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PubMedDate not supplied

Molecular profiling of temporal lobe epilepsy: comparison of data from human tissue samples and animal models.

The advent of gene chip technology and the era of functional genomics have initially been accompanied by huge anticipations to quickly unravel the molecular pathogenesis of multifactorial diseases. Expectations have, today, given way to some concerns about this non-hypothesis driven approach. However, the careful and controlled application of expression microarrays in concert with refined bioinformatic tools may provide novel insights in major disorders particularly of highly complex organs such as the central nervous system (CNS). Epilepsies are among the most frequent CNS disorders affecting approximately 1.5% of the population worldwide. In temporal lobe epilepsy (TLE), the seizure origin typically involves the hippocampal formation, a structure located in the mesial temporal lobe. Many TLE patients develop pharmacoresistance, i.e. seizures can no more be controlled by antiepileptic drugs. In order to achieve seizure control, surgical removal of the epileptogenic focus has been established as successful therapeutic strategy. Hippocampal biopsy tissue of pharmacoresistant TLE patients represents an excellent substrate to analyze molecular mechanisms related to structural and cellular reorganization in epilepsy. The complexity of alterations in TLE hippocampi suggests numerous genes and signaling cascades to be involved in the pathogenesis. By microarrays, genome wide expression profiles can be constituted from TLE tissues. However, hippocampi of pharmacoresistant TLE patients represent an advanced stage of the disease. Early stages of epilepsy development are not available for functional genome analysis in humans. Animal models of TLE appear particularly helpful to study molecular mechanisms of highly dynamic processes such as the development of hyperexcitability and pharmacoresistance. In this review, we summarize recent data of gene expression profiles in human and experimental TLE and discuss the relevance of novel tools for bioinformatic analysis and data mining.

Animals↗
PubMed1979

Molecular clocks, molecular profiles, and optimum diets: three approaches to the problem of aging.

It has been hypothesized that the deamidation of glutaminyl and asparaginyl residues serves as a molecular clock for many biological processes including protein turnover, development, and aging. At present, this hypothesis has passed some experimental tests which are necessary but not sufficient for its acceptance. The current state of evidence about deamidation as a molecular clock is discussed. In addition, since the molecular biology of aging, especially in humans, is only partly understood, it is of value to develop quantitative, empirical measures of physiological human age and to use these measures to evaluate alternative human living conditions, especially easily adopted alternatives like variations in diet. This may allow some decrease in the suffering and loss from human aging until such time as molecular biology provides superior and more intellectually satisfying answers. An empirical system which consists of quantitative measurement of several hundred human chemical constituents followed by computerized pattern recognition is described. It is hoped that this system will eventually become an aid in the minimization of the rate of human aging through changes in diet and other factors.

Adult↗
PubMed2026

Integrated morphologic, immunophenotypic, and molecular profiling of advanced upper tract urothelial carcinoma across tumor compartments supports biopsy-based testing.

Upper tract urothelial carcinoma (UTUC) is an aggressive malignancy with limited molecular characterization in advanced disease. FGFR3 alterations are well established in low-grade urothelial carcinoma, but their prevalence, stability, and biological significance in locally advanced and metastatic UTUC remain only partially defined. We performed an integrated morphologic, immunohistochemical, and molecular analysis of 24 locally advanced and/or metastatic UTUC from 20 patients. FGFR3 status was assessed by RT-PCR across multiple tumor compartments, including biopsies, primary tumors, lymph-node metastases, and distant metastatic sites. Immunohistochemistry included CK20, CK5, GATA3, p53, and mismatch repair proteins. Targeted next-generation sequencing (NGS) was used to characterize co-occurring genomic alterations and to assess concordance with p53 immunophenotype. FGFR3 alterations were identified in 50% of patients and in 54.2% of analyzed tumors. FGFR3 status showed high intra-patient stability, with concordance between primary tumors and distant metastases in 90% of cases, whereas concordance with lymph node metastases was lower (50%), suggesting site-specific clonal divergence. Despite advanced stage, 92.3% of FGFR3-altered tumors displayed papillary urothelial carcinoma morphology, and most showed a luminal immunophenotype (61.5% by CK20/CK5 and 69.2% by GATA3/CK5). Targeted NGS revealed additional pathogenic alterations in 75% of patients, most frequently involving RTK/RAS/MAPK signaling (70%), cell-cycle regulation (25%), and PI3K/AKT pathway components (10%). TP53 mutations co-occurred with FGFR3 alterations in 60% of FGFR3-mutated patients and showed 90.4% concordance with p53 immunohistochemistry. Finally, a few cases exhibited complex, multi-site FGFR3 mutational patterns, consistent with intratumoral clonal evolutions. In conclusion, FGFR3 alterations are frequent and remarkably stable in advanced UTUC, even in high-grade and metastatic disease. These findings support the reliability of FGFR3 testing on limited diagnostic material and reinforce its relevance for therapeutic stratification. UTUC emerges as a molecularly dynamic disease in which early oncogenic drivers such as FGFR3 continue to shape tumor biology and therapeutic vulnerability at advanced stages.

