class that is necessary for primordial germ cell migration in Drosophila melanogaster embryos. In this study, we employ molecular genetic approaches to identify residues in Tre1 that are critical to its functions in germ cell migration. METHODOLOGY/PRINCIPAL FINDINGS: First, we show that the previously reported scattershot mutation is an allele of tre1. The scattershot allele results in an in-frame deletion of 8 amino acids at the junction of the third transmembrane domain and the second intracellular loop of Tre1 that dramatically impairs the function of this GPCR in germ cell migration. To further refine the molecular basis for this phenotype, we assayed the effects of single amino acid substitutions in transgenic animals and determined that the arginine within the evolutionarily conserved E/N/DRY motif is critical for receptor function in mediating germ cell migration within an intact developing embryo. CONCLUSIONS/SIGNIFICANCE: These structure-function studies of GPCR signaling in native contexts will inform future studies into the basic biology of this large and clinically important family of receptors