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Supplementary Figure 10 from Molecular Classifiers for Gastric Cancer and Nonmalignant Diseases of the Gastric Mucosa

Supplementary Figure 7 from Molecular Classifiers for Gastric Cancer and Nonmalignant Diseases of the Gastric Mucosa

Supplementary Figure 2 from Molecular Classifiers for Gastric Cancer and Nonmalignant Diseases of the Gastric Mucosa

Supplementary Figure 5 from Molecular Classifiers for Gastric Cancer and Nonmalignant Diseases of the Gastric Mucosa

Supplementary Figure 6 from Molecular Classifiers for Gastric Cancer and Nonmalignant Diseases of the Gastric Mucosa

Supplementary Figure 9 from Molecular Classifiers for Gastric Cancer and Nonmalignant Diseases of the Gastric Mucosa

Supplementary Figure 4 from Molecular Classifiers for Gastric Cancer and Nonmalignant Diseases of the Gastric Mucosa

High incidence of gastric cancer-related death is mainly due to diagnosis at an advanced stage in addition to the lack of adequate neoadjuvant therapy. Hence, new tools aimed at early diagnosis would have a positive impact in the outcome of the disease. Using cDNA arrays having 376 genes either identified previously as altered in gastric tumors or known to be altered in human cancer, we determined expression signature of 99 tissue fragments representing normal gastric mucosa, gastritis, intestinal metaplasia, and adenocarcinomas. We first validated the array by identifying molecular markers that are associated with intestinal metaplasia, considered as a transition stage of gastric adenocarcinomas of the intestinal type as well as markers that are associated with diffuse type of gastric adenocarcinomas. Next, we applied Fisher’s linear discriminant analysis in an exhaustive search of trios of genes that could be used to build classifiers for class distinction. Many classifiers could distinguish between normal and tumor samples, whereas, for the distinction of gastritis from tumor and for metaplasia from tumor, fewer classifiers were identified. Statistical validations showed that trios that discriminate between normal and tumor samples are powerful classifiers to distinguish between tumor and nontumor samples. More relevant, it was possible to identify samples of intestinal metaplasia that have expression signature resembling that of an adenocarcinoma and can now be used for follow-up of patients to determine their potential as a prognostic test for malignant transformation.

BackgroundSince the emergence of the COVID-19, health officials have struggled to devise strategies to counteract the speed of the pandemic’s spread across the globe. It became imperative to implement accurate diagnostic tests for the detection of SARS-CoV-2 RNA on respiratory samples. In many places, however, besides the limited availability of test reagents, laboratory personnel face the challenge of adapting their working routines to manipulate highly infective clinical samples. Here, we proposed the use of a virus-inactivating solution as part of a sample collection kit to decrease the infectious potential of the collected material without affecting the integrity of RNA samples used in diagnostic tests based on RT-qPCR.MethodsNasopharyngeal and oropharyngeal swab samples were collected from SARS-CoV-2-infected patients and from laboratory personnel using a commercially available viral transport solution (VTM) and the denaturing solution (DS) described here. RNA extracted from all samples was tested by RT-qPCR using probes for viral and human genes. Exposure of laboratory personnel to infective viruses was also accessed using ELISA tests.FindingsThe use of the DS did not interfere with the detection of viral genome or the endogenous human mRNA, since similar results were obtained from samples collected with VTM or DS. In addition, all tests of laboratory personnel for the presence of viral RNA and IgG antibodies against SARS-CoV-2 were negative.InterpretationThe methodology described here provides a strategy that allow high diagnostic accuracy as well as safe manipulation of clinical samples by those involved with diagnostic procedures.FundingCAPES, FAPEMIG, CNPq, MCTIC, FIOCRUZ and the UK Global Challenges Research Fund (GCRF).

