Reverse transcription-PCR Approach Research Articles

DAS-ELISA-based detection and RT-PCR validation for precise and timely analysis of Citrus tristeza virus infection in Nepalese citrus species

Citrus viral infections pose a substantial threat to the global citrus industry, resulting in multitudinous economic losses annually. Timely and precise identification of citrus viruses offers a promising avenue for proactive insights preemptively before the instigation of extensive infections. The present study was devised to expedite the prompt identification of the presence of Citrus tristeza virus (CTV) within Nepalese citrus species utilizing a Double Antibody Sandwich-Enzyme-Linked Immunosorbent Assay (DAS-ELISA), followed by its validation using reverse transcription PCR (RT-PCR). Samplings extracted from plants harvested from 74 out of the 77 administrative districts within Nepal provide substantial evidence of extensive propagation of viral contagion among citrus cultivars across 64 districts (86.48%) of the nation. A comprehensive analysis of the samples obtained from diverse geographical localities revealed that 36 districts (48.64%) exhibit mild infestation, while 9 districts (12.16%) evince moderate infestation. Moreover, samples harvested from 19 districts (25.67%) were highly infected; intriguingly, specimens from 10 districts (13.51%) manifest an absence of viral intrusion. The results have been validated with RT-PCR assay. Given the profound significance of citrus as a pivotal repository of vitamin C and its essential role in conferring advantageous health attributes to humankind, alongside its substantial contribution to the economic paradigm, a meticulous examination is imperative to safeguard these plants against an array of viral infections. Collectively, the DAS-ELISA methodology along with RT-PCR approaches constitutes an exquisitely sensitive and meticulously specific modality, poised to accelerate the expeditious detection of citrus-associated viruses within burgeoning economies like Nepal. This advancement supports timely and precise detection and diagnosis of agriculturally virulent conditions, thereby forestalling the eruption of viral epidemics and concomitantly curbing their financial repercussions.

An Enhanced Quantitative Reverse Transcription-PCR Approach for Measuring Circulating MicroRNAs.

Short (22nt) RNAs called microRNAs (miRNAs) bind to and inhibit target messenger RNAs in gene regulatory networks. Recent study suggests that miRNAs circulate in a stable, cell-free form and that particular miRNAs in plasma or serum may be biomarkers for cancer and other diseases. Circulating miRNAs as biomarkers provide distinct challenges including pre-analytic variance and data standardization. We describe our qRT-PCR approach for measuring circulating miRNAs as biomarkers, as well as sample preparation, experimental design, and data processing issues.

Distinct gene expression patterns of SOX2 and SOX2OT variants in different types of brain tumours

Numerous investigations have been recently published on the dysregulated expression of long-noncoding RNAs (lncRNAs) in various cancer types, emphasizing that abnormal lncRNA expression is a major contributor to tumourigenesis. A broad spectrum of lncRNAs is expressed in the central nervous system, where these RNAs seem to play key roles in brain development and function. In addition to expressing SOX2, a master regulator of pluripotency that lies within its third intron, lncRNA SOX2OT has a proposed role in regulating neural development. Based on our previous studies, alternative splicing of SOX2OT generates two alternatively spliced variants (SOX2OT-S1 and SOX2OT-S2). The present study investigated the expression patterns of SOX2OT variants and SOX2 in three principal types of brain tumours (gliomas, meningiomas and pituitary adenomas) and in four brain tumour cell lines (U87-MG, 1321N1, A172 and DAOY). Total RNAwas extracted from 34 human brain tumour specimens, and the expression profile of target genes was measured using a real-time reverse transcription PCR approach. Our data revealed distinct expression patterns for SOX2OT variants and SOX2 in the brain tumour samples, indicating their potential involvement in brain tumourigenesis. Moreover, our results highlighted the potential usefulness of SOX2OT-S1, SOX2OT-S2, and SOX2 in molecular diagnosis and brain tumour classification.