Humans↗
PubMed2003

Immune profiling: molecular monitoring in renal transplantation.

Molecular techniques have become a mainstay for most biomedical research. In particular, sensitive methods for gene transcript detection and advanced flow cytometry have been crucial in fostering our understanding of the basic mechanisms promoting allosensitization and adaptive immune regulation. These technologies have been validated in vitro, and in pre-clinical settings, and as such their clinical application is now clearly appropriate. It is becoming increasingly clear that these robust techniques hold much promise to better elucidate human transplant biology, and more importantly, guide clinical decision making with mechanistically-based information. This article will discuss our laboratory's use of several novel technologies, including gene polymorphism analysis, real-time polymerase chain reaction transcript quantification, and multi-color flow cytometry in clinical human renal transplantation. Specific technical methodology will be presented outlining keys for effective clinical application. Clinical correlations will be presented as examples of how these techniques may have clinical relevance. Suggestions for the adaptation of these methods for therapeutic intervention will be given. We propose that clinical transplantation should proceed in close step with modern molecular diagnostics.

Animals↗
PubMed2005

Molecular profiling of ulcerative colitis-associated neoplastic progression.

Fundamental differences exist between ulcerative colitis (UC)-associated and sporadic forms of colorectal cancer, including preexisting inflammation, type of dysplasia, and timing of molecular events in carcinogenesis. Transcriptional alterations that occur in UC-associated neoplasia in the progression from normal mucosa through dysplastic epithelium to invasive cancer have not been described. We used Affymetrix U95Av2 microarrays to assess differential gene expression in the neoplastic progression of UC tissue from the colonic mucosa of individuals with benign UC, UC-dysplasia-associated lesions or masses, and UC adenocarcinoma. By correlating transcript alterations across tissue types using a mixed statistical model, we identified 699 genes exhibiting altered expression with dysplasia development. A different expression profile was observed in progression to adenocarcinoma with 392 transcripts exhibiting differential expression. There were 224 transcripts common to both dysplasia and adenocarcinoma. Most of the differentially expressed genes described herein were not previously known to play a role in neoplastic progression in UC, including transcripts affecting cell proliferation and apoptosis, signal transduction and signaling, and DNA repair. The altered expression of five transcripts was confirmed by quantitative real-time reverse-transcription polymerase chain reaction. Based on comparisons with previous studies on sporadic colorectal carcinoma, several similarities were found. There were, however, important differences that suggest that different molecular events may occur in the development of UC-associated neoplasia. Several of these genes demonstrated similar changes in dysplastic and cancerous tissue and may be involved in early cancer formation. Identification of these genes as potential clinical biomarkers may lead to improved early disease diagnosis.

Adenocarcinoma↗
PubMed2004

Molecular profiling of primary and metastatic neoplasms in the lung using cytologic material obtained by fine-needle aspiration: report of two cases.

Two cases are presented in which molecular analyses of cytologic material obtained by fine-needle aspiration were helpful in establishing relationships between morphologically similar neoplasms in the same patient. For appropriate clinical management, it is important to ascertain whether the tumors represent independent primaries or metastases. Alcohol-fixed cytologic material prepared as cell blocks and formalin-fixed paraffin-embedded tissue were microdissected and analyzed for allelic loss of heterozygosity at multiple preselected genetic loci. The first case illustrates a 69-yr-old man with multiple intrapulmonary nodules involving the upper and lower lobes of the left lung. Genomic analysis showed that the neoplasms in the left upper and lower lung lobes were independent primaries, because the loss of heterozygosity (LOH) patterns were substantially different. By contrast, the second case is that of a 58-yr-old man with a right thyroid nodule and multiple pulmonary tumors. LOH analysis confirmed that a sampled pulmonary tumor represented a metastasis from the thyroid primary, as similar LOH patterns involving locus D9S252 were observed on comparison of the thyroid and pulmonary neoplasms. These cases illustrate the practical diagnostic utility of genomic analysis using cytologic material in the assessment of primary and metastatic malignancies.

