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Fight against cancer in countries with limited resources: the post-genomic era scenario.

The enormous advances in science and technology in the 20th century have facilitated the process of globalization with the aim of a better quality of life for all. Paradoxically, the gap between the rich and the poor, for both nations and people, is constantly widening. The actual trends in human genome research are leading towards promising genomic medicine, but it will be expensive and inaccessible for many. Also, it may not offer a quick fix "cure" for various types of cancers. The biggest challenge before the clinicians now is the management of the rising incidence of cancer in developing countries, with little prospect of more resources becoming available to fight the disease. The death rate from cancer in the developing countries is set to rise at least 3-fold by the year 2025 largely due to the increased life expectancy, containment of infectious diseases and changing lifestyles. It is estimated that about 50% of cancers are curable if they are detected early and treated appropriately. Screening has a major role in early diagnosis. However, in the developing world around 80% of cancer patients have late stage incurable disease when they are diagnosed. Moreover, in a developing country like India, about 70% of the population obtain medical help from private practitioners. Nearly half of those who seek medical help utilize alternative and traditional systems of medicine. Appalling poverty, poor hygiene and complex social dynamics, pose major hurdles in this regard. Many in the private sector who call themselves doctors have no medical degree. By 2030 tobacco is expected to kill 10 million people worldwide, out of which 70% of the deaths will occur in the developing countries. Control of usage of tobacco has still not achieved a conducive atmosphere. It is now realized that the research information and knowledge generated in the west may neither be relevant nor applicable to developing countries, due to differences in social and cultural attitudes, lifestyles and lack of sophisticated technologies. Though the sequencing of the human genome will have a major impact on the prevention, diagnosis, treatment, monitoring, and outcome of cancer, the cancer scenario in the developing countries for the next 20 years is likely to be more or less the same, rather than presenting a radically different picture. Cancer awareness and screening programs for early detection thus should be continue to be given utmost attention.

Antineoplastic Agents↗

Genome-wide ORFeome cloning and analysis of Arabidopsis transcription factor genes.

Here, we report our effort in generating an ORFeome collection for the Arabidopsis transcription factor (TF) genes. In total, ORFeome clones representing 1,282 Arabidopsis TF genes have been obtained in the Gateway high throughput cloning pENTR vector, including 411 genes whose annotation lack cDNA support. All the ORFeome inserts have also been mobilized into a yeast expression destination vector, with an estimated 85% rate of expressing the respective proteins. Sequence analysis of these clones revealed that 34 of them did not match with either the reported cDNAs or current predicted open-reading-frame sequences. Among those, novel alternative splicing of TF gene transcripts is responsible for the observed differences in at least five genes. However, those alternative splicing events do not appear to be differentially regulated among distinct Arabidopsis tissues examined. Lastly, expression of those TF genes in 17 distinct Arabidopsis organ types and the cultured cells was profiled using a 70-mer oligo microarray.

Alternative Splicing↗

DARFA: a novel technique for studying differential gene expression and bacterial comparative genomics.

We have developed a powerful method, named differential analysis of restriction fragments amplification (DARFA), which enables researchers to perform comprehensive transcriptome analysis as well as bacterial DNA fingerprinting. The key feature of this novel technique lies within the usage of a type IIS enzyme, Hpy188III, which cleaves cDNA or genomic DNA at a TC/NNGA recognition sequence. Cleavage at this particular site results in the production of a pool of restriction fragments which can be divided into 120 subsets based on the 2-nt 5'-overhang sequence. Each subset of restriction fragments is then selectively amplified by PCR after ligation with a pair of hairpin adaptors containing 2-nt overhangs which are complementary to those in the subset of fragments that are to be analyzed. The results obtained from the analysis of strain-specific and tissue-specific differences using DARFA and further confirmation by DNA sequencing and Northern analysis have demonstrated that the DARFA technique provides a novel tool for expression profiling, as well as bacterial DNA fingerprinting.

Bacteria↗

Detection and measurement of alternative splicing using splicing-sensitive microarrays.