MiR-30-HNF4γ and miR-194-NR2F2 regulatory networks contribute to the upregulation of metaplasia markers in the stomach

ObjectiveIntestinal metaplasia and spasmolytic polypeptide-expressing metaplasia (SPEM) are considered neoplastic precursors of gastric adenocarcinoma and are both marked by gene expression alterations in comparison to normal stomach. Since miRNAs are...

A Novel Three-Colour Fluorescence in Situ Hybridization Approach for the Detection of t(7;12)(q36;p13) in Acute Myeloid Leukaemia Reveals New Cryptic Three Way Translocation t(7;12;16)

The t(7;12)(q36;p13) translocation is a recurrent chromosome abnormality that involves the ETV6 gene on chromosome 12 and has been identified in 20–30% of infant patients with acute myeloid leukaemia (AML). The detection of t(7;12) rearrangements relies on the use of fluorescence in situ hybridization (FISH) because this translocation is hardly visible by chromosome banding methods. Furthermore, a fusion transcript HLXB9-ETV6 is found in approximately 50% of t(7;12) cases, making the reverse transcription PCR approach not an ideal screening method. Considering the report of few cases of variant translocations harbouring a cryptic t(7;12) rearrangement, we believe that the actual incidence of this abnormality is higher than reported to date. The clinical outcome of t(7;12) patients is believed to be poor, therefore an early and accurate diagnosis is important in the clinical management and treatment. In this study, we have designed and tested a novel three-colour FISH approach that enabled us not only to confirm the presence of the t(7;12) in a number of patients studied previously, but also to identify a cryptic t(7;12) as part of a complex rearrangement. This new approach has proven to be an efficient and reliable method to be used in the diagnostic setting.

Hepatitis E virus genotype 3 in wild rats, United States.

The role of rodents in the epidemiology of zoonotic hepatitis E virus (HEV) infection has been a subject of considerable debate. Seroprevalence studies suggest widespread HEV infection in commensal Rattus spp. rats, but experimental transmission has been largely unsuccessful and recovery of zoonotic genotype 3 HEV RNA from wild Rattus spp. rats has never been confirmed. We surveyed R. rattus and R. norvegicus rats from across the United States and several international populations by using a hemi-nested reverse transcription PCR approach. We isolated HEV RNA in liver tissues from 35 of 446 rats examined. All but 1 of these isolates was relegated to the zoonotic HEV genotype 3, and the remaining sequence represented the recently discovered rat genotype from the United States and Germany. HEV-positive rats were detected in urban and remote localities. Genetic analyses suggest all HEV genotype 3 isolates obtained from wild Rattus spp. rats were closely related.

Comprehensive MicroRNA Profiling for Head and Neck Squamous Cell Carcinomas

The objective of this study is to investigate the significance of microRNAs (miRNA) in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). A global miRNA profiling was done on 51 formalin-fixed archival HNSCC samples using quantitative reverse transcription-PCR approach, correlated with patients' clinical parameters. Functional characterization of HNSCC-associated miRNAs was conducted on three HNSCC cell lines. Cell viability and proliferation were investigated using MTS and clonogenic assays, respectively; cell cycle analyses were assessed using flow cytometry. Thirty-eight of the 117 (33%) consistently detected miRNAs were significantly differentially expressed between malignant versus normal tissues. Concordant with previous reports, overexpression of miR-21, miR-155, let-7i, and miR-142-3p and underexpression of miR-125b and miR-375 were detected. Upregulation of miR-423, miR-106b, miR-20a, and miR-16 as well as downregulation of miR-10a were newly observed. Exogenous overexpression of miR-375 in HNSCC cell lines reduced proliferation and clonogenicity and increased cells in sub-G(1). Similar cellular effects were observed in knockdown studies of the miR-106b-25 cluster but with accumulation of cells in G(1) arrest. No major difference was detected in miRNA profiles among laryngeal, oropharyngeal, or hypopharyngeal cancers. miR-451 was found to be the only significantly overexpressed miRNA by 4.7-fold between nonrelapsed and relapsed patients. We have identified a group of aberrantly expressed miRNAs in HNSCC and showed that underexpression of miR-375 and overexpression of miR-106b-25 cluster might play oncogenic roles in this disease. Further detailed examinations of miRNAs will provide opportunities to dissect the complex molecular abnormalities driving HNSCC progression.