Aged↗
PubMed2007

Molecular profiles of mitogen activated protein kinase signaling pathways in orofacial development.

BACKGROUND: Formation of the mammalian orofacial region involves multiple signaling pathways regulating sequential expression of and interaction between molecular signals during embryogenesis. The present study examined the expression patterns of members of the MAPK family in developing murine orofacial tissue. METHODS: Total RNA was extracted from developing embryonic orofacial tissue during gestational days (GDs) 12-14 and used to prepare biotinylated cDNA probes, which were then denatured and hybridized to murine MAPK signaling pathways gene arrays. RESULTS: Expression of a number of genes involved in the (ERK1/2) cascade transiently increased in the embryonic orofacial tissue over the developmental period examined. Numerous members of the SAPK/JNK cascade were constitutively expressed in the tissue. Genes known to play a role in p38 MAPK signaling exhibited constitutive expression during orofacial development. Western blot analysis demonstrated that ERK2/1, p38, and SAPK/JNK kinases are present in embryonic orofacial tissue on each of GD 12, 13, and 14. By using phospho-specific antibodies, active ERK was shown to be temporally regulated during orofacial development. Minimal amounts of active p38 and active SAPK/JNK were detected in orofacial tissue during GDs 12-14. CONCLUSIONS: Our study documents specific expression patterns of genes coding for proteins belonging to the ERK1/2, p38, and SAPK/JNK MAPK families in embryonic orofacial tissue. We also demonstrate that active, phosphorylated forms of ERK1/2 only were detected in the embryonic tissue investigated, suggesting a more central role for members of this family in embryonic orofacial development.

Animals↗
PubMed2006

Phospholipid molecular profiles in the seed kernel from different sunflower (Helianthus annuus) mutants.

Phospholipids are essential components of plant cell membranes whose acyl composition appears to be influenced by oil composition in the sunflower. In the current study, we have determined the diacylglycerol profile of the main phospholipids using phospholipase C degradation and separation of the diacylglycerols by HPLC and GLC. The main polar lipid molecular species were defined in different classes of sunflower kernel: PC, PE, and PI. The proportions of each were determined at different stages of development in order to define the point at which the mutations carried by each sunflower line affected the phospholipid composition of the seeds. The results indicated that modifications to intraplastidial de novo FA synthesis affected the seed phospholipid profile during the whole period of the seed formation, including accumulation and maturation, whereas the influence of mutations in the endoplasmic reticulum desaturases were more readily detected at later stages of development. These results are discussed in terms of the pathways involved in glycerolipid synthesis and phospholipid conversion in sunflower seeds.

Helianthus↗
PubMed1981

Molecular profiles of human melanoma-associated antigens.

Melanoma-associated antigens (MAA) shed into spent culture medium of intrinsically radiolabeled melanoma cells react specifically with monoclonal and polyclonal anti-melanoma xenoantiserums and are represented by two glycoproteins with molecular weights of 240,000 (240K) and 94,000 (94K): 240K is present only on melanoma cells whereas 94k is also found on carcinoma cells and on fetal melanocytes. Both 240K and 94K have been obtained radiochemically pure by utilizing cellulose ion-exchange and antibody affinity chromatography. The two antigens have different charge properties, as 240K binds to CM-cellulose while 94K does not. A difference in carbohydrate moieties is also indicated since 240K binds selectively to lentil lectin and 94K to ricin lectin. Two-dimensional gel electrophoresis an tryptic peptide maps of the two antigens reveal distinct and characteristics profiles. Subunit structure determination of both antigens suggests 94K to be a single chain whereas 240K appears to be a subunit of a larger molecular structure.

Antibodies, Neoplasm↗
PubMed2004

Molecular profiling of inflammatory breast cancer: identification of a poor-prognosis gene expression signature.

PURPOSE: Inflammatory breast cancer (IBC) is a rare but particularly aggressive form of primary breast cancer. The molecular mechanisms responsible for IBC are largely unknown. EXPERIMENTAL DESIGN: To obtain further insight into the molecular pathogenesis of IBC, we used real-time quantitative reverse transcription (RT)-PCR to quantify the mRNA expression of 538 selected genes in IBC relative to non-IBC. RESULTS: Twenty-seven (5.0%) of the 538 genes were significantly up-regulated in IBC compared with non-IBC. None were down-regulated. The 27 up-regulated genes mainly encoded transcription factors (JUN, EGR1, JUNB, FOS, FOSB, MYCN, and SNAIL1), growth factors (VEGF, DTR/HB-EGF, IGFBP7, IL6, ANGPT2, EREG, CCL3/MIP1A, and CCL5/RANTES) and growth factor receptors (TBXA2R, TNFRSF10A/TRAILR1, and ROBO2). We also identified a gene expression profile, based on MYCN, EREG, and SHH, which discriminated subgroups of IBC patients with good, intermediate, and poor outcome. CONCLUSION: Our study has identified a limited number of signaling pathways that require inappropriate activation for IBC development. Some of the up-regulated genes identified here could offer useful diagnostic or prognostic markers and could form the basis of novel therapeutic strategies.

