Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing >= 50% inhibition property against CHIKV at 10 mu M were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC50 values between 2.2 mu M and 7.1 mu M. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity -inhibition of virus-induced CPE - likely by targeting kinases involved in apoptosis.

High-throughput sugarcane leaf analysis using a low cost closed-vessel conductively heated digestion system and inductively coupled plasma optical emission spectroscopy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A high-throughput screening assay for distinguishing nitrile hydratases from nitrilases

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

High-throughput non-destructive nuclear magnetic resonance method to measure intramuscular fat content in beef

High intake of saturated fat from meats has been associated with cardiovascular disease, cancer, diabetes, and others diseases. In this paper, we are introducing a simple, high-throughput, and non-destructive low-resolution nuclear magnetic resonance method that has the potential to analyze the intramuscular fat content (IMF) in more than 1,000 beef portions per hour. The results can be used in nutritional fact labels, replacing the currently used average value. The method is based on longitudinal (T(1)) and transverse (T(2)) relaxation time information obtained by a continuous wave-free precession (CWFP) sequence. CWFP yields a higher correlation coefficient (r=0.9) than the conventional Carr-Purcell-Meiboom- Gill (CPMG) method (r=-0.25) for IMF in beef and is just as fast and a simpler pulse sequence than CPMG. The method can also be applied to other meat products.

Application of an active attachment model as a high-throughput demineralization biofilm model

Objectives: To investigate the potential of an active attachment biofilm model as a highthroughput demineralization biofilm model for the evaluation of caries-preventive agents. Methods: Streptococcus mutans UA159 biofilms were grown on bovine dentine discs in a highthroughput active attachment model. Biofilms were first formed in a medium with high buffer capacity for 24 h and then subjected to various photodynamic therapies (PACT) using the combination of Light Emitting Diodes (LEDs, Biotable (R)) and Photogem (R). Viability of the biofilms was evaluated by plate counts. To investigate treatment effects on dentine lesion formation, the treated biofilms were grown in a medium with low buffer capacity for an additional 24 h. Integrated mineral loss (IML) and lesion depth (LD) were assessed by transversal microradiography. Calcium release in the biofilm medium was measured by atomic absorption spectroscopy. Results: Compared to the water treated control group, significant reduction in viability of S. mutans biofilms was observed when the combination of LEDs and Photogem (R) was applied. LEDs or Photogem (R) only did not result in biofilm viability changes. Similar outcomes were also found for dentine lesion formation. Significant lower IML and LD values were only found in the group subjected to the combined treatment of LEDs and Photogem (R). There was a good correlation between the calcium release data and the IML or LD values. Conclusions: The high-throughput active attachment biofilm model is applicable for evaluating novel caries-preventive agents on both biofilm and demineralization inhibition. PACT had a killing effect on 24 h S. mutans biofilms and could inhibit the demineralization process. (C) 2011 Elsevier Ltd. All rights reserved.

High-throughput sequencing of black pepper root transcriptome

Background: Black pepper (Piper nigrum L.) is one of the most popular spices in the world. It is used in cooking and the preservation of food and even has medicinal properties. Losses in production from disease are a major limitation in the culture of this crop. The major diseases are root rot and foot rot, which are results of root infection by Fusarium solani and Phytophtora capsici, respectively. Understanding the molecular interaction between the pathogens and the host's root region is important for obtaining resistant cultivars by biotechnological breeding. Genetic and molecular data for this species, though, are limited. In this paper, RNA-Seq technology has been employed, for the first time, to describe the root transcriptome of black pepper. Results: The root transcriptome of black pepper was sequenced by the NGS SOLiD platform and assembled using the multiple-k method. Blast2Go and orthoMCL methods were used to annotate 10338 unigenes. The 4472 predicted proteins showed about 52% homology with the Arabidopsis proteome. Two root proteomes identified 615 proteins, which seem to define the plant's root pattern. Simple-sequence repeats were identified that may be useful in studies of genetic diversity and may have applications in biotechnology and ecology. Conclusions: This dataset of 10338 unigenes is crucially important for the biotechnological breeding of black pepper and the ecogenomics of the Magnoliids, a major group of basal angiosperms.

Translocation capture sequencing: A method for high throughput mapping of chromosomal rearrangements

Chromosomal translocations require formation and joining of DNA double strand breaks (DSBs). These events disrupt the integrity of the genome and are involved in producing leukemias, lymphomas and sarcomas. Translocations are frequent, clonal and recurrent in mature B cell lymphomas, which bear a particularly high DNA damage burden by virtue of activation-induced cytidine deaminase (AID) expression. Despite the ubiquity of genomic rearrangements, the forces that underlie their genesis are not well understood. Here, we provide a detailed description of a new method for studying these events, translocation capture sequencing (TC-Seq). TC-Seq provides the means to document chromosomal rearrangements genome-wide in primary cells, and to discover recombination hotspots. Demonstrating its effectiveness, we successfully estimate the frequency of c-myc/IgH translocations in primary B cells, and identify hotspots of AID-mediated recombination. Furthermore. TC-Seq can be adapted to generate genome-wide rearrangement maps in any cell type and under any condition. (C) 2011 Elsevier B.V. All rights reserved.