Milk Protein Fragments Induce the Biosynthesis of Macedocin, the Lantibiotic Produced by Streptococcus macedonicus ACA-DC 198

The aim of the present work was to study the mode of the induction of the biosynthesis of macedocin, the lantibiotic produced by Streptococcus macedonicus ACA-DC 198. Macedocin was produced when the strain was grown in milk but not in MRS or M17 broth. No autoinduction mechanism was observed. Production did not depend on the presence of lactose or galactose in the culture medium or on a coculture of the producer strain with macedocin-sensitive or macedocin-resistant strains. Induction seemed to depend on the presence of one or more heat-stable protein components produced when S. macedonicus ACA-DC 198 was grown in milk. The partial purification of the induction factor was performed by a combination of chromatography methods, and its activity was confirmed by a reverse transcription-PCR approach (RT-PCR). Mass spectrometric (MS) and tandem mass spectrometric (MS/MS) analyses of an induction-active fraction showed the presence of several peptides of low molecular mass corresponding to fragments of alpha(S1)- and beta-casein as well as beta-lactoglobulin. The chemically synthesized alpha(S1)-casein fragment 37-55 (2,253.65 Da) was proven to be able to induce macedocin biosynthesis. This is the first time that milk protein degradation fragments are reported to exhibit a bacteriocin induction activity.

Large-scale Real-time Reverse Transcription-PCR Approach of Angiogenic Pathways in Human Transitional Cell Carcinoma of the Bladder: Identification of VEGFA as a Major Independent Prognostic Marker

Actors of the angiogenesis pathways are targets for the new promising targeted therapies already used in several malignancies. In bladder cancer, antiangiogenic molecules could also add to already existing treatment options. To evaluate the involvement of angiogenesis pathways in bladder carcinogenesis and identify new molecular markers having a clinical implication. Expression levels of 40 genes involved in angiogenesis were assessed by quantitative real time RT-PCR in 157 urothelial tumour bladder samples obtained from patients who underwent transurethral bladder resection or radical cystectomy between 2001 and 2005. Pathologic tumour staging showed: 73 non-muscle-invasive bladder tumours (30 low-grade pTa, 14 high-grade pTa, and 29 high-grade pT1), and 84 muscle-invasive tumours (> or = pT2), all of high grade. RT-PCR results were associated with a survival analysis. VEGFA, MET, CXCR4, and IL8 were significantly overexpressed in tumour samples as compared to normal bladder tissue. VEGFA overexpressions were found in 89% of non-muscle-invasive and 66% of muscle-invasive tumour samples. In univariate analysis, for invasive tumours, VEGFA overexpression was associated with a poorer outcome in both overall and disease-free survival (p=0.011 and 0.026 respectively) at a 13-mo median follow-up. Multivariate analysis retained T stage, N status, and VEGFA overexpression as independent prognostic factors in both overall and disease-free survival (p=0.02 and p=0.04, respectively, for VEGFA). This study shows that, in bladder cancer, VEGFA status could be used as a prognostic factor at the individual level. VEGFA overexpression could guide a rationalized use of the costly antiangiogenic therapies which could therefore become part of the treatment options in bladder cancer.