Breast Neoplasms↗
PubMed2004

Spinal cord molecular profiling provides a better understanding of amyotrophic lateral sclerosis pathogenesis.

Research efforts in amyotrophic lateral sclerosis (ALS) have not yet provided a comprehensive explanation of the disease pathogenesis, which is emerging as a complex interaction between multiple factors. Gene expression studies traditionally based on single mRNA specie analysis have recently progressed to allow entire transcriptional profiles of affected tissues to be obtained through array-based methods. This experimental approach has significantly improved our understanding of the molecular changes occurring in ALS, although its limitations in the detection of low-abundance transcripts in tissues with a high level of complexity are becoming increasingly recognized. In this paper, experimental findings based on an expression study in post-mortem spinal cord from sporadic ALS individuals will be discussed in light of recently published data using array analysis in an animal model of the disease. Previous expression data obtained using conventional techniques are also compared. Through the analysis of the information arising from ALS post-mortem and animal model tissues studies, we have identified a pattern of molecular events in which factors implicated in the immune response, cytoprotection and growth-differentiation are differentially regulated in a time-dependent way from early to advanced stages of disease progression.

Amyotrophic Lateral Sclerosis↗
PubMed2000

Molecular profiling of human cancer.

Traditionally, tumours have been categorized on the basis of histology. However, the staining pattern of cancer cells viewed under the microscope is insufficient to reflect the complicated underlying molecular events that drive the neoplastic process. By surveying thousands of genes at once, using DNA arrays, it is now possible to read the molecular signature of an individual patient's tumour. When the signature is analysed with clustering algorithms, new classes of cancer emerge that transcend distinctions based on histological appearance alone. Using DNA arrays, protein arrays and appropriate experimental models, the ultimate goal is to move beyond correlation and classification to achieve new insights into disease mechanisms and treatment targets.

Gene Expression Profiling↗
PubMed2005

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of pancreatic malignancy and other biological phenomena. This chapter reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed-over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification. High-quality tissue microdissection does not necessarily mean high-quality samples to analyze. The quality of biomaterials obtained for analysis is highly dependent on steps upstream and downstream from tissue microdissection. We provide protocols for each of these steps, and encourage you to improve upon these. It is worth the effort of every laboratory to optimize and document its technique at each stage of the process, and we provide a starting point for those willing to spend the time to optimize. In our view, poor documentation of tissue and cell type of origin and the use of nonoptimized protocols is a source of inefficiency in current life science research. Even incremental improvement in this area will increase productivity significantly.

Coloring Agents↗
PubMed2005

Molecular profiling of CD34+ cells identifies low expression of CD7, along with high expression of proteinase 3 or elastase, as predictors of longer survival in patients with CML.

Although most patients with chronic myeloid leukemia (CML) have the same initial molecular abnormality, the BCR-ABL fusion gene, the duration of chronic phase (CP) varies widely. To identify the possible molecular basis of this heterogeneity, we studied CD34+ cells collected at diagnosis from 68 patients with CML-CP. By using oligonucleotide microarray screening, we performed gene-expression profiling on 2 subsets of patients, one comprising patients with an "aggressive disease" who developed blastic transformation (BT) within 3 years of diagnosis (n = 10) and, at the other extreme, patients with an "indolent disease" whose BT occurred 7 or more years from diagnosis (n = 9). This screening revealed 20 genes differentially expressed in patients with aggressive and indolent disease, which were validated by quantitative reverse transcriptase/polymerase chain reaction (Q-RT/PCR). A multivariate Cox regression model identified the combination of low CD7 expression with high expression of proteinase 3 or elastase as associated with longer survival in the complete cohort of 68 patients. This differential pattern of gene expression probably reflects the intrinsic heterogeneity of the disease; if so, assessing expression levels of selected genes at diagnosis may be valuable in predicting duration of survival in patients treated with imatinib and the newer tyrosine kinase inhibitors.

Antigens, CD34↗
PubMedDate not supplied

Effect of hepatitis C virus core protein on the molecular profiling of human B lymphocytes.

