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[Preliminary analysis of gene expression profiles in oral cancer with microarray technique].

OBJECTIVE: To study the difference in gene expression between oral squamous cell carcinoma tissue and their surrounding normal tissue by microarray so as to investigate the preliminary mechanism of pathogenesis of oral cancer. METHODS: The tissues from 5 patients with oral squamous cell carcinoma tissue and their surrounding normal tissue from the same patients were analyzed by cDNA microarray technology(including 4124 genes). Total RNAs were isolated from two tissues, and then were reversely transcribed to cDNAs with the incorporations of fluorescent dUTP,for preparing the hybridization probes. The mixed probes were then hybridized to the cDNA microarray. After high-stringent washing, the cDNA microarray was scanned for the fluorescent signals and showed the differences between the two tissues. Bioinformatical analysis of those genes had been performed. RESULTS: Among the 4124 target genes, there were 37(0.89%)genes whose expression levels differed between the carcinoma and their surrounding normal tissues in all 5 cases. Bioinformatical analysis of those genes suggested that they may be related to the multistep process of carcinogenesis. CONCLUSION: cDNA microarray technique can simultaneously screen the different expressions of genes from 2 different kinds of tissue. Further analysis of the obtained genes will help to understand the molecular mechanism of malignant carcinoma.

Aged↗

[Cloning and bioinformatics of human REV3 gene promoter region and its response to carcinogen N-methyl-N'-nitro-N-nitrosoguanidine].

OBJECTIVE: To understand the up regulatory mechanism of human REV3 gene induced by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). METHODS: Bioinformatic analysis of human REV3 gene promoter region was based on BLAST alignment, promoter prediction software and recognition of transcriptional factor binding sites. Cloning of human REV3 gene promoter region was performed by nested PCR. Response of human REV3 gene promoter to the chemical carcinogen MNNG was measured by transient transfection assay based on the dual luciferase reporter assay system. RESULT: Bioinformatic analysis showed that human REV3 gene promoter region was located on chromosome 6 PAC clone RP3-415N12, and that the hypothetical promoter region contained promoter sequences, rich CpG islands, and putative recognition sites for several transcriptional factors, including AP-1/c-Jun/c-Fos, AP-2, STAT, CREBP, and NF-kappaB. Reconstructed reporter plasmid pGL3- 2582 was established by inserting 2582 nucleotides from the promoter region into the luciferase reporter vector pGL3-Basic. Transient transfection assay showed the hypothetical REV3 promoter region had promoter function, and it responded to MNNG treatment (P<0.01). CONCLUSION: Human mutator REV3 gene promoter region has been successfully cloned. The response of REV3 promoter region to MNNG suggests that REV3 gene can be regulated at transcriptional level under conditions of genotoxic stress.

Base Sequence↗

A qualitative study of the implementation of a bioinformatics tool in a biological research laboratory.

OBJECTIVE: To explore how the implementation of a comprehensive new bioinformatics analysis system would affect workflow, collaboration and information management in a small genetic research lab. DESIGN: This was a longitudinal qualitative study of seven individuals involved in genomic and proteomic research. The study data were gathered using the illuminative/responsive approach of immersion in the environment. Additional qualitative data were gathered using informal semi-structured interviews, participant observation in lab meetings, and direct observation of lab researchers engaged in specific tasks. MEASUREMENTS: Interview, observation and field note data were coded and analyzed based on three analysis perspectives. A subset of the data was independently evaluated by an external researcher to enhance the trustworthiness of results. RESULTS: Three reoccurring themes were observed in the study. (1) Satisfaction and acceptance of software tools tended to be role and goal specific. (2) The system was seen primarily as a measurement system rather than a "total laboratory analysis system". (3) Lab meetings deemphasized the system, preferring more traditional data analysis techniques. These themes support the observations that the system was not used to its full potential in the lab. CONCLUSION: Themes identified in this study suggest that sophisticated genetic researchers face similar problems of technology implementation as do professionals in other fields. We recommend that leadership support and on-going training and evolution of academic curricula can improve chances of bioinformatics analysis systems becoming used more effectively.

Computational Biology↗

Complete mutation analysis panel of the 39 human HOX genes.