Editorial Comment on: Large-scale Real-time Reverse Transcription-PCR Approach of Angiogenic Pathways in Human Transitional Cell Carcinoma of the Bladder: Identification of VEGFA as a Major Independent Prognostic Marker

Abstract Background Actors of the angiogenesis pathways are targets for the new promising targeted therapies already used in several malignancies. In bladder cancer, antiangiogenic molecules could also add to already existing treatment options. Objective To evaluate the involvement of angiogenesis pathways in bladder carcinogenesis and identify new molecular markers having a clinical implication. Design, setting, and participants Expression levels of 40 genes involved in angiogenesis were assessed by quantitative real time RT-PCR in 157 urothelial tumour bladder samples obtained from patients who underwent transurethral bladder resection or radical cystectomy between 2001 and 2005. Pathologic tumour staging showed: 73 non-muscle-invasive bladder tumours (30 low-grade pTa, 14 high-grade pTa, and 29 high-grade pT1), and 84 muscle-invasive tumours (≥ pT2), all of high grade. RT-PCR results were associated with a survival analysis. Results and limitations VEGFA , MET , CXCR4 , and IL8 were significantly overexpressed in tumour samples as compared to normal bladder tissue. VEGFA overexpressions were found in 89% of non-muscle-invasive and 66% of muscle-invasive tumour samples. In univariate analysis, for invasive tumours, VEGFA overexpression was associated with a poorer outcome in both overall and disease-free survival ( p =0.011 and 0.026 respectively) at a 13-mo median follow-up. Multivariate analysis retained T stage, N status, and VEGFA overexpression as independent prognostic factors in both overall and disease-free survival ( p =0.02 and p =0.04, respectively, for VEGFA ). Conclusions This study shows that, in bladder cancer, VEGFA status could be used as a prognostic factor at the individual level. VEGFA overexpression could guide a rationalized use of the costly antiangiogenic therapies which could therefore become part of the treatment options in bladder cancer.

Multiple Molecular Analyses From Minimal Cell Quantities by Sequential Isolation and Preamplification of DNA and RNA

Molecular analyses of single or very few cells are often handicapped by low amounts of DNA or RNA from starting material. Specimens like biopsies, embryonic stem cells, or laser-microdissected tissues often do not provide nucleic acid quantities required for robust amplification and verification by diagnostic polymerase chain reaction (PCR)-based analyses. A simultaneous isolation procedure for both RNA and DNA from a single sample would greatly facilitate demanding molecular studies. Here, we describe a new method that combines the isolation of mRNA using commercially available oligo (dT)-bound magnetic beads with the precipitation of DNA from the remaining cell lysate. Both types of nucleic acids can then be separately preamplified by improved primer extension preamplification and subsequently used for multiple molecular analyses. With this procedure we were able to reliably gain DNA for PCR from as few as 5 cells. RNA suitable for various reverse transcription-PCR approaches and sequencing analyses could simultaneously be isolated even from single cell preparations.

Viral Load and Crimean-Congo Hemorrhagic Fever

Viral Load and Crimean-Congo Hemorrhagic Fever

Isoprenoid biosynthesis authenticates the classification of the green alga Mesostigma viride as an ancient streptophyte

Land plants harbor two essential and completely different metabolic pathways for isoprenoid synthesis. The cytosolic mevalonate pathway (MVA) is shared with heterotrophic eukaryotes, whereas the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway has a cyanobacterial origin and was recruited after primary endosymbiosis. Terrestrial plants and green algae have a common evolutionary ancestry, but biochemical as well as genome analyses indicate that the cytosolic MVA pathway is generally absent from Chlorophyta. We investigated the distribution of genes for both pathways in the green alga Mesostigma viride, a key species at the basis of streptophycean (charophycean green algae, land plant) evolution. Ten of altogether twelve generally weakly expressed genes for isoprenoid biosynthesis, including three for the cytosolic MVA pathway, were amplified using a reverse transcription PCR approach with individually designed degenerate primers. Two full length cDNA clones for the first enzyme of the MVA pathway (HMGS) were additionally established from the charophycean green alga Chara vulgaris by library screening. The presence of the MVA pathway in these advanced green algae indicates a universal distribution among Streptophyta, and our phylogenetic HMGS analyses substantiate the recent classification of Mesostigma basal to charophytes and land plants. We identified each of the five cytosolic MVA genes/cDNAs in the genome of the rhodophyte Galdieria sulphuraria and, furthermore, amplified four of them from the glaucophyte Cyanophora paradoxa. Our data indicate that the MVA pathway is a characteristic trait of Plantae in general and propose that it was specifically lost in a common ancestor of Chlorophyta.