Individuals with asymptomatic infection due to Plasmodium vivax are posited to be important reservoirs of malaria transmission in endemic regions. Here we studied a cohort of P. vivax malaria patients in a suburban area in the Brazilian Amazon. Overall 1,120 individuals were screened for P. vivax infection and 108 (9.6%) had parasitemia detected by qPCR but not by microscopy. Asymptomatic individuals had higher levels of antibodies against P. vivax and similar hematological and biochemical parameters compared to uninfected controls. Blood from asymptomatic individuals with very low parasitemia transmitted P. vivax to the main local vector, Nyssorhynchus darlingi. Lower mosquito infectivity rates were observed when blood from asymptomatic individuals was used in the membrane feeding assay. While blood from symptomatic patients infected 43.4% (199/458) of the mosquitoes, blood from asymptomatic infected 2.5% (43/1,719). However, several asymptomatic individuals maintained parasitemia for several weeks indicating their potential role as an infectious reservoir. These results suggest that asymptomatic individuals are an important source of malaria parasites and Science and Technology for Vaccines granted by Conselho Nacional de may contribute to the transmission of P. vivax in low-endemicity areas of malaria., Author summary Malaria still poses as one of the most important parasitic diseases in the world. The advance of molecular diagnosis brought to light the existence of asymptomatic infections, which may represent most of the infections in some areas. Importantly, the role of asymptomatic carriers in the natural history of malaria is not completely understood. Herein we describe the general characteristics of asymptomatic individuals infected with Plasmodium vivax, and provide evidence of their potential as parasitic reservoirs, even when molecular methods fail to detect the infection. Our findings reinforce the need for better diagnostic tests and open a new window of complexity to be considered in control programs.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-CoV-2 RNA is an essential test to monitor the occurrence of COVID-19. A methodology is proposed for the determination of maximum pool size and adjustments of cut-off values of cycle threshold (Ct in RT-qPCR pool testing, to compensate for the dilution caused by pooling. The trade-off between pool size and test sensitivity is stated explicitly. The procedure was designed to ensure that samples that would be detectable in individual testing remain detectable in pool testing. The proposed relaxation in cut-off is dependent on the pool size, allowing a relatively tight correction to avoid loss of detection of positive samples. The methodology was evaluated in a study of pool testing of adults attending a public emergency care unit, reference for COVID-19 in Belo Horizonte, Brazil, and presenting flu-like symptoms. Even samples on the edge of detectability in individual testing were detected correctly. The proposed procedure enhances the consistency of RT-qPCR pool testing by enforcing that the scales of detectability in pool processing and in individual sample processing are compatible. This may enhance the contribution of pool testing to large-scale testing for COVID-19.

Corynebacterium pseudotuberculosis is a pathogenic bacterium which has been rapidly spreading all over the world, causing economic losses in the agricultural sector and sporadically infecting humans. Six C. pseudotuberculosis strains were isolated from goats, sheep, and horses with distinct abscess locations. For the first time, Mexican genomes of this bacterium were sequenced and studied in silico. All strains were sequenced using Ion Personal Genome Machine sequencer, assembled using Newbler and SPAdes software. The automatic genome annotation was done using the software RAST and in-house scripts for transference, followed by manual curation using Artemis software and BLAST against NCBI and UniProt databases. The six genomes are publicly available in NCBI database. The analysis of nucleotide sequence similarity and the generated phylogenetic tree led to the observation that the Mexican strains are more similar between strains from the same host, but the genetic structure is probably more influenced by transportation of animals between farms than host preference. Also, a putative drug target was predicted and in silico analysis of 46 strains showed two gene clusters capable of differentiating the biovars equi and ovis: Restriction Modification system and CRISPR-Cas cluster. Electronic supplementary material The online version of this article (10.1186/s40793-018-0325-z) contains supplementary material, which is available to authorized users.

Corynebacterium pseudotuberculosis has been widely studied in an effort to understand its biological evolution. Transcriptomics has revealed possible candidates for virulence and pathogenicity factors of strain 1002 (biovar Ovis). Because C. pseudotuberculosis is classified into two biovars, Ovis and Equi, it was interesting to assess the transcriptional profile of biovar Equi strain 258, the causative agent of ulcerative lymphangitis. The genome of this strain was re-sequenced; the reassembly was completed using optical mapping technology, and the sequence was subsequently re-annotated. Two growth conditions that occur during the host infection process were simulated for the transcriptome: the osmotic and acid medium. Genes that may be associated with the microorganism's resilience under unfavorable conditions were identified through RNAseq, including genes present in pathogenicity islands. The RT-qPCR was performed to confirm the results in biological triplicate for each condition for some genes. The results extend our knowledge of the factors associated with the spread and persistence of C. pseudotuberculosis during the infection process and suggest possible avenues for studies related to the development of vaccines, diagnosis, and therapies that might help minimize damage to agribusinesses. Fil: Gomide, Anne Cybelle Pinto. Universidade Federal de Minas Gerais; Brasil Fil: Ibraim, Izabela Coimbra. Universidade Federal de Minas Gerais; Brasil Fil: Alves, Jorianne T. C.. Universidade Federal do Pará; Brasil Fil: de Sá, Pablo Gomes. Federal Rural University Of Amazonia; Brasil Fil: de Oliveira Silva, Yuri Rafael. Universidade Federal do Pará; Brasil Fil: Santana, Mariana Passos. Universidade Federal de Minas Gerais; Brasil Fil: Marques Da Silva, Wanderson. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Folador, Edson Luiz. Universidade Federal Da Paraiba; Brasil Fil: Mariano, Diego C. B.. Universidade Federal de Minas Gerais; Brasil Fil: de Paula Castro, Thiago Luiz. Universidade Federal da Bahia; Brasil Fil: Barbosa, Silvanira. Universidade Federal do Pará; Brasil Fil: Dorella, Fernanda Alves. Universidade Federal de Minas Gerais; Brasil Fil: Carvalho, Alex F.. Universidade Federal de Minas Gerais; Brasil Fil: Pereira, Felipe L.. Universidade Federal de Minas Gerais; Brasil Fil: Leal, Carlos A. G.. Universidade Federal de Minas Gerais; Brasil Fil: Figueiredo, Henrique C. P.. Universidade Federal de Minas Gerais; Brasil Fil: Azevedo, Vasco. Universidade Federal de Minas Gerais; Brasil Fil: Silva, Artur. Universidade Federal do Pará; Brasil Fil: Folador, Adriana Ribeiro Carneiro. Universidade Federal do Pará; Brasil

