Vaccination with Helicobacter pylori attachment proteins protects against gastric cancer.

Most cases of gastric cancer are caused by chronic Helicobacter pylori infection, but the lack of early onco-diagnostics and a high risk for antibiotic resistance hampers early intervention through eradication of H. pylori infection by antibiotics. We reported on a protective mechanism where H. pylori gastric mucosal attachment can be reduced by natural antibodies that block the binding of its attachment protein BabA. Here we show that challenge infection with H. pylori induced response of such blocking antibodies in both human volunteers and in rhesus macaques, that mucosal vaccination with BabA protein antigen induced blocking antibodies in rhesus macaques, and that vaccination in a mouse model induced blocking antibodies that reduced gastric mucosal inflammation, preserved the gastric juice acidity, and fully protected the mice from gastric cancer caused by H. pylori.

Shewanella septentrionalis sp. nov. and Shewanella holmiensis sp. nov., isolated from Baltic Sea water and sediments.

Two bacterial strains, SP1W3T and SP1S2-7T, were isolated from samples of water and sediments collected in Vaxholm, a town located on the Stockholm archipelago in the Baltic Sea, in November 2021. The strains were identified as novel genomic species within the genus Shewanella, based upon comparative analysis of whole genome sequence data. Strain SP1W3T (genome size, 5.20 Mbp; G+C content, 46.0 mol%), isolated from water, was determined to be most closely related to S. hafniensis ATCC-BAA 1207T and S. baltica NCTC 10735T, with digital DNA-DNA hybridization (dDDH) values of 61.7% and 60.4 %, respectively. Strain SP1S2-7T (genome size, 4.26 Mbp; G+C content, 41.5 mol%), isolated from sediments, was observed to be most closely related to S. aestuarii JCM17801T, with a pairwise dDDH value of 33.8 %. Polyphasic analyses of physiological and phenotypic characteristics, in addition to genomic analyses, confirmed that each of these two strains represent distinct, novel species within the genus Shewanella, for which the names Shewanella septentrionalis sp. nov. (type strain SP1W3T=CCUG 76164T=CECT 30651T) and Shewanella holmiensis sp. nov. (type strain SP1S2-7T=CCUG 76165T=CECT 30652T) are proposed.

Genomic differentiation within East Asian Helicobacter pylori.

The East Asian region, including China, Japan and Korea, accounts for half of gastric cancer deaths. However, different areas have contrasting gastric cancer incidences and the population structure of Helicobacter pylori in this ethnically diverse region is yet unknown. We aimed to investigate genomic differences in H. pylori between these areas to identify sequence polymorphisms associated with increased cancer risk. We analysed 381 H. pylori genomes collected from different areas of the three countries using phylogenetic and population genetic tools to characterize population differentiation. The functional consequences of SNPs with a highest fixation index (Fst) between subpopulations were examined by mapping amino acid changes on 3D protein structure, solved or modelled. Overall, 329/381 genomes belonged to the previously identified hspEAsia population indicating that import of bacteria from other regions of the world has been uncommon. Seven subregional clusters were found within hspEAsia, related to subpopulations with various ethnicities, geographies and gastric cancer risks. Subpopulation-specific amino acid changes were found in multidrug exporters (hefC), transporters (frpB-4), outer membrane proteins (hopI) and several genes involved in host interaction, such as a catalase site, involved in H2O2 entrance, and a flagellin site mimicking host glycosylation. Several of the top hits, including frpB-4, hefC, alpB/hopB and hofC, have been found to be differentiated within the Americas in previous studies, indicating that a handful of genes may be key to local geographic adaptation. H. pylori within East Asia are not homogeneous but have become differentiated geographically at multiple loci that might have facilitated adaptation to local conditions and hosts. This has important implications for further evaluation of these changes in relation to the varying gastric cancer incidence between geographical areas in this region.

FLAME: long-read bioinformatics tool for comprehensive spliceome characterization.

Comprehensive characterization of differentially spliced RNA transcripts with nanopore sequencing is limited by bioinformatics tools that are reliant on existing annotations. We have developed FLAME, a bioinformatics pipeline for alternative splicing analysis of gene-specific or transcriptome-wide long-read sequencing data. FLAME is a Python-based tool aimed at providing comprehensible quantification of full-length splice variants, reliable de novo recognition of splice sites and exons, and representation of consecutive exon connectivity in the form of a weighted adjacency matrix. Notably, this workflow circumvents issues related to inadequate reference annotations and allows for incorporation of short-read sequencing data to improve the confidence of nanopore sequencing reads. In this study, the Epstein-Barr virus long noncoding RNA RPMS1 was used to demonstrate the utility of the pipeline. RPMS1 is ubiquitously expressed in Epstein-Barr virus associated cancer and known to undergo ample differential splicing. To fully resolve the RPMS1 spliceome, we combined gene-specific nanopore sequencing reads from a primary gastric adenocarcinoma and a nasopharyngeal carcinoma cell line with matched publicly available short-read sequencing data sets. All previously reported splice variants, including putative ORFs, were detected using FLAME. In addition, 32 novel exons, including two intron retentions and a cassette exon, were discovered within the RPMS1 gene.