High-throughput sequencing of small RNA transcriptomes reveals critical biological features targeted by microRNAs in cell models used for squamous cell cancer research

Abstract Background The implication of post-transcriptional regulation by microRNAs in molecular mechanisms underlying cancer disease is well documented. However, their interference at the cellular level is not fully explored. Functional in vitro studies are fundamental for the comprehension of their role; nevertheless results are highly dependable on the adopted cellular model. Next generation small RNA transcriptomic sequencing data of a tumor cell line and keratinocytes derived from primary culture was generated in order to characterize the microRNA content of these systems, thus helping in their understanding. Both constitute cell models for functional studies of microRNAs in head and neck squamous cell carcinoma (HNSCC), a smoking-related cancer. Known microRNAs were quantified and analyzed in the context of gene regulation. New microRNAs were investigated using similarity and structural search, ab initio classification, and prediction of the location of mature microRNAs within would-be precursor sequences. Results were compared with small RNA transcriptomic sequences from HNSCC samples in order to access the applicability of these cell models for cancer phenotype comprehension and for novel molecule discovery. Results Ten miRNAs represented over 70% of the mature molecules present in each of the cell types. The most expressed molecules were miR-21, miR-24 and miR-205, Accordingly; miR-21 and miR-205 have been previously shown to play a role in epithelial cell biology. Although miR-21 has been implicated in cancer development, and evaluated as a biomarker in HNSCC progression, no significant expression differences were seen between cell types. We demonstrate that differentially expressed mature miRNAs target cell differentiation and apoptosis related biological processes, indicating that they might represent, with acceptable accuracy, the genetic context from which they derive. Most miRNAs identified in the cancer cell line and in keratinocytes were present in tumor samples and cancer-free samples, respectively, with miR-21, miR-24 and miR-205 still among the most prevalent molecules at all instances. Thirteen miRNA-like structures, containing reads identified by the deep sequencing, were predicted from putative miRNA precursor sequences. Strong evidences suggest that one of them could be a new miRNA. This molecule was mostly expressed in the tumor cell line and HNSCC samples indicating a possible biological function in cancer. Conclusions Critical biological features of cells must be fully understood before they can be chosen as models for functional studies. Expression levels of miRNAs relate to cell type and tissue context. This study provides insights on miRNA content of two cell models used for cancer research. Pathways commonly deregulated in HNSCC might be targeted by most expressed and also by differentially expressed miRNAs. Results indicate that the use of cell models for cancer research demands careful assessment of underlying molecular characteristics for proper data interpretation. Additionally, one new miRNA-like molecule with a potential role in cancer was identified in the cell lines and clinical samples.

Sviluppo di un modulo VHDL per interfacciamento Ethernet-FPGA in applicazioni di high throughput screening

In molti settori della ricerca in campo biologico e biomedico si fa ricorso a tecniche di High Throughput Screening (HTS), tra cui studio dei canali ionici. In questo campo si studia la conduzione di ioni attraverso una membrana cellulare durante fenomeni che durano solo alcuni millisecondi. Allo scopo sono solitamente usati sensori e convertitori A/D ad elevata velocità insieme ad opportune interfacce di comunicazione, ad elevato bit-rate e latenza ridotta. In questa tesi viene descritta l'implementazione di un modulo VHDL per la trasmissione di dati digitali provenienti da un sistema HTS attraverso un controller di rete integrato dotato di un'interfaccia di tipo Ethernet, individuando le possibili ottimizzazioni specifiche per l'applicazione di interesse.

Wide-scale population genomics of Atlantic bluefin tuna (Thunnus thynnus) inferred by novel high-throughput technology

My PhD project was focused on Atlantic bluefin tuna, Thunnus thynnus, a fishery resource overexploited in the last decades. For a better management of stocks, it was necessary to improve scientific knowledge of this species and to develop novel tools to avoid collapse of this important commercial resource. To do this, we used new high throughput sequencing technologies, as Next Generation Sequencing (NGS), and markers linked to expressed genes, as SNPs (Single Nucleotide Polymorphisms). In this work we applied a combined approach: transcriptomic resources were used to build cDNA libreries from mRNA isolated by muscle, and genomic resources allowed to create a reference backbone for this species lacking of reference genome. All cDNA reads, obtained from mRNA, were mapped against this genome and, employing several bioinformatics tools and different restricted parameters, we achieved a set of contigs to detect SNPs. Once a final panel of 384 SNPs was developed, following the selection criteria, it was genotyped in 960 individuals of Atlantic bluefin tuna, including all size/age classes, from larvae to adults, collected from the entire range of the species. The analysis of obtained data was aimed to evaluate the genetic diversity and the population structure of Thunnus thynnus. We detect a low but significant signal of genetic differentiation among spawning samples, that can suggest the presence of three genetically separate reproduction areas. The adult samples resulted instead genetically undifferentiated between them and from the spawning populations, indicating a presence of panmictic population of adult bluefin tuna in the Mediterranean Sea, without different meta populations.