Streptococcus agalactiae is one of the most important pathogens associated with streptococcosis outbreaks in Nile tilapia farms worldwide. High water temperature (above 27°C) has been described as a predisposing factor for the disease in fish. At low temperatures (below 25°C), fish mortalities are not usually observed in farms. Temperature variation can modulate the expression of genes and proteins involved in metabolism, adaptation, and bacterial pathogenicity, thus increasing or decreasing the ability to infect the host. This study aimed to evaluate the transcriptome and proteome of a fish-pathogenic S. agalactiae strain SA53 subjected to in vitro growth at different temperatures using a microarray and label-free shotgun LC-HDMSE approach. Biological triplicates of isolates were cultured in BHIT broth at 22 or 32°C for RNA and protein isolation and submitted for transcriptomic and proteomic analyses. In total, 1,730 transcripts were identified in SA53, with 107 genes being differentially expressed between the temperatures evaluated. A higher number of genes related to metabolism, mainly from the phosphotransferase system (PTS) and ATP-binding cassette (ABC) transport system, were upregulated at 32°C. In the proteome analysis, 1,046 proteins were identified in SA53, of which 81 were differentially regulated between 22 and 32°C. Proteins involved in defense mechanisms, lipid transport and metabolism, and nucleotide transport and metabolism were upregulated at 32°C. A higher number of interactions were observed in proteins involved in nucleotide transport and metabolism. We observed a low correlation between the transcriptome and proteome datasets. Our study indicates that the transcriptome and proteome of a fish-adapted S. agalactiae strain are modulated by temperature, particularly showing differential expression of genes/proteins involved in metabolism, virulence factors, and adaptation.

The species Corynebacterium renale, Corynebacterium pilosum, and Corynebacterium cystitidis were initially thought to be the same species C. renale, but with different immunological types. These bacteria are the causative agent of cystitis, urethritis and pyelonephritis and are found usually as constituents of the normal flora in the lower urogenital tract of cattle. Therefore, we present the draft genome sequences of two pathogenic Corynebacterium species: C. renale CIP 52.96 and C. pilosum CIP 103422. The genome sequences of these species have 2,322,762 bp with 2,218 protein encoding genes and 2,548,014 bp with 2,428 protein encoding genes, respectively. These genomes can help clarify the virulence mechanisms of these unknown bacteria and enable the development of more effective methods for control.

We present here the genome sequence of the attenuated Corynebacterium pseudotuberculosis strain T1. The sequencing was performed with an Ion Torrent Personal Genome Machine platform. The genome is a circular chromosome of 2,337,201 bp, with a G+C content of 52.85% and a total of 2,125 coding sequences (CDSs), 12 rRNAs, 49 tRNAs, and 24 pseudogenes.

Brucellosis is still a widespread zoonotic disease. Very little is known about the interaction between Brucella abortus and trophoblastic cells, which is essential for better understanding the pathogenesis of the Brucella -induced placentitis and abortion, a key event for transmission of the disease. The goal of this study was to evaluate the profile of gene expression by bovine trophoblastic cells during infection with B. abortus . Explants of chorioallantoic membranes were inoculated with B. abortus strain 2308. Microarray analysis was performed at 4 h after infection, and expression of cytokines and chemokines by trophoblastic cells was assessed by real-time reverse transcription-PCR at 6 and 12 h after inoculation. In addition, cytokine and chemokine expression in placentomes from experimentally infected cows was evaluated. Expression of proinflammatory genes by trophoblastic cells was suppressed at 4 h after inoculation, whereas a significant upregulation of CXC chemokines, namely, CXCL6 (GCP-2) and CXCL8 (interleukin 8), was observed at 12 but not at 6 h after inoculation. Placentomes of experimentally infected cows had a similar profile of chemokine expression, with upregulation of CXCL6 and CXCL8. Our data indicate that B. abortus modulates the innate immune response by trophoblastic cells, suppressing the expression of proinflammatory mediators during the early stages of infection that is followed by a delayed and mild expression of proinflammatory chemokines, which is similar to the profile of chemokine expression in the placentomes of experimentally infected cows. This trophoblastic response is likely to contribute to the pathogenesis of B. abortus -induced placentitis.