BACKGROUND: The HOX gene family consists of highly conserved transcription factors that specify the identity of the body segments along the anteroposterior axis of the embryo. Because the phenotypes of mice with targeted disruptions of Hox genes resemble some patterns of human malformations, mutations in HOX genes have been expected to be associated with a significant number of human malformations. Thus far, however, mutations have been documented in only three of the 39 human HOX genes (HOXD13, HOXA13, and HOXA11) partly because current knowledge on the complete coding sequence and genome structure is limited to only 20 of the 39 human HOX genes. METHODS: Taking advantage of the human and mouse draft genome sequences, we attempted to characterize the remaining 19 human HOX genes by bioinformatic analysis including phylogenetic footprinting, the probabilistic prediction method, and comparison of genomic sequences with the complete set of the human anonymous cDNA sequences. RESULTS: We were able to determine the full coding sequences of 19 HOX genes and their genome structure and successfully designed a complete set of PCR primers to amplify the entire coding region of each of the 39 HOX genes from genomic DNA. CONCLUSIONS: Our results indicate the usefulness of bioinformatic analysis of the draft genome sequences for clinically oriented research projects. It is hoped that the mutation panel provided here will serve as a launchpad for a new discourse on the genetic basis of human malformations.

Animals↗

Discovering the interactome, functions, and clinical relevance of enhancer RNAs in kidney renal clear cell carcinoma.

Enhancer RNA (eRNA) has emerged as a key player in cancer biology, influencing various aspects of tumor development and progression. In this study, we investigated the role of eRNAs in kidney renal clear cell carcinoma (KIRC), the most common subtype of renal cell carcinoma. Leveraging high-throughput sequencing data and bioinformatics analysis, we identified differentially expressed eRNAs in KIRC and constructed eRNA-centric regulatory networks. Our findings revealed that up-regulated eRNAs in KIRC potentially regulate immune response and hypoxia pathways, while down-regulated eRNAs may impact ion transport, cell cycle, and metabolism. Furthermore, we developed a diagnostic prediction model based on eRNA expression profiles, demonstrating its effectiveness in KIRC diagnosis. Finally, we elucidated the regulatory mechanism of an eRNA (ENSR00000305834) on the expression of SLC15A2, a potential prognostic biomarker in KIRC, through bioinformatics analysis and in vitro validation experiments. In summary, Our study highlights the clinical significance of eRNAs in KIRC and underscores their potential as therapeutic targets.

Carcinoma, Renal Cell↗

PCSK9 as a Key Gene of Metastasis in Lung Adenocarcinoma: A Multi-omics and Experimental Validation Study.

BACKGROUND: Lung adenocarcinoma (LUAD) is the most common form of lung cancer. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is abnormally expressed in various tumor tissues and is associated with malignant phenotypes. However, the clinical significance, function, and mechanism of LUAD invasion and metastasis remain unclear. METHODS: We retrospectively enrolled 100 patients with LUAD in this study. Initially, qRT-PCR was performed to detect PCSK9 levels in clinical tissues. Subsequently, bioinformatics analysis of scRNA-seq and The Cancer Genome Atlas Program (TCGA) datasets was performed to predict the role of PCSK9 in tumor cell malignancy and its potential downstream pathways. These predictions were validated experimentally using the CCK-8 assay, TUNEL staining, wound healing, transwell invasion assay, and an in vivo lung metastasis model. Finally, Western blotting and an AKT inhibitor (MK2206) were used to verify the underlying mechanism. RESULTS: PCSK9 was significantly upregulated in LUAD tissues compared to paracancerous tissues and was associated with poorer OS and DFS. Bioinformatics analysis of scRNA-seq data and TCGA analysis predicted that PCSK9 is highly enriched in tumor cells and is involved in EMT, and that the PI3K/AKT pathway plays a significant role in LUAD development. Experiments confirmed that PCSK9 markedly promoted LUAD cell proliferation, migration, and invasion in vitro and lung metastasis in vivo. PCSK9 overexpression significantly upregulated p-AKT, p-PI3K, and p-mTOR levels. Furthermore, the AKT inhibitor, MK2206, reversed the promoting effects of PCSK9. CONCLUSIONS: PCSK9 expression is associated with the prognosis and diagnosis of LUAD. This molecule activates the PI3K/AKT signaling pathway, thereby driving invasion, metastasis, and proliferation in LUAD.