A 500-year tale of co-evolution, adaptation, and virulence: Helicobacter pylori in the Americas.

Helicobacter pylori is a common component of the human stomach microbiota, possibly dating back to the speciation of Homo sapiens. A history of pathogen evolution in allopatry has led to the development of genetically distinct H. pylori subpopulations, associated with different human populations, and more recent admixture among H. pylori subpopulations can provide information about human migrations. However, little is known about the degree to which some H. pylori genes are conserved in the face of admixture, potentially indicating host adaptation, or how virulence genes spread among different populations. We analyzed H. pylori genomes from 14 countries in the Americas, strains from the Iberian Peninsula, and public genomes from Europe, Africa, and Asia, to investigate how admixture varies across different regions and gene families. Whole-genome analyses of 723 H. pylori strains from around the world showed evidence of frequent admixture in the American strains with a complex mosaic of contributions from H. pylori populations originating in the Americas as well as other continents. Despite the complex admixture, distinctive genomic fingerprints were identified for each region, revealing novel American H. pylori subpopulations. A pan-genome Fst analysis showed that variation in virulence genes had the strongest fixation in America, compared with non-American populations, and that much of the variation constituted non-synonymous substitutions in functional domains. Network analyses suggest that these virulence genes have followed unique evolutionary paths in the American populations, spreading into different genetic backgrounds, potentially contributing to the high risk of gastric cancer in the region.

Acinetobacter portensis sp. nov. and Acinetobacter guerrae sp. nov., isolated from raw meat.

The taxonomic status of six strains of Acinetobacter obtained from meat samples, collected from supermarkets in Porto, Portugal, was investigated using polyphasic analysis. Partial rpoB sequence similarities lower than 95 % to other Acinetobacter species with validly published names led to the hypothesis that these strains represented novel species. This was confirmed based on comparative multilocus sequence analysis, which included the gyrB, recA and 16S rRNA genes, revealing that these strains represented two coherent lineages that were distinct from each other and from all known species. The names Acinetobacter portensis sp. nov. (comprising four strains) and Acinetobacter guerrae sp. nov. (comprising two strains) are proposed for these novel species. The species status of these two groups was confirmed by low (below 95 %) whole-genome sequence average nucleotide identity values and low (below 70 %) digital DNA-DNA hybridization similarities between the whole-genome sequences of the proposed type strains of each novel species and the representatives of the known Acinetobacter species. Phylogenomic treeing from core genome analysis supported these results. The coherence of each new species lineage was supported by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry differentiation of the species at the protein level, by cellular fatty acid profiles, and by unique and differential combinations of metabolic and physiological properties shared by each novel species. The type strain of A. portensis sp. nov. is AC 877T (=CCUG 68672T=CCM 8789T) and the type strain of A. guerrae sp. nov. is AC 1271T (=CCUG 68674T=CCM 8791T).

Isolates from Colonic Spirochetosis in Humans Show High Genomic Divergence and Potential Pathogenic Features but Are Not Detected Using Standard Primers for the Human Microbiota.

Colonic spirochetosis, diagnosed based on the striking appearance in histological sections, still has an obscure clinical relevance, and only a few bacterial isolates from this condition have been characterized to date. In a randomized, population-based study in Stockholm, Sweden, 745 healthy individuals underwent colonoscopy with biopsy sampling. Of these individuals, 17 (2.3%) had colonic spirochetosis, which was associated with eosinophilic infiltration and a 3-fold-increased risk for irritable bowel syndrome (IBS). We aimed to culture the bacteria and perform whole-genome sequencing of the isolates from this unique representative population sample. From 14 out of 17 individuals with spirochetosis we successfully isolated, cultured, and performed whole-genome sequencing of in total 17 isolates, including the Brachyspira aalborgi type strain, 513A. Also, 16S analysis of the mucosa-associated microbiota was performed in the cases and nonspirochetosis controls. We found one isolate to be of the species Brachyspira pilosicoli; all remaining isolates were of the species Brachyspira aalborgi Besides displaying extensive genetic heterogeneity, the isolates harbored several mucin-degrading enzymes and other virulence-associated genes that could confer a pathogenic potential in the human colon. We also showed that 16S amplicon sequencing using standard primers for human microbiota studies failed to detect Brachyspira due to primer incompatibility.IMPORTANCE This is the first report of whole-genome analysis of clinical isolates from individuals with colonic spirochetosis. This characterization provides new opportunities in understanding the physiology and potentials of these bacteria that densely colonize the gut in the individuals infected. The observation that standard 16S amplicon primers fail to detect colonic spirochetosis may have major implications for studies searching for associations between members of the microbiota and clinical conditions such as irritable bowel syndrome (IBS) and should be taken into consideration in project design and interpretation of gastrointestinal tract microbiota in population-based and clinical settings.