We present the complete genome sequence of Corynebacterium pseudotuberculosis strain N1. The sequencing was performed with the Ion Torrent Personal Genome Machine system. The genome is a circular chromosome with 2,337,845 bp, a G+C content of 52.85%, and a total of 2,045 coding sequences, 12 rRNAs, 49 tRNAs, and 58 pseudogenes.

Here, we present the genome sequence of Corynebacterium ulcerans strain FRC11. The genome includes one circular chromosome of 2,442,826 bp (53.35% G+C content), and 2,210 genes were predicted, 2,146 of which are putative protein-coding genes, with 12 rRNAs and 51 tRNAs; 1 pseudogene was also identified.

Using cDNA microarrays with 3800 cDNA fragments, we determined the expression profile of normal thyroid tissue, goiter, adenoma and papillary carcinoma (10 samples from each class). After background correction and statistical analysis, we identified a set of 160 genes as being differentially expressed in all pair-wise comparisons. Here we demonstrate that, at least on the basis of these differentially expressed genes, a positive correlation between goiter and papillary carcinomas could be observed. We identified a common set of genes whose expression is diminished in both goiter and papillary carcinomas as compared to normal thyroid tissue. Moreover, no genes with inverse correlation in samples from goiter and papillary carcinomas could be detected. Using Real-Time PCR and/or tissue microarrays, we confirmed the altered expression of some of the identified genes. Of notice, we demonstrate that the reduced mRNA levels of p27(kip1) observed in papillary carcinomas as compared to either goiter or normal thyroid tissues (P

During the last 5 years, the number of papers describing data obtained by microarray technology increased exponentially with about 3000 papers in 2003. Undoubtedly, cancer is by far the disease that received most of the attention as far as the amount of data generated. As array technology is rather new and highly dependent on bioinformatics, mathematics and statistics, a clear understanding of the knowledge and information derived from array-based experiments is not widely appreciated. We shall review herein some of the issues related to the construction of DNA arrays, quantities and heterogeneity of probes and targets, the consequences of the physical characteristics of the probes, data extraction and data analysis as well as the applications of array technology. Our goal is to bring to the general audience, some of the basics of array technology and its possible application in oncology. By discussing some of the basic aspects of the methodology, we hope to stimulate criticism concerning the conclusions proposed by authors, especially in the light of the very low degree of reproducibility already proven when commercially available platforms were compared . Regardless of its pitfalls, it is unquestionable that array technology will have a great impact in the management of cancer and its applications will range from the discovery of new drug targets, new molecular tools for diagnosis and prognosis as well as for a tailored treatment that will take into account the molecular determinants of a given tumor. Hence, we shall also highlight some of the already available and promising applications of array technology on the day-to-day practice of oncology.

Using DDRT-PCR, we compared the mRNA content of untreated and TNF-treated mouse embryonic fibroblasts (MEFs). Among differentially represented fragments, we identified and cloned a novel TNF-stimulated gene named Tsg-5. This gene, mapped to mouse chromosome 14, has three exons that can be alternatively spliced giving rise to two mRNA species, one spanning three exons and another that skips the second exon. Analysis of full-length Tsg-5 cDNA revealed a potential start codon within exon 2 encoding an ORF of 40 amino-acids. No homology with known mouse or human sequences, neither at the nucleotide nor at the amino-acid level could be found in public databases. In MEFs, Tsg-5 is induced by tumor necrosis factor-alpha (TNF) and IL-1 beta, albeit with distinct kinetics. TNF-induced Tsg-5 expression is NF-kappa B-dependent as it was inhibited by MG132, lactacystin, Bay 11-7083, and Bay 11-7085. Analysis of Tsg-5 expression in vivo revealed that the gene and its encoded polypeptide are constitutively expressed in the thymus and ovary, whereas, in LPS-treated mice, Tsg-5 mRNA can be detected in the spleen, lung, and brain. Our data suggest that Tsg-5 encodes a new, rare transcript, with a very tight regulation of expression and differential splicing.

Here, we describe the identification of three human genes with altered expression in thyroid diseases. One of them corresponds to insulin-like growth factor binding protein 5 (IGFBP5), which has already been described as over expressed in other cancers and, for the first time, is identified as overexpressed in thyroid tumors. The other genes, named 44 and 199, are ESTs with yet unknown function and were mapped on human chromosomes seven and four, respectively. We determined by RT-PCR the expression level of these genes in ten samples of disease-free thyroid, ten of goiter, nine of papillary carcinoma, ten of adenoma and seven of follicular carcinoma and the significance of observed differences was statistically determined. IGFBP-5 and gene 44 were significantly overexpressed in papillary carcinoma when compared to normal and goiter. Genes 44 and 199 were differentially expressed in follicular carcinoma and adenoma when compared to normal thyroid tissue.