Humans↗

Identification and characterization of the peroxiredoxin gene family in chickens.

Peroxiredoxin (PRX) is a crucial antioxidant protein that protects against endogenously produced peroxides in prokaryotes to eukaryotes. To date, 6 different isoforms have been identified in mammals. In this study, we describe the first members of the PRX protein family to be characterized in Chicken. Through bioinformatics analysis, we observed that at least 4 different classes of PRX protein have been evolutionarily conserved in chickens. Furthermore, in vitro functional assays of the candidate chicken PRX proteins demonstrated that they had levels of antioxidant activity similar to those of the mammalian enzymes. The expression patterns of the PRX transcript in several chicken tissues were not tissue specific, suggesting that they might play an essential role as a housekeeping gene in all tissues to protect against oxidative damage. In conclusion, the sequences of the putative members of this functional gene family in chickens could be effectively retrieved in silico through bioinformatics analysis, and the functionality of their gene products evaluated by in vitro comparative assay.

Animals↗

Membrane-associated transcripts in Arabidopsis; their isolation and characterization by DNA microarray analysis and bioinformatics.

Membrane-associated, integral membrane and secreted proteins are of key importance in many cellular processes. For most of the 28,952 predicted proteins in Arabidopsis, the actual subcellular localization has not been demonstrated experimentally. So far, their potential membrane-association has been deduced from algorithms that predict transmembrane domains and signal peptides. However, the comprehensiveness and accuracy of these algorithms is still limited. The majority of membrane-associated and secreted proteins is synthesized on membrane-bound polysomes. Therefore, the isolation and characterization of mRNA associated with membrane-bound polysomes offers an experimental tool for the genome-wide identification of these proteins. Here we describe an efficient method to isolate mRNA from membrane-bound polysomes and report on the validation of the method to enrich for transcripts encoding membrane-associated and secreted proteins. The sensitivity and reproducibility of the isolation method was investigated by DNA microarray analysis. Pearson correlations between transcript levels obtained from three replicate isolations showed that the method is highly reproducible. A significant enrichment for mRNAs encoding proteins containing predicted transmembrane domains and signal peptides was observed in the membrane-bound polysomal fraction. In this fraction, 301 transcripts were classified by gene ontologies as 'cellular component unknown', and potentially encode previously unrecognized secreted or membrane-associated proteins.

Algorithms↗

Use of toxicogenomics for identifying genetic markers of pulmonary oedema.

This study was undertaken primarily to identify genetic markers of oedema and inflammation. Mild pulmonary injury was induced following the instillation of the oedema-producing agent, bleomycin (0.5 units). Oedema was then confirmed by conventional toxicology (lavage protein levels, free cell counts and lung/body weight ratios) and histology 3 days post-bleomycin instillation. The expression profile of 1176 mRNA species was determined for bleomycin-exposed lung (Clontech Atlas macroarray, n=9). To obtain pertinent results from these data, it was necessary to develop a simple, effective method for bioinformatic analysis of altered gene expression. Data were log10 transformed followed by global normalisation. Differential gene expression was accepted if: (a) genes were statistically significant (P < or = 0.05) from a two-tailed t test; (b) genes were consistently outside a two standard deviation (SD) range from control levels. A combination of these techniques identified 31 mRNA transcripts (approximately 3%) which were significantly altered in bleomycin treated tissue. Of these genes, 26 were down-regulated whilst only five were up-regulated. Two distinct clusters were identified, with 17 genes classified as encoding hormone receptors, and nine as encoding ion channels. Both these clusters were consistently down-regulated. The magnitude of the changes in gene expression were quantified and confirmed by Q-PCR (n = 6), validating the macroarray data and the bioinformatic analysis employed. In conclusion, this study has developed a suitable macroarray analysis procedure and provides the basis for a better understanding of the gene expression changes occurring during the early phase of drug-induced pulmonary oedema. This work has been presented orally, in part at the British Association for Lung Research Summer Meeting, University of Brighton, 3-5 September, 2003 and in full at the British Toxicology Society Annual Congress, Heriot Watt University, Edinburgh, 21-24 April 2004.

Animals↗

Protein intrinsic disorder and human papillomaviruses: increased amount of disorder in E6 and E7 oncoproteins from high risk HPVs.