Splicing and alternative splicing are major processes in the interpretation and expression of genetic information for metazoan organisms. The study of splicing is moving from focused attention on the regulatory mechanisms of a selected set of paradigmatic alternative splicing events to questions of global integration of splicing regulation with genome and cell function. For this reason, parallel methods for detecting and measuring alternative splicing are necessary. We have adapted the splicing-sensitive oligonucleotide microarrays used to estimate splicing efficiency in yeast to the study of alternative splicing in vertebrate cells and tissues. We use gene models incorporating knowledge about splicing to design oligonucleotides specific for discriminating alternatively spliced mRNAs from each other. Here we present the main strategies for design, application, and analysis of spotted oligonucleotide arrays for detection and measurement of alternative splicing. We demonstrate these strategies using a two-intron yeast gene that has been altered to produce different amounts of alternatively spliced RNAs, as well as by profiling alternative splicing in NCI 60 cancer cell lines.

Alternative Splicing↗

Nutrigenomics: goals and strategies.

Nutrigenomics is the application of high-throughput genomics tools in nutrition research. Applied wisely, it will promote an increased understanding of how nutrition influences metabolic pathways and homeostatic control, how this regulation is disturbed in the early phase of a diet-related disease and to what extent individual sensitizing genotypes contribute to such diseases. Ultimately, nutrigenomics will allow effective dietary-intervention strategies to recover normal homeostasis and to prevent diet-related diseases.

Animals↗

Small evolutionarily conserved RNA, resembling C/D box small nucleolar RNA, is transcribed from PWCR1, a novel imprinted gene in the Prader-Willi deletion region, which Is highly expressed in brain.

Prader-Willi syndrome is a complex neurodevelopmental disorder caused by the inactivation or deletion of imprinted, paternally expressed genes in chromosome band 15q11.2. We report the identification and characterization of PWCR1, a novel imprinted gene within that region, and its mouse orthologue, Pwcr1, which was mapped to the conserved syntenic region on mouse chromosome 7. Expressed only from the paternal allele, both genes require the imprinting-center regulatory element for expression and are transcribed from the same strand. They are intronless and do not appear to encode a protein product. High human/mouse sequence similarity (87% identity) is limited to a 99-bp region called "HMCR" (for "human-mouse conserved region"). The HMCR sequence has features of a C/D box small nucleolar RNA (snoRNA) and is represented in an abundant small transcript in both species. Located in nucleoli, snoRNAs serve as methylation guidance RNAs in the modification of ribosomal RNA and other small nuclear RNAs. In addition to the nonpolyadenylated small RNAs, larger polyadenylated PWCR1 transcripts are found in most human tissues, whereas expression of any Pwcr1 RNAs is limited to mouse brain. Genomic sequence analysis reveals the presence of multiple copies of PWCR1 and Pwcr1 that are organized within local tandem-repeat clusters. On a multispecies Southern blot, hybridization to an HMCR probe encoding the putative snoRNA is limited to mammals.

Alleles↗

A comprehensive transcript index of the human genome generated using microarrays and computational approaches.

BACKGROUND: Computational and microarray-based experimental approaches were used to generate a comprehensive transcript index for the human genome. Oligonucleotide probes designed from approximately 50,000 known and predicted transcript sequences from the human genome were used to survey transcription from a diverse set of 60 tissues and cell lines using ink-jet microarrays. Further, expression activity over at least six conditions was more generally assessed using genomic tiling arrays consisting of probes tiled through a repeat-masked version of the genomic sequence making up chromosomes 20 and 22. RESULTS: The combination of microarray data with extensive genome annotations resulted in a set of 28,456 experimentally supported transcripts. This set of high-confidence transcripts represents the first experimentally driven annotation of the human genome. In addition, the results from genomic tiling suggest that a large amount of transcription exists outside of annotated regions of the genome and serves as an example of how this activity could be measured on a genome-wide scale. CONCLUSIONS: These data represent one of the most comprehensive assessments of transcriptional activity in the human genome and provide an atlas of human gene expression over a unique set of gene predictions. Before the annotation of the human genome is considered complete, however, the previously unannotated transcriptional activity throughout the genome must be fully characterized.

Chromosomes, Human, Pair 20↗

Applications of molecular methods for Leishmania control.