Using cDNA fragments from the FAPESP/lICR Cancer Genome Project, we constructed a cDNA array having 4512 elements and determined gene expression in six normal and six tumor gastric tissues. Using t-statistics, we identified 80 cDNAs whose expression in normal and tumor samples differed more than 3.5 sample standard deviations. Using Self-Organizing Map, the expression profile of these cDNAs allowed perfect separation of malignant and non-malignant samples. Using the supervised learning procedure Support Vector Machine, we identified trios of cDNAs that could be used to classify samples as normal or tumor, based on single-array analysis. Finally, we identified genes with altered linear correlation when their expression in normal and tumor samples were compared. Further investigation concerning the function of these genes could contribute to the understanding of gastric carcinogenesis and may prove useful in molecular diagnostics.

We describe here the genome sequencing and annotation of Weissella ceti strains WS74 and WS105, isolated from diseased rainbow trout in Brazil. The two genomes were sequenced with an Ion Torrent personal genome machine (PGM) using a fragment library. The genomes of strains WS74 and WS105 consist of circular chromosomes 1,389,513 bp and 1,390,396 bp long, respectively, both presenting a G+C content of 40.75%.

We report here the complete genome sequence of Weissella ceti strain WS08, an emerging pathogen to farm-raised rainbow trout. The genome of strain WS08 is composed of a circular chromosome with 1,355,853 bp and a G+C content of 40.78%.

Background The White spot syndrome virus (WSSV) and Penaeus stylirostris penstyldensovirus 1 (previously named Infectious hypodermal and hematopoietic necrosis virus-IHHNV) are two of the most important viral pathogens of penaeid shrimp. Different methods have been applied for diagnosis of these viruses, including Real-time PCR (qPCR) assays. A duplex qPCR method allows the simultaneous detection of two viruses in the same sample, which is more cost-effective than assaying for each virus separately. Currently, an assay for the simultaneous detection of the WSSV and the PstDV1 in shrimp is unavailable. The aim of this study was to develop and standardize a duplex qPCR assay for the simultaneous detection of the WSSV and the PstDV1 in clinical samples of diseased L. vannamei. In addition, to evaluate the performance of two qPCR master mixes with regard to the clinical sensitivity of the qPCR assay, as well as, different methods for qPCR results evaluation. Results The duplex qPCR assay for detecting WSSV and PstDV1 in clinical samples was successfully standardized. No difference in the amplification of the standard curves was observed between the duplex and singleplex assays. Specificities and sensitivities similar to those of the singleplex assays were obtained using the optimized duplex qPCR. The analytical sensitivities of duplex qPCR were two copies of WSSV control plasmid and 20 copies of PstDV1 control plasmid. The standardized duplex qPCR confirmed the presence of viral DNA in 28 from 43 samples tested. There was no difference for WSSV detection using the two kits and the distinct methods for qPCR results evaluation. High clinical sensitivity for PstDV1 was obtained with TaqMan Universal Master Mix associated with relative threshold evaluation. Three cases of simultaneous infection by the WSSV and the PstDV1 were identified with duplex qPCR. Conclusion The standardized duplex qPCR was shown to be a robust, highly sensitive, and feasible diagnostic tool for the simultaneous detection of the WSSV and the PstDV1 in whiteleg shrimp. The use of the TaqMan Universal Master Mix and the relative threshold method of data analysis in our duplex qPCR method provided optimal levels of sensitivity and specificity.

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription–PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1,181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan . ( http://genes.mit.edu/GENSCAN.html ).

This study investigated whether primary culture of human amniotic membrane cells (PCHAM) could be used as an in vitro model system for the study of interferon (IFN) production. PCHAM cells infected with Newcastle disease virus (NDV) produced the two antigenic types of IFN, previously shown in a amniotic membrane cells (HAM) system. PCHAM IFN was detected as early as 2 h after NDV infection and was composed by two antigenically distinct fractions, one neutralized with anti-HuIFN beta antibody and another that is not related to IFN beta, -alpha and -gamma. These fractions correspond respectively to 80 and 20 per cent of the IFN produced 4 h after virus induction, 55 and 45 per cent of the IFN produced from 4 to 12 h and 67 and 33 per cent of the IFN produced 12 h after virus induction. A cDNA library, established from PCHAM with or without NDV infection, was screened for IFN alpha and -beta using specific primers. The PCR product, amplified by IFN beta primers, was cloned, sequenced and expressed in Escherichia coli M15. The sequences of several cloned cDNAs were identical to HuIFN beta gene and the antiviral activity of the expressed protein was neutralized only by antiHuIFN-beta antibody. The other IFN fraction not neutralized by polyclonal antibodies anti-IFN beta, -alpha and -gamma is now being studied.