Identification of novel genes that co-cluster with estrogen receptor alpha in breast tumor biopsy specimens, using a large-scale real-time reverse transcription-PCR approach

The estrogen receptor alpha (ERalpha) plays a critical role in the pathogenesis and clinical behavior of breast cancer. To obtain further insights into the molecular basis of estrogen-dependent forms of this malignancy, we used real-time quantitative reverse transcription (RT)-PCR to compare the mRNA expression of 560 selected genes in ERalpha-positive and ERalpha-negative breast tumors. Fifty-one (9.1%) of the 560 genes were significantly upregulated in ERalpha-positive breast tumors compared with ERalpha-negative breast tumors. In addition to well-known ERalpha-induced genes (PGR, TFF1/PS2, BCL2, ERBB4, CCND1, etc.) and genes recently identified by cDNA microarray-based approaches (GATA3, TFF3, MYB, STC2, HPN/HEPSIN, FOXA1, XBP1, SLC39A6/LIV-1, etc.), an appreciable number of novel genes were identified, many of, which were weakly expressed. This validates the use of large-scale real-time RT-PCR as a method complementary to cDNA microarrays for molecular tumor profiling. Most of the new genes identified here encoded secreted proteins (SEMA3B and CLU), growth factors (BDNF, FGF2 and EGF), growth factor receptors (IL6ST, PTPRT, RET, VEGFR1 and FGFR2) or metabolic enzymes (CYP2B6, CA12, ACADSB, NAT1, LRBA, SLC7A2 and SULT2B1). Importantly, we also identified a large number of genes encoding proteins with either pro-apoptotic (PUMA, NOXA and TATP73) or anti-apoptotic properties (BCL2, DNTP73 and TRAILR3). Surprisingly, only a small proportion of the 51 genes identified in breast tumor biopsy specimens were confirmed to be ERalpha-regulated and/or E2-regulated in vitro (cultured cell lines). Therefore, this study identified a limited number of genes and signaling pathways, which better delineate the role of ERalpha in breast cancer. Some of the genes identified here could be useful for diagnosis or for predicting endocrine responsiveness, and could form the basis for novel therapeutic strategies.

Molecular Interactions between the Specialist Herbivore Manduca sexta (Lepidoptera, Sphingidae) and Its Natural Host Nicotiana attenuata. VII. Changes in the Plant's Proteome

When Manduca sexta attacks Nicotiana attenuata, fatty acid-amino acid conjugates (FACs) in the larvae's oral secretions (OS) are introduced into feeding wounds. These FACs trigger a transcriptional response that is similar to the response induced by insect damage. Using two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization-time of flight, and liquid chromatography-tandem mass spectrometry, we characterized the proteins in phenolic extracts and in a nuclear fraction of leaves elicited by larval attack, and/or in leaves wounded and treated with OS, FAC-free OS, and synthetic FACs. Phenolic extracts yielded approximately 600 protein spots, many of which were altered by elicitation, whereas nuclear protein fractions yielded approximately 100 spots, most of which were unchanged by elicitation. Reproducible elicitor-induced changes in 90 spots were characterized. In general, proteins that increased were involved in primary metabolism, defense, and transcriptional and translational regulation; those that decreased were involved in photosynthesis. Like the transcriptional defense responses, proteomic changes were strongly elicited by the FACs in OS. A semiquantitative reverse transcription-PCR approach based on peptide sequences was used to compare transcript and protein accumulation patterns for 17 candidate proteins. In six cases the patterns of elicited transcript accumulation were consistent with those of elicited protein accumulation. Functional analysis of one of the identified proteins involved in photosynthesis, RuBPCase activase, was accomplished by virus-induced gene silencing. Plants with decreased levels of RuBPCase activase protein had reduced photosynthetic rates and RuBPCase activity, and less biomass, responses consistent with those of herbivore-attacked plants. We conclude that the response of the plant's proteome to herbivore elicitation is complex, and integrated transcriptome-proteome-metabolome analysis is required to fully understand this ubiquitous ecological interaction.