This article reviews the recent advances made in the field of human leishmaniasis. Special emphasis is placed upon the application of various molecular tools for accurate and rapid diagnosis, understanding the mechanisms of drug resistance and identification of vaccine candidates. The focus will be on the major role played by recombinant antigens in the immunoserodiagnosis and progress of the Leishmania genome project, which has enabled researchers to design better PCR primers and molecular probes for microarrays. A special interest is placed on the recombinant antigen (rK39) cloned from the Leishmania chagasi kinesin gene and a very recently cloned recombinant antigen (KE16) from the Old World Leishmania donovani species with high sensitivity and specificity. Advances made in the specific PCR primer designed to diagnose and differentiate various species and strains of Leishmania causing visceral and post-kala-azar-dermal leishmaniasis have been covered. Molecular methods (e.g., DNA and protein microarrays) applied to understanding the pathobiology of the parasite, mechanism of host invasion, drug interaction and drug resistance to develop effective therapeutic molecules, gene expression profiling studies that have opened doors to understand many host-parasite relations, effective therapy and vaccine candidates are extensively covered in this review.

Animals↗

Acute drug-induced vascular injury in beagle dogs: pathology and correlating genomic expression.

Acute vascular injury that leads to vascular inflammation is a common finding in the preclinical toxicity testing of drugs in rats and dogs. However, the relevance of this finding for risk to humans is unclear. Concern about the safety of these drugs is heightened by the current lack of noninvasive clinical methods to predict the onset of vascular damage in animals or humans. Determining the relevance of this poorly understood preclinical outcome for humans requires a better understanding of the molecular mechanisms of injury in addition to the development of sensitive and specific leading biomarkers for the clinical diagnosis of acute vascular damage. Most molecular research on this toxicity has been performed in rats, but recent development of canine gene expression microarrays makes transcriptomic studies now possible in the dog. In this study, we investigated the molecular mechanisms of drug-induced vascular injury in dogs using gene arrays. After treating Beagles with toxic doses of CI-947, an adenosine receptor agonist, we profiled gene expression in the coronary arteries and correlated those changes with histopathology at 16 and 24 hours after dosing. The results demonstrated that pathobiological processes such as stimulation of the innate immune response, increased extracellular matrix turnover and oxidative stress were active at times of very early injury.

Acute Disease↗

GeneHuggers: database mining and application connectivity tools for subsequence analyses of the human genome.

UNLABELLED: GeneHuggers is a collection of program modules that enables precise selection of subsequence regions from records of the RefSeq human genome database. Subsequence regions can be selected based on diverse criteria, including feature addresses, annotations from LocusLink and UniGene, and results obtained from analyses with homologous subsequence detection programs. GeneHuggers provides functionality to the UNIX operating system that allows customized bioinformatics program development. AVAILABILITY: GeneHuggers source code is available under the GNU general public license and can be downloaded from ftp://ftp.scripps.edu/pub/genehuggers/gh.tar.gz

Abstracting and Indexing↗

Gene expression analysis in burn wounds of rats.

The events occurring early in the burn wound trigger a sequence of local and systemic responses that influence cell and tissue survival and, consequently, wound healing and recovery. Using high-density oligonucleotide arrays we identified gene expression patterns in skin samples taken from a region of injury in the burn rat model. The associated genomic events include the differential expression of genes involved in cell survival and death, cell growth regulation, cell metabolism, inflammation, and immune response. The functional gene cluster detected and their time appearance matched the time sequence known to occur in burn wound healing.

Animals↗

Reproductive biology: delivering spermatozoan RNA to the oocyte.

Even though the genetic fingerprint of human sperm has been defined, its role in orchestrating fertilization and the development of the early embryo remains vague. Here we show that human male gametes pass over more to the oocyte than just the haploid male genome--paternal messenger RNAs are also delivered to the egg at fertilization. If these transcripts, previously thought to be left-overs from spermatogenesis, are important in early development, our findings may have implications for the success of somatic-cell nuclear transfer in cloning technology and the identification of components leading to unexplained male-factor infertility.

Animals↗

Recurrent genomic alterations with impact on survival in colorectal cancer identified by genome-wide array comparative genomic hybridization.