The pathogenesis of the Brucella-induced inflammatory response in the bovine placenta is not completely understood. In this study we evaluated the role of the B. abortus Type IV secretion system and the anti-inflammatory factor BtpB in early interactions with bovine placental tissues. Transcription profiles of chorioallantoic membrane (CAM) explants inoculated with wild type (strain 2308), ΔvirB2 or ΔbtpB Brucella abortus were compared by microarray analysis at 4 hours post infection. Transcripts with significant variation (>2 fold change; P

Lactococcus lactis subsp. lactis NCDO 2118 is a nondairy lactic acid bacterium, a xylose fermenter, and a gamma-aminobutyric acid (GABA) producer isolated from frozen peas. Here, we report the complete genome sequence of L. lactis NCDO 2118, a strain with probiotic potential activity.

Background A wide variety of high-throughput microarray platforms have been used to identify molecular targets associated with biological and clinical tumor phenotypes by comparing samples representing distinct pathological states. Methods The gene expression profiles of human cutaneous melanomas were determined by cDNA microarray analysis. Next, a robust analysis to determine functional classifications and make predictions based on data-oriented hypotheses was performed. Relevant networks that may be implicated in melanoma progression were also considered. Results In this study we aimed to analyze coordinated gene expression changes to find molecular pathways involved in melanoma progression. To achieve this goal, ontologically-linked modules with coordinated expression changes in melanoma samples were identified. With this approach, we detected several gene networks related to different modules that were induced or repressed during melanoma progression. Among them we observed high coordinated expression levels of genes involved in a) cell communication (KRT4, VWF and COMP); b) epidermal development (KLK7, LAMA3 and EVPL); and c) functionally related to kallikreins (EVPL, KLK6, KLK7, KLK8, SERPINB13, SERPING1 and SLPI). Our data also indicated that hKLK7 protein expression was significantly associated with good prognosis and survival. Conclusions Our findings, derived from a different type of analysis of microarray data, highlight the importance of analyzing coordinated gene expression to find molecular pathways involved in melanoma progression.

Background Cancer shows a great diversity in its clinical behavior which cannot be easily predicted using the currently available clinical or pathological markers. The identification of pathways associated with lymph node metastasis (N+) and recurrent head and neck squamous cell carcinoma (HNSCC) may increase our understanding of the complex biology of this disease. Methods Tumor samples were obtained from untreated HNSCC patients undergoing surgery. Patients were classified according to pathologic lymph node status (positive or negative) or tumor recurrence (recurrent or non-recurrent tumor) after treatment (surgery with neck dissection followed by radiotherapy). Using microarray gene expression, we screened tumor samples according to modules comprised by genes in the same pathway or functional category. Results The most frequent alterations were the repression of modules in negative lymph node (N0) and in non-recurrent tumors rather than induction of modules in N+ or in recurrent tumors. N0 tumors showed repression of modules that contain cell survival genes and in non-recurrent tumors cell-cell signaling and extracellular region modules were repressed. Conclusions The repression of modules that contain cell survival genes in N0 tumors reinforces the important role that apoptosis plays in the regulation of metastasis. In addition, because tumor samples used here were not microdissected, tumor gene expression data are represented together with the stroma, which may reveal signaling between the microenvironment and tumor cells. For instance, in non-recurrent tumors, extracellular region module was repressed, indicating that the stroma and tumor cells may have fewer interactions, which disable metastasis development. Finally, the genes highlighted in our analysis can be implicated in more than one pathway or characteristic, suggesting that therapeutic approaches to prevent tumor progression should target more than one gene or pathway, specially apoptosis and interactions between tumor cells and the stroma.

Acute expression of E7 oncogene from human papillomavirus (HPV) 16 or HPV18 is sufficient to overcome tumor necrosis factor (TNF)-alpha cytostatic effect on primary human keratinocytes. In the present study, we investigated the molecular basis of E7-induced TNF resistance through a comparative analysis of the effect of this cytokine on the proliferation and global gene expression of normal and E7-expressing keratinocytes. Using E7 functional mutants, we show that E7-induced TNF resistance correlates with its ability to mediate pRb degradation and cell transformation. On the other hand, this effect does not depend on E7 sequences required to override DNA damage-induced cell cycle arrest or extend keratinocyte life span. Furthermore, we identified a group of 66 genes whose expression pattern differs between normal and E7-expressing cells upon cytokine treatment. These genes are mainly involved in cell cycle regulation suggesting that their altered expression may contribute to sustained cell proliferation even in the presence of a cytostatic stimulus. Differential expression of TCN1 (transcobalamin I), IFI44 (Interferon-induced protein 44), HMGB2 (high-mobility group box 2) and FUS [Fusion (involved in t(12;16) in malignant liposarcoma)] among other genes were further confirmed by western-blot and/or real-time polymerase chain reaction. Moreover, FUS upregulation was detected in HPV-positive cervical high-grade squamous intraepithelial lesions when compared with normal cervical tissue. Further evaluation of the role of such genes in TNF resistance and HPV-associated disease development is warranted.