Multiplex network analysis with application to biological high-throughput data

In questa tesi vengono studiate alcune caratteristiche dei network a multiplex; in particolare l'analisi verte sulla quantificazione delle differenze fra i layer del multiplex. Le dissimilarita sono valutate sia osservando le connessioni di singoli nodi in layer diversi, sia stimando le diverse partizioni dei layer. Sono quindi introdotte alcune importanti misure per la caratterizzazione dei multiplex, che vengono poi usate per la costruzione di metodi di community detection . La quantificazione delle differenze tra le partizioni di due layer viene stimata utilizzando una misura di mutua informazione. Viene inoltre approfondito l'uso del test dell'ipergeometrica per la determinazione di nodi sovra-rappresentati in un layer, mostrando l'efficacia del test in funzione della similarita dei layer. Questi metodi per la caratterizzazione delle proprieta dei network a multiplex vengono applicati a dati biologici reali. I dati utilizzati sono stati raccolti dallo studio DILGOM con l'obiettivo di determinare le implicazioni genetiche, trascrittomiche e metaboliche dell'obesita e della sindrome metabolica. Questi dati sono utilizzati dal progetto Mimomics per la determinazione di relazioni fra diverse omiche. Nella tesi sono analizzati i dati metabolici utilizzando un approccio a multiplex network per verificare la presenza di differenze fra le relazioni di composti sanguigni di persone obese e normopeso.

High-throughput procedure for tick surveys of tick-borne encephalitis virus and its application in a national surveillance study in Switzerland

Tick-borne encephalitis (TBE), a viral infection of the central nervous system, is endemic in many Eurasian countries. In Switzerland, TBE risk areas have been characterized by geographic mapping of clinical cases. Since mass vaccination should significantly decrease the number of TBE cases, alternative methods for exposure risk assessment are required. We established a new PCR-based test for the detection of TBE virus (TBEV) in ticks. The protocol involves an automated, high-throughput nucleic acid extraction method (QIAsymphony SP system) and a one-step duplex real-time reverse transcription-PCR (RT-PCR) assay for the detection of European subtype TBEV, including an internal process control. High usability, reproducibility, and equivalent performance for virus concentrations down to 5 x 10(3) viral genome equivalents/microl favor the automated protocol compared to the modified guanidinium thiocyanate-phenol-chloroform extraction procedure. The real-time RT-PCR allows fast, sensitive (limit of detection, 10 RNA copies/microl), and specific (no false-positive test results for other TBEV subtypes, other flaviviruses, or other tick-transmitted pathogens) detection of European subtype TBEV. The new detection method was applied in a national surveillance study, in which 62,343 Ixodes ricinus ticks were screened for the presence of TBE virus. A total of 38 foci of endemicity could be identified, with a mean virus prevalence of 0.46%. The foci do not fully agree with those defined by disease mapping. Therefore, the proposed molecular test procedure constitutes a prerequisite for an appropriate TBE surveillance. Our data are a unique complement of human TBE disease case mapping in Switzerland.

High-throughput mutation profiling of CTCL samples reveals KRAS and NRAS mutations sensitizing tumors toward inhibition of the RAS/RAF/MEK signaling cascade

Cutaneous T-cell lymphomas (CTCLs) are malignancies of skin-homing lymphoid cells, which have so far not been investigated thoroughly for common oncogenic mutations. We screened 90 biopsy specimens from CTCL patients (41 mycosis fungoides, 36 Sézary syndrome, and 13 non-mycosis fungoides/Sézary syndrome CTCL) for somatic mutations using OncoMap technology. We detected oncogenic mutations for the RAS pathway in 4 of 90 samples. One mycosis fungoides and one pleomorphic CTCL harbored a KRAS(G13D) mutation; one Sézary syndrome and one CD30(+) CTCL harbored a NRAS(Q61K) amino acid change. All mutations were found in stage IV patients (4 of 42) who showed significantly decreased overall survival compared with stage IV patients without mutations (P = .04). In addition, we detected a NRAS(Q61K) mutation in the CTCL cell line Hut78. Knockdown of NRAS by siRNA induced apoptosis in mutant Hut78 cells but not in CTCL cell lines lacking RAS mutations. The NRAS(Q61K) mutation sensitized Hut78 cells toward growth inhibition by the MEK inhibitors U0126, AZD6244, and PD0325901. Furthermore, we found that MEK inhibitors exclusively induce apoptosis in Hut78 cells. Taken together, we conclude that RAS mutations are rare events at a late stage of CTCL, and our preclinical results suggest that such late-stage patients profit from MEK inhibitors.