BACKGROUND & AIMS: Although genetic aspects of tumorigenesis in colorectal cancer (CRC) have been well studied, reliable biomarkers predicting prognosis are scarce. We aimed to identify recurrently altered genomic regions (RAR) in CRC with high resolution, to investigate their implications on survival and to explore novel cancer-related genes in prognosis-associated RARs. METHODS: A 1-Mb resolution microarray-based comparative genomic hybridization (array CGH) was applied to 59 CRCs. RARs, defined as genomic alterations, detected in more than 10 cases were identified and analyzed for their association with survival. Expression levels of genes in prognosis-associated RARs were examined by real-time quantitative polymerase chain reaction. RESULTS: Twenty-seven RARs were identified. Eleven high-level amplifications and 2 homozygous deletions also were detected, but they were not as common as RARs. Multivariate analysis revealed RAR-L1 (loss on 1p36; hazard ratio = 8.15, P = .002) and RAR-L20 (loss on 21q22; hazard ratio = 3.53, P = .034) are independent indicators of poor prognosis. Expression of CAMTA1, located in RAR-L1, was reduced frequently in CRCs, and low CAMTA1 expression was associated significantly with poor prognosis, which indicates that CAMTA1 may play a role as a tumor suppressor in CRC. Five pairs of RARs were correlated significantly to each other and 3 pairs share genes involved in the same biological functions, suggesting possible collaborative roles in tumorigenesis. CONCLUSIONS: We identified recurrent genomic changes in 59 CRCs. RARs could be more important in sporadic tumors where the effect of genomic changes on tumorigenesis is relatively smaller than in familial cancer. Our results and analysis strategy will be helpful to elucidate pathogenesis of CRCs or to develop biomarkers for predicting prognosis.

Adult↗

PGAGENE: integrating quantitative gene-specific results from the NHLBI programs for genomic applications.

SUMMARY: PGAGENE is a web-based gene-specific genomic data search engine, which allows users to search over 5.9 million pieces of collective genetic and genomic data from the NHLBI supported Programs for Genomic Applications. This data includes microarray measurements, SNPs, and mutations, and data may be found using symbols, parts of gene names or products, Affymetrix probe IDs, GenBank accession numbers, UniGene IDs, dbSNP IDs, and others. The PGAGENE indexing agent periodically maps all publicly available gene-specific PGA data onto LocusLink using dynamically generated cross-referencing tables.

Base Sequence↗

A novel design of whole-genome microarray probes for Saccharomyces cerevisiae which minimizes cross-hybridization.

BACKGROUND: Numerous DNA microarray hybridization experiments have been performed in yeast over the last years using either synthetic oligonucleotides or PCR-amplified coding sequences as probes. The design and quality of the microarray probes are of critical importance for hybridization experiments as well as subsequent analysis of the data. RESULTS: We present here a novel design of Saccharomyces cerevisiae microarrays based on a refined annotation of the genome and with the aim of reducing cross-hybridization between related sequences. An effort was made to design probes of similar lengths, preferably located in the 3'-end of reading frames. The sequence of each gene was compared against the entire yeast genome and optimal sub-segments giving no predicted cross-hybridization were selected. A total of 5660 novel probes (more than 97% of the yeast genes) were designed. For the remaining 143 genes, cross-hybridization was unavoidable. Using a set of 18 deletant strains, we have experimentally validated our cross-hybridization procedure. Sensitivity, reproducibility and dynamic range of these new microarrays have been measured. Based on this experience, we have written a novel program to design long oligonucleotides for microarray hybridizations of complete genome sequences. CONCLUSIONS: A validated procedure to predict cross-hybridization in microarray probe design was defined in this work. Subsequently, a novel Saccharomyces cerevisiae microarray (which minimizes cross-hybridization) was designed and constructed. Arrays are available at Eurogentec S. A. Finally, we propose a novel design program, OliD, which allows automatic oligonucleotide design for microarrays. The OliD program is available from authors.

Base Sequence↗

Application of array-based comparative genomic hybridization to clinical diagnostics.