It is recognized now that many functional proteins or their long segments are devoid of stable secondary and/or tertiary structure and exist instead as very dynamic ensembles of conformations. They are known by different names including natively unfolded, intrinsically disordered, intrinsically unstructured, rheomorphic, pliable, and different combinations thereof. Many important functions and activities have been associated with these intrinsically disordered proteins (IDPs), including molecular recognition, signaling, and regulation. It is also believed that disorder of these proteins allows function to be readily modified through phosphorylation, acetylation, ubiquitination, hydroxylation, and proteolysis. Bioinformatics analysis revealed that IDPs comprise a large fraction of different proteomes. Furthermore, it is established that the intrinsic disorder is relatively abundant among cancer-related and other disease-related proteins and IDPs play a number of key roles in oncogenesis. There are more than 100 different types of human papillomaviruses (HPVs), which are the causative agents of benign papillomas/warts, and cofactors in the development of carcinomas of the genital tract, head and neck, and epidermis. With respect to their association with cancer, HPVs are grouped into two classes, known as low (e.g., HPV-6 and HPV-11) and high-risk (e.g., HPV-16 and HPV-18) types. The entire proteome of HPV includes six nonstructural proteins [E1, E2, E4, E5, E6, and E7 (the latter two are known to function as oncoproteins in the high-risk HPVs)] and two structural proteins (L1 and L2). To understand whether intrinsic disorder plays a role in the oncogenic potential of different HPV types, we have performed a detailed bioinformatics analysis of proteomes of high-risk and low-risk HPVs with the major focus on E6 and E7 oncoproteins. The results of this analysis are consistent with the conclusion that high-risk HPVs are characterized by the increased amount of intrinsic disorder in transforming proteins E6 and E7.

Algorithms↗

Triple primary synchronous liver cancer in one patient: the first case report and origin speculation through bioinformatics.

INTRODUCTION: A diagnosis of multiple primary liver tumors is extremely rare. Preoperative diagnosis based on imaging findings is difficult. Moreover, the clinical benefits of treatment strategies for multiple liver cancers remain unclear. Here, we report a case of three synchronous primary liver tumors with three distinct pathological types-hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and combined hepatocellular-cholangiocarcinoma (cHCC&#x2011;CCA)-in a single patient. Bioinformatics analysis supported at least two clonal origins, with cHCC&#x2011;CCA and ICC sharing a common lineage based on identical HBV integration sites. CASE PRESENTATION: A 63-year-old female with a history of hepatitis B for several years presented with three lesions in hepatic segment VIII. Multiphase magnetic resonance imaging with gadolinium ethoxybenzyl diethylenetriaminepentaacetic acid revealed a diagnosis of multiple lesions, namely, cHCC&#x2011;CCA, with multiple intrahepatic metastases. The AFP level was normal, while the CA 19&#x2009;-&#x2009;9 level was mildly elevated (normal range&#x2009;&#x2264;&#x2009;30.00 U/ml). Hepatectomy was performed, and postoperative assessment confirmed that the large lesion was cHCC&#x2011;CCA. However, the small lesions close to the large lesion were HCC and ICC. Gene testing revealed distinct mutational profiles among the three tumors. Similar gene mutations were detected in cHCC&#x2011;CCA and ICC. We also found that gene fragments of hepatitis B virus-C (HBV-C) were inserted into the genomes of ICC and cHCC&#x2011;CCA rather than that of HCC. The genomic integration site of HBV-C in cHCC&#x2011;CCA and ICC was the same. CONCLUSION: We report an extremely rare case of three synchronous primary liver tumors with three distinct pathological types (HCC, ICC, and cHCC&#x2011;CCA) in a single patient. Bioinformatics analysis supported at least two clonal origins, with cHCC&#x2011;CCA and ICC sharing a common lineage based on identical HBV integration sites. Hepatectomy represents a potential radical strategy for the treatment of multiple PLCs.

Humans↗

Temporal regulation of viral transcription during development of Thermus thermophilus bacteriophage phiYS40.