The speciation and hybridization history of the genus Salmonella.

Bacteria and archaea make up most of natural diversity, but the mechanisms that underlie the origin and maintenance of prokaryotic species are poorly understood. We investigated the speciation history of the genus Salmonella, an ecologically diverse bacterial lineage, within which S. enterica subsp. enterica is responsible for important human food-borne infections. We performed a survey of diversity across a large reference collection using multilocus sequence typing, followed by genome sequencing of distinct lineages. We identified 11 distinct phylogroups, 3 of which were previously undescribed. Strains assigned to S. enterica subsp. salamae are polyphyletic, with two distinct lineages that we designate Salamae A and B. Strains of the subspecies houtenae are subdivided into two groups, Houtenae A and B, and are both related to Selander's group VII. A phylogroup we designate VIII was previously unknown. A simple binary fission model of speciation cannot explain observed patterns of sequence diversity. In the recent past, there have been large-scale hybridization events involving an unsampled ancestral lineage and three distantly related lineages of the genus that have given rise to Houtenae A, Houtenae B and VII. We found no evidence for ongoing hybridization in the other eight lineages, but detected subtler signals of ancient recombination events. We are unable to fully resolve the speciation history of the genus, which might have involved additional speciation-by-hybridization or multi-way speciation events. Our results imply that traditional models of speciation by binary fission and divergence are not sufficient to account for Salmonella evolution.

A GWAS on Helicobacter pylori strains points to genetic variants associated with gastric cancer risk.

BACKGROUND: Helicobacter pylori are stomach-dwelling bacteria that are present in about 50% of the global population. Infection is asymptomatic in most cases, but it has been associated with gastritis, gastric ulcers and gastric cancer. Epidemiological evidence shows that progression to cancer depends upon the host and pathogen factors, but questions remain about why cancer phenotypes develop in a minority of infected people. Here, we use comparative genomics approaches to understand how genetic variation amongst bacterial strains influences disease progression. RESULTS: We performed a genome-wide association study (GWAS) on 173 H. pylori isolates from the European population (hpEurope) with known disease aetiology, including 49 from individuals with gastric cancer. We identified SNPs and genes that differed in frequency between isolates from patients with gastric cancer and those with gastritis. The gastric cancer phenotype was associated with the presence of babA and genes in the cag pathogenicity island, one of the major virulence determinants of H. pylori, as well as non-synonymous variations in several less well-studied genes. We devised a simple risk score based on the risk level of associated elements present, which has the potential to identify strains that are likely to cause cancer but will require refinement and validation. CONCLUSION: There are a number of challenges to applying GWAS to bacterial infections, including the difficulty of obtaining matched controls, multiple strain colonization and the possibility that causative strains may not be present when disease is detected. Our results demonstrate that bacterial factors have a sufficiently strong influence on disease progression that even a small-scale GWAS can identify them. Therefore, H. pylori GWAS can elucidate mechanistic pathways to disease and guide clinical treatment options, including for asymptomatic carriers.

In Vivo Analysis of the Viable Microbiota and Helicobacter pylori Transcriptome in Gastric Infection and Early Stages of Carcinogenesis.

Emerging evidence shows that the human microbiota plays a larger role in disease progression and health than previously anticipated. Helicobacter pylori, the causative agent of gastric cancer and duodenal and gastric ulcers, was early associated with gastric disease, but it has also been proposed that the accompanying microbiota in Helicobacter pylori-infected individuals might affect disease progression and gastric cancer development. In this study, the composition of the transcriptionally active microbial community and H. pylori gene expression were determined using metatranscriptomic RNA sequencing of stomach biopsy specimens from individuals with different H. pylori infection statuses and premalignant tissue changes. The results show that H. pylori completely dominates the microbiota not only in infected individuals but also in most individuals classified as H. pylori uninfected using conventional methods. Furthermore, H. pylori abundance is positively correlated with the presence of Campylobacter, Deinococcus, and