BACKGROUND Nodules of the thyroid gland are observed frequently in patients who undergo ultrasound studies. The majority of these nodules are benign, corresponding to goiters or adenomas, and only a small fraction corresponds to carcinomas. Among thyroid tumors, the diagnosis of follicular adenocarcinomas by preoperative fine-needle aspiration biopsy is a major challenge, because it requires inspection of the entire capsule to differentiate it from adenoma. Consequently, large numbers of patients undergo unnecessary thyroidectomy. METHODS Using data from gene expression analysis, the authors applied Fisher linear discriminant analysis and searched for expression signatures of individual samples of adenomas and follicular carcinomas that could be used as molecular classifiers for the precise classification of malignant and nonmalignant lesions. RESULTS Fourteen trios of genes were described that fulfilled the criteria for the correct classification of 100% of samples. The robustness of these trios was verified by using leave-1-out cross-validation and bootstrap analyses. The results demonstrated that, by combining trios, better classifiers could be generated that correctly classified >92% of samples. CONCLUSIONS The strategy of classifiers based on individual signatures was a useful strategy for distinguishing between samples with very similar expression profiles. Cancer 2006. © 2006 American Cancer Society.

High incidence of gastric cancer-related death is mainly due to diagnosis at an advanced stage in addition to the lack of adequate neoadjuvant therapy. Hence, new tools aimed at early diagnosis would have a positive impact in the outcome of the disease. Using cDNA arrays having 376 genes either identified previously as altered in gastric tumors or known to be altered in human cancer, we determined expression signature of 99 tissue fragments representing normal gastric mucosa, gastritis, intestinal metaplasia, and adenocarcinomas. We first validated the array by identifying molecular markers that are associated with intestinal metaplasia, considered as a transition stage of gastric adenocarcinomas of the intestinal type as well as markers that are associated with diffuse type of gastric adenocarcinomas. Next, we applied Fisher’s linear discriminant analysis in an exhaustive search of trios of genes that could be used to build classifiers for class distinction. Many classifiers could distinguish between normal and tumor samples, whereas, for the distinction of gastritis from tumor and for metaplasia from tumor, fewer classifiers were identified. Statistical validations showed that trios that discriminate between normal and tumor samples are powerful classifiers to distinguish between tumor and nontumor samples. More relevant, it was possible to identify samples of intestinal metaplasia that have expression signature resembling that of an adenocarcinoma and can now be used for follow-up of patients to determine their potential as a prognostic test for malignant transformation.

Whereas genome sequencing defines the genetic potential of an organism, transcript sequencing defines the utilization of this potential and links the genome with most areas of biology. To exploit the information within the human genome in the fight against cancer, we have deposited some two million expressed sequence tags (ESTs) from human tumors and their corresponding normal tissues in the public databases. The data currently define ≈23,500 genes, of which only ≈1,250 are still represented only by ESTs. Examination of the EST coverage of known cancer-related (CR) genes reveals that

Limiting amounts of RNA is a major issue in cDNA microarray, especially when one is dealing with fresh tissue samples. Here we describe a protocol based on template switch and T7 amplification that led to efficient and linear amplification of 1300×. Using a glass-array containing 368 genes printed in three or six replicas covering a wide range of expression levels and ratios, we determined quality and reproducibility of the data obtained from one nonamplified and two independently amplified RNAs (aRNA) derived from normal and tumor samples using replicas with dye exchange (dye-swap measurements). Overall, signal-to-noise ratio improved when we used aRNA (1.45-fold for channel 1 and 2.02-fold for channel 2), increasing by 6% the number of spots with meaningful data. Measurements arising from independent aRNA samples showed strong correlation among themselves ( r 2 =0.962) and with those from the nonamplified sample ( r 2 =0.975), indicating the reproducibility and fidelity of the amplification procedure. Measurement differences, i.e, spots with poor correlation between amplified and nonamplified measurements, did not show association with gene sequence, expression intensity, or expression ratio and can, therefore, be compensated with replication. In conclusion, aRNA can be used routinely in cDNA microarray analysis, leading to improved quality of data with high fidelity and reproducibility.