Regulation of gene expression of lytic bacteriophage varphiYS40 that infects the thermophilic bacterium Thermus thermophilus was investigated and three temporal classes of phage genes, early, middle, and late, were revealed. varphiYS40 does not encode a (RNAP) and must rely on host RNAP for transcription of its genes. Bioinformatic analysis using a model of Thermus promoters predicted 43 putative sigma(A)-dependent -10/-35 class phage promoters. A randomly chosen subset of those promoters was shown to be functional in vivo and in vitro and to belong to the early temporal class. Macroarray analysis, primer extension, and bioinformatic predictions identified 36 viral middle and late promoters. These promoters have a single common consensus element, which resembles host sigma(A) RNAP holoenzyme -10 promoter consensus element sequence. The mechanism responsible for the temporal control of the three classes of promoters remains unknown, since host sigma(A) RNAP holoenzyme purified from either infected or uninfected cells efficiently transcribed all varphiYS40 promoters in vitro. Interestingly, our data showed that during infection, there is a significant increase and decrease of transcript amounts of host translation initiation factors IF2 and IF3, respectively. This finding, together with the fact that most middle and late varphiYS40 transcripts were found to be leaderless, suggests that the shift to late viral gene expression may also occur at the level of mRNA translation.

Bacteriophages↗

GeneX Va: VBC open source microarray database and analysis software.

Developed by the Virginia Bioinformatics Consortium (VBC), GeneX Va is an open source, freeware database and bioinformatics analysis software for archiving and analyzing Affymetrix GeneChip data. It provides an integrated framework for management, documentation, and analysis of microarray experiments and data to support a range of users, from individual research laboratories to institutional microarray facilities. GeneX Va also provides web-based access to a PostgreSQL relational database system with a comprehensive security system. Data can be extracted from the database and delivered to interactive or scriptable statistical analysis protocols. The security system allows each investigator to manage their own array data and analysis output files and also provides custom access privileges for other users, groups, and internal/external collaborators. The analysis interface uses "Analysis Trees," an innovative user interface that allows researchers to interactively create a tree-structured flow chart of analysis routines. The latest GeneX Va software is available from and can be freely downloaded at the Sourceforge web site http://va-genex.sourceforge.net. To allow researchers to access the database and analysis capabilities of the GeneX Va system, microarray data from many VBC GeneChip experiments have been deposited into a public section of the GeneX Va system at the University of Virginia. The VBC GeneX Va sites, which include documentation, are at http://genes.med.virginia.edu/ of the University of Virginia and at http://genex.csbc.vcu.edu/ of the Virginia Commonwealth University.

Computer Security↗

Genetic analysis and fine mapping of a rice brown planthopper (Nilaparvata lugens Stål) resistance gene bph19(t).

Genetic analysis and fine mapping of a resistance gene against brown planthopper (BPH) biotype 2 in rice was performed using two F(2) populations derived from two crosses between a resistant indica cultivar (cv.), AS20-1, and two susceptible japonica cvs., Aichi Asahi and Lijiangxintuanheigu. Insect resistance was evaluated using F(1) plants and the two F(2) populations. The results showed that a single recessive gene, tentatively designated as bph19(t), conditioned the resistance in AS20-1. A linkage analysis, mainly employing microsatellite markers, was carried out in the two F(2) populations through bulked segregant analysis and recessive class analysis (RCA), in combination with bioinformatics analysis (BIA). The resistance gene locus bph19(t) was finely mapped to a region of about 1.0 cM on the short arm of chromosome 3, flanked by markers RM6308 and RM3134, where one known marker RM1022, and four new markers, b1, b2, b3 and b4, developed in the present study were co-segregating with the locus. To physically map this locus, the bph19(t)-linked markers were landed on bacterial artificial chromosome or P1 artificial chromosome clones of the reference cv., Nipponbare, released by the International Rice Genome Sequencing Project. Sequence information of these clones was used to construct a physical map of the bph19(t) locus, in silico, by BIA. The bph19(t) locus was physically defined to an interval of about 60 kb. The detailed genetic and physical maps of the bph19(t) locus will facilitate marker-assisted gene pyramiding and cloning.

Animals↗

From genomes to vaccines: Leishmania as a model.