Identification and Clinical Significance of Circulating Endothelial Progenitor Cells in Human Non–Small Cell Lung Cancer

Until recently, it was generally accepted that vascularization of tumors arises exclusively from endothelial sprouting. Whether circulating bone marrow-derived endothelial progenitor cells (EPC) participate in the progression of non-small cell lung cancer (NSCLC) has not yet been evaluated. EPCs labeled with CD34, CD133, and vascular endothelial growth factor receptor-2 (VEGFR2) antibodies were counted by flow cytometry in the peripheral blood of 53 NSCLC patients. Furthermore, by means of a quantitative reverse transcription-PCR approach, we measured VEGFR2, CD133, CD34, and VE-cadherin mRNA in the peripheral blood samples of the same patient population. EPCs in tumor samples were identified by confocal microscopy using CD31, CD34, CD133, and VEGFR2 antibodies. Although immunofluorescent labeling of microvessels made clear that incorporation of EPCs is a rare phenomenon in NSCLC tissue (9 of 22 cases), circulating EPC levels before therapeutic intervention were increased in NSCLC patients (P < 0.002, versus healthy controls), and high pretreatment circulating EPC numbers correlated with poor overall survival (P < 0.001). Furthermore, in the subgroup of responders to treatment, the posttreatment EPC numbers in the peripheral blood were significantly lower compared with nonresponding patients. Interestingly, pretreatment mRNA levels of CD133, VE-cadherin, and CD34 were not significantly increased in NSCLC patients, whereas VEGFR2 expression was increased by 80-fold. Moreover, posttreatment VEGFR2 mRNA level in the peripheral blood was significantly higher in the subgroup of nonresponding patients when compared with posttreatment level of patients responding to antitumor therapy. Circulating levels of bone marrow-derived EPCs are significantly increased in NSCLC patients and correlate with clinical behavior.

Expression of ATP-Sensitive K+ Channel Subunits during Perinatal Maturation in the Mouse Heart

Prevailing data suggest that sarcolemmal ATP-sensitive (K(ATP)) channels in the adult heart consist of Kir6.2 and SUR2A subunits, but the expression of other K(ATP) channel subunits (including SUR1, SUR2B, and Kir6.1) is poorly defined. The situation is even less clear for the immature heart, which shows a remarkable resistance to hypoxia and metabolic stress. The hypoxia-induced action potential shortening and opening of sarcolemmal K(ATP) channels that occurs in adults is less prominent in the immature heart. This might be due in part to the different biophysical and pharmacological properties of K(ATP) channels of immature and adult K(ATP) channels. Because these properties are largely conferred by subunit composition, it is important to examine the relative expression levels of the various K(ATP) channel subunits during maturation. We therefore used RNAse protection assays, reverse transcription-PCR approaches, and Western blotting to characterize the mRNA and protein expression profiles of K(ATP) channel subunits in fetal, neonatal, and adult mouse heart. Our data indicate that each of the K(ATP) channel subunits (Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B) is expressed in the mouse heart at all of the developmental time points studied. However, the expression level of each of the subunits is low in the fetal heart and progressively increases with maturation. Each of the subunits seems to be expressed in ventricular myocytes with a subcellular expression pattern matching that found in the adult. Our data suggest that the K(ATP) channel composition may change during maturation, which has important implications for K(ATP) channel function in the developing heart.