Hepatitis C virus (HCV) core protein features many intriguing properties and plays a pivotal role in cellular immunity, cell growth, apoptosis, cell transformation, and eventually in tumor development. However, the role of B cells, the primary players in the humoral immune response, during HCV infection is largely unknown. To explore the molecular effects of HCV core on human B cells, we conducted gene expression profiling of serial RNA samples from B cells that were infected with adenovirus harboring full-length HCV core protein and beta-galactosidase as a reference using a microarray platform containing 22,149 human oligo probes. The entire experiment was performed in duplicate in B lymphocytes that were isolated from two individual donors and incubated for up to 3 days after infection with adenovirus expressing HCV core protein to identify dynamic gene expression patterns. Differential expression of representative genes was validated by quantitative RT-PCR. We found that HCV core significantly inhibited B-lymphocyte apoptosis. We showed a dramatic downregulation of MHC class II molecules in B cells expressing HCV core, whereas the expression of immunoglobulin genes was not significantly altered. Moreover, genes associated with leukemia and B-lymphoma were consistently upregulated by HCV core. In contrast, downregulation of caspase-1 and caspase-4 was found to be associated with core's ability to prevent B-lymphocyte apoptosis. In summary, we have identified several clusters of genes that are differentially expressed in human B lymphocytes expressing HCV core, suggesting a potential impairment of antigen processing and presentation, which may provide more insights into HCV infection in B lymphocytes.

Adenoviridae↗
PubMed2006

Transcriptome profiling, molecular biological, and physiological studies reveal a major role for ethylene in cotton fiber cell elongation.

Upland cotton (Gossypium hirsutum) produces the most widely used natural fibers, yet the regulatory mechanisms governing fiber cell elongation are not well understood. Through sequencing of a cotton fiber cDNA library and subsequent microarray analysis, we found that ethylene biosynthesis is one of the most significantly upregulated biochemical pathways during fiber elongation. The 1-Aminocyclopropane-1-Carboxylic Acid Oxidase1-3 (ACO1-3) genes responsible for ethylene production were expressed at significantly higher levels during this growth stage. The amount of ethylene released from cultured ovules correlated with ACO expression and the rate of fiber growth. Exogenously applied ethylene promoted robust fiber cell expansion, whereas its biosynthetic inhibitor l-(2-aminoethoxyvinyl)-glycine (AVG) specifically suppressed fiber growth. The brassinosteroid (BR) biosynthetic pathway was modestly upregulated during this growth stage, and treatment with BR or its biosynthetic inhibitor brassinazole (BRZ) also promoted or inhibited, respectively, fiber growth. However, the effect of ethylene treatment was much stronger than that of BR, and the inhibitory effect of BRZ on fiber cells could be overcome by ethylene, but the AVG effect was much less reversed by BR. These results indicate that ethylene plays a major role in promoting cotton fiber elongation. Furthermore, ethylene may promote cell elongation by increasing the expression of sucrose synthase, tubulin, and expansin genes.

Amino Acids, Cyclic↗
PubMed2002

Molecular profiling of human chondrosarcomas for matrix production and cancer markers.

Chondrosarcoma is the second most common malignant bone tumor, characterized by production of abundant extracellular matrix resembling hyaline cartilage. To better understand the molecular pathogenesis of chondrosarcoma, we analyzed 12 chondrosarcomas for their production of connective tissue components and SOX9, a key regulator of normal chondrocyte differentiation. Furthermore, 10 chondrosarcoma samples were screened for additional changes in gene expression using cDNA array analysis. In Northern analysis, several tumors were found to express type II collagen mRNA at levels comparable to fetal cartilage used as a control. Interestingly, the highest levels of type II collagen mRNA were seen in 2 of the 3 grade 3 chondrosarcomas, which also exhibited the highest mRNA levels of SOX9 and "prechondrogenic" pro alpha 1(IIA) collagen. Expression of SOX9 in human chondrosarcomas is novel and suggests that chondrosarcomas originate from a multipotent stem cell committed to differentiation along the chondrogenic pathway. Results of the cDNA array analyses emphasize the heterogeneous nature of chondrosarcoma as no single transcript was systematically up- or downregulated in all tumors analyzed. Among the interesting changes observed was upregulation of decorin mRNA in 7 of the 10 tumors analyzed. Further studies are needed to determine whether decorin plays a role in the pathogenesis of chondrosarcoma. The cDNA arrays also revealed discrepancies from Northern and RNase protection analyses in transcript levels of matrix components, emphasizing the need to validate cDNA array data with other techniques.

Adult↗