The 35 Mb genome of Leishmania should be sequenced by late 2002. It contains approximately 8500 genes that will probably translate into more than 10 000 proteins. In the laboratory we have been piloting strategies to try to harness the power of the genome-proteome for rapid screening of new vaccine candidate. To this end, microarray analysis of 1094 unique genes identified using an EST analysis of 2091 cDNA clones from spliced leader libraries prepared from different developmental stages of Leishmania has been employed. The plan was to identify amastigote-expressed genes that could be used in high-throughput DNA-vaccine screens to identify potential new vaccine candidates. Despite the lack of transcriptional regulation that polycistronic transcription in Leishmania dictates, the data provide evidence for a high level of post-transcriptional regulation of RNA abundance during the developmental cycle of promastigotes in culture and in lesion-derived amastigotes of Leishmania major. This has provided 147 candidates from the 1094 unique genes that are specifically upregulated in amastigotes and are being used in vaccine studies. Using DNA vaccination, it was demonstrated that pooling strategies can work to identify protective vaccines, but it was found that some potentially protective antigens are masked by other disease-exacerbatory antigens in the pool. A total of 100 new vaccine candidates are currently being tested separately and in pools to extend this analysis, and to facilitate retrospective bioinformatic analysis to develop predictive algorithms for sequences that constitute potentially protective antigens. We are also working with other members of the Leishmania Genome Network to determine whether RNA expression determined by microarray analyses parallels expression at the protein level. We believe we are making good progress in developing strategies that will allow rapid translation of the sequence of Leishmania into potential interventions for disease control in humans.

Animals↗

Sequence analysis for assessing potential allergenicity.

Sequence analysis plays an important role in assessing the potential allergenicity of proteins used in transgenic foods, particularly for proteins that have not previously been part of the food supply. Sequence comparisons are used to indicate potential unexpected cross reactivity to existing allergens and to assess the potential for developing new sensitivities. Although the concept of using sequence analysis is straightforward, implementing a bioinformatic analysis that is accurate and complete can be complex. Several factors need to be considered, including the design and content of the sequence database, the analysis strategy, and the criteria for evaluating the results.

Algorithms↗

Evolutionarily conserved ELOVL4 gene expression in the vertebrate retina.

PURPOSE: The gene elongation of very long chain fatty acids-4 (ELOVL4) has been shown to underlie phenotypically heterogeneous forms of autosomal dominant macular degeneration. In this study, the extent of evolutionary conservation and the existence and localization of retinal expression of this gene was investigated across a wide variety of species. METHODS: Southern blot analysis of genomic DNA and bioinformatic analysis using the human ELOVL4 cDNA and protein sequences, respectively, were performed to identify species in which ELOVL4 orthologues and/or homologues are present. Retinal RNA and protein extracts derived from different species were assessed by Northern hybridization and immunoblot techniques to assess evolutionary conservation of gene expression. Immunohistochemical analysis of tissue sections prepared from various mammalian retinas was performed to determine the distribution of ELOVL4 and homologous proteins within specific retinal cell layers. RESULTS: The existence of ELOVL4 sequence orthologues and homologues was confirmed by both Southern blot analysis and in silico searches of protein sequence databases. Phylogenetic analysis places ELOVL4 among a large family of known and putative fatty acid elongase proteins. Northern blot analysis revealed the presence of multiple transcripts corresponding to ELOVL4 homologues expressed in the retina of several different mammalian species. Conserved proteins were also detected among retinal extracts of different mammals and were found to localize predominantly to the photoreceptor cell layer within retinal tissue preparations. CONCLUSIONS: The ELOVL4 gene is highly conserved throughout evolution and is expressed in the photoreceptor cells of the retina in a variety of different species, which suggests that it plays a critical role in retinal cell biology.

Amino Acid Sequence↗

Accurate prediction of scorpion toxin functional properties from primary structures.

Scorpion toxins are common experimental tools for studies of biochemical and pharmacological properties of ion channels. The number of functionally annotated scorpion toxins is steadily growing, but the number of identified toxin sequences is increasing at much faster pace. With an estimated 100,000 different variants, bioinformatic analysis of scorpion toxins is becoming a necessary tool for their systematic functional analysis. Here, we report a bioinformatics-driven system involving scorpion toxin structural classification, functional annotation, database technology, sequence comparison, nearest neighbour analysis, and decision rules which produces highly accurate predictions of scorpion toxin functional properties.

Algorithms↗