Genetic Characterization of the Bile Salt Response in Lactobacillus plantarum and Analysis of Responsive Promoters In Vitro and In Situ in the Gastrointestinal Tract

In this paper we describe the growth, morphological, and genetic responses of Lactobacillus plantarum WCFS1 to bile. Growth experiments revealed that a stepwise increase in the porcine bile concentration led to a gradual decrease in the maximal growth rate. Moreover, the final density reached by an L. plantarum culture growing in MRS containing 0.1% bile was approximately threefold lower than that in MRS lacking bile. The morphology of the cells grown in MRS containing 0.1% bile was investigated by scanning electron microscopy, which revealed that cells clumped together and had rough surfaces and that some of the cells had a shrunken and empty appearance, which clearly contrasted with the characteristic rod-shaped, smooth-surface morphology of L. plantarum cells grown in MRS without bile. An alr complementation-based genome-wide promoter screening analysis was performed with L. plantarum, which led to identification of 31 genes whose expression was potentially induced by 0.1% porcine bile. Remarkably, 11 membrane- and cell wall-associated functions appeared to be induced by bile, as were five functions involved in redox reactions and five regulatory factors. Moreover, the lp_0237 and lp_0775 genes, identified here as genes that are inducible by bile in vitro, were previously identified in our laboratory as important for L. plantarum in vivo during passage in the mouse gastrointestinal tract (P. A. Bron, C. Grangette, A. Mercenier, W. M. de Vos, and M. Kleerebezem, J. Bacteriol. 186:5721-5729, 2004). A quantitative reverse transcription-PCR approach focusing on these two genes confirmed that the expression level of lp_0237 and lp_0775 was significantly higher in cells grown in the presence of bile and cells isolated from the mouse duodenum than in cells grown on laboratory medium without bile.

Twelve Group I introns in the same pre-rRNA transcript of the myxomycete Fuligo septica: RNA processing and evolution.

The ribosomal DNA region of the myxomycete Fuligo septica was investigated and found to contain 12 group I introns (four in the small subunit and eight in the large subunit ribosomal RNAs). We have performed molecular and phylogenetic analyses to provide insight into intron structure and function, intron-host biology, and intron origin and evolution. The introns vary in size from 398 to 943 nt, all lacking detectable open reading frames. Secondary structure models revealed considerable structural diversity, but all, except one (subclass IB), represent the common group IC1 intron subclass. In vitro splicing analysis revealed that 10 of the 12 introns were able to self-splice as naked RNA, but all 12 introns were able to splice out from the precursor rRNA in vivo as evaluated by reverse transcription PCR analysis on total F. septica RNA. Furthermore, RNA processing analyses in vitro and in vivo showed that 10 of 12 introns perform hydrolytic cleavage at the 3' splice site, as well as intron circularization. Full-length intron RNA circles were detected in vivo. The order of splicing was analyzed by a reverse transcription PCR approach on cellular RNA, but no strict order of intron excision could be detected. Phylogenetic analysis indicated that most Fuligo introns were distantly related to each other and were independently gained in ribosomal DNA during evolution.

Assessing tumor angiogenesis: increased circulating VE-cadherin RNA in patients with cancer indicates viability of circulating endothelial cells.

No markers are currently available to indicate the angiogenic profile of a specific malignant disease nor to predict response to antiangiogenic therapies. Nevertheless, many different antiangiogenic drugs are presently being tested in many clinical trials, with an obvious scarcity of useful endpoints for treatment outcome beside survival. By means of a quantitative reverse transcription-PCR approach, we measured VE-cadherin (VE-C), Tie-2, vascular endothelial growth factor receptor 2 and CD133 RNA in the blood of 14 healthy controls, 3 pregnant women, and 84 newly diagnosed (or relapsed) cancer patients. Circulating VE-C RNA was increased in pregnant women and cancer patients (P = 0.0002). VE-C RNA was particularly increased in patients affected by hematological malignancies and decreased to normal values in patients achieving complete remission. Conversely, circulating RNA levels of other endothelial or progenitor cell-specific markers Tie-2, vascular endothelial growth factor receptor 2, and CD133 were not significantly increased in either pregnant women or cancer patients. Comparison of various surrogate angiogenesis markers indicated a switch toward increased plasma vascular endothelial growth factor (VEGF) levels, viable circulating endothelial cells, and circulating VE-C RNA levels in patients affected by hematological malignancies. Taken together, our data indicate that the quantitative evaluation of circulating VE-C RNA is a specific and highly promising tool with which to investigate the angiogenic phenotype of cancer patients.