Microarray-based comparative genomic hybridization (array CGH) is a revolutionary platform that was recently adopted in the clinical laboratory. This technology was first developed as a research tool for the investigation of genomic alterations in cancer. It allows for a high-resolution evaluation of DNA copy number alterations associated with chromosome abnormalities. Array CGH is based on the use of differentially labeled test and reference genomic DNA samples that are simultaneously hybridized to DNA targets arrayed on a glass slide or other solid platform. In this review, we examine the technology and its transformation from a research tool into a maturing diagnostic instrument. We also evaluate the various approaches that have shaped the current platforms that are used for clinical applications. Finally, we discuss the advantages and shortcomings of "whole-genome" arrays and compare their diagnostic use to "targeted" arrays. Depending on their design, microarrays provide distinct advantages over conventional cytogenetic analysis because they have the potential to detect the majority of microscopic and submicroscopic chromosomal abnormalities. This new platform is poised to revolutionize modern cytogenetic diagnostics and to provide clinicians with a powerful tool to use in their increasingly sophisticated diagnostic capabilities.

Chromosome Aberrations↗

"A system biology" approach to bioinformatics and functional genomics in complex human diseases: arthritis.

Human and other annotated genome sequences have facilitated generation of vast amounts of correlative data, from human/animal genetics, normal and disease-affected tissues from complex diseases such as arthritis using gene/protein chips and SNP analysis. These data sets include genes/proteins whose functions are partially known at the cellular level or may be completely unknown (e.g. ESTs). Thus, genomic research has transformed molecular biology from "data poor" to "data rich" science, allowing further division into subpopulations of subcellular fractions, which are often given an "-omic" suffix. These disciplines have to converge at a systemic level to examine the structure and dynamics of cellular and organismal function. The challenge of characterizing ESTs linked to complex diseases is like interpreting sharp images on a blurred background and therefore requires a multidimensional screen for functional genomics ("functionomics") in tissues, mice and zebra fish model, which intertwines various approaches and readouts to study development and homeostasis of a system. In summary, the post-genomic era of functionomics will facilitate to narrow the bridge between correlative data and causative data by quaint hypothesis-driven research using a system approach integrating "intercoms" of interacting and interdependent disciplines forming a unified whole as described in this review for Arthritis.

Animals↗

Evolutionary and resistance dynamics in oligometastatic and oligoprogressive cancer treated with stereotactic radiotherapy and systemic therapies: A systematic review and focused meta-analysis.

BACKGROUND: Oligometastatic and oligoprogressive disease treated with stereotactic ablative radiotherapy (SABR) represents a clinically heterogeneous entity. Increasing evidence suggests that anatomical definitions alone may not adequately capture underlying biological diversity. This systematic review aimed to synthesize translational evidence exploring evolutionary dynamics, resistance mechanisms, and biomarker-driven stratification in patients treated with SABR. METHODS: A systematic literature review was performed including prospective and retrospective studies evaluating translational biomarkers in oligometastatic or oligoprogressive settings treated with SABR. Studies assessing genomic, transcriptomic, circulating or immune-related biomarkers were included. Data were summarized qualitatively according to predefined translational domains: (i) evolutionary dynamics under systemic therapy pressure, (ii) baseline biological stratification, (iii) longitudinal circulating biomarkers, and (iv) systemic immune remodeling. Exploratory quantitative visual syntheses were performed using reported hazard ratios when conceptually comparable endpoints were available. RESULTS: 19 studies comprising 1527 patients were included. Across tumor types and treatment contexts, translational analyses consistently indicated that anatomically defined oligometastatic states encompass biologically distinct subgroups with different risks of systemic progression. Studies evaluating oligoprogression under ongoing systemic therapy suggested a distinction between spatially constrained resistance and systemic molecular escape, supported by circulating tumor DNA and tissue- or plasma-based molecular profiling (including genomic and transcriptomic analyses). Baseline biological features, including adverse genomic signatures and circulating biomarkers, were associated with inferior progression outcomes despite metastasis-directed therapy. Longitudinal biomarkers provided early signals of treatment response and systemic control. Immune remodeling after SABR showed context-dependent effects, both systemic immune activation and treatment-related immunosuppression reported across studies.

Humans↗