Background: Accurate pathological analysis of biopsies is crucial for conclusive and precise disease diagnosis, prognosis and therapeutic guidelines. Evaluation of estrogen receptor (ER) expression in breast cancer (BC) is imperative in determining the disease prognosis, and patients' eligibility for hormonotherapy. Herein, ERα status was evaluated in 61 BC cases using two different anti-ERα monoclonal antibodies, Sp1 (rabbit) and 1D5 (mouse). Methods: Primary BC cases registered in two reference hospitals, with correspondent written informed consent, were used to generate in house tissue microarray platforms. ERα expression was accessed by immunohistochemistry using the monoclonal antibodies anti-ERα, SP1 and 1D5. Staining scoring followed the Allred method (PS, proportion score or percentage of stained cells; IS, intensity score). Concordance and correlation parameters were calculated using Kappa factor; Pearson, Spearman's, or intraclass correlation coefficient (CF). Staining patterns were compared by paired T-Test and Wilcoxon test. Findings: SP1 and 1D5 provided equivalent results (Concordance rate, 96.7%; Kappa factor, 0.921), whereas SP1 was more prone for positive results than 1D5 (2 samples diverged). Total concordance of PS was obtained (Pearson and intraclass CF, 0.7351 and 0.6193, respectively); however, concordance between the antibodies seems more accurate in higher PS values. Excellent SI correlation between antibodies was observed throughout the population (Spearman's CF, α=0.9150). Following the Allred score, 17 out of 42 positive BC samples diverged; always pointing 1D5 to weaker staining than SP1. When calculating Spearman's CF of Total Score (TS) within the population, excellent correlation between both the antibodies (α=0.9238) was noted; nonetheless, results were less concordant result among the BC positive cases (α=0.7743). Indeed, 20 samples were differentially classified using the antibodies (only 3 had higher TS with 1D5). Considering the mean TS of all samples, or of invasive ductal carcinoma, SP1 provided higher scores than 1D5 (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3600. doi:1538-7445.AM2012-3600

Uterine leiomyoma (UL) are tumors that arise from the uterine smooth muscle wall. Despite their nonmalignant nature, UL represent a major concern in women's health through their induction of significant morbidity in more than 25% of reproductive-age. The etiology of UL remains unknown. Cytogenetic studies have been identified recurrent aberrations involving the t(12;14); del(7)(q22q32); trisomy 12; and 6p21 rearrangements. The aim of this study was integrate genomic and gene expression microarray data to identify genes with altered function due to genomic instability. DNA and RNA were extracted from 24 UL from women at proliferative (n=14) and secretory (n=10) phase of the menstrual cycle. Normal control RNA was obtained from adjacent myometrium from all cases. Array CGH and gene expression microarray was done using the Human 44K Agilent oligoarrays according to manufacturer's instructions. Data were extracted and flagged with Feature Extraction software (version 10.1.1.1) and processed using Genomic Workbench Standard (version.5.0.14). Non-supervised analysis of gene expression revealed clustering of all cases independent of the phase of menstrual cycle. It was observed a significant correlation between deletion and down expression at 1q32.2-q44 and 7q11.22-q33. Although deletions at 1q and 7q had been described previously as involved in UL, the genes mapped at these regions were poorly studied. These genes are involved in Wnt signaling pathway, induction of apoptosis, transforming growth factor signaling pathway, fumarate hydratase activity, and fibrinolysis. Furthermore, it was observed correlation between amplification and increased gene expression at 11p15.5, 14q32.33, and 16p13.3. These regions harboring genes with function associated with regulation of cell proliferation and regulation of JNK cascade. Our results provide strong circumstantial evidence that different patterns of genomic imbalances contribute to their specific transcriptomic profiles. In the future, new therapies based on these findings can be applied in a subgroup of patients with specific genomic imbalances. Supported by the FAPESP (07/51643-8) and CNPq (485032/2007-4), Brazil. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2144.

Background The ongoing efforts to sequence the honey bee genome require additional initiatives to define its transcriptome. Towards this end, we employed the Open Reading frame ESTs (ORESTES) strategy to generate profiles for the life cycle of Apis mellifera workers. Results Of the 5,021 ORESTES, 35.2% matched with previously deposited Apis ESTs. The analysis of the remaining sequences defined a set of putative orthologs whose majority had their best-match hits with Anopheles and Drosophila genes. CAP3 assembly of the Apis ORESTES with the already existing 15,500 Apis ESTs generated 3,408 contigs. BLASTX comparison of these contigs with protein sets of organisms representing distinct phylogenetic clades revealed a total of 1,629 contigs that Apis mellifera shares with different taxa. Most (41%) represent genes that are in common to all taxa, another 21% are shared between metazoans (Bilateria), and 16% are shared only within the Insecta clade. A set of 23 putative genes presented a best match with human genes, many of which encode factors related to cell signaling/signal transduction. 1,779 contigs (52%) did not match any known sequence. Applying a correction factor deduced from a parallel analysis performed with Drosophila melanogaster ORESTES, we estimate that approximately half of these no-match ESTs contigs (22%) should represent Apis-specific genes. Conclusions The versatile and cost-efficient ORESTES approach produced minilibraries for honey bee life cycle stages. Such information on central gene regions contributes to genome annotation and also lends itself to cross-transcriptome comparisons to reveal evolutionary trends in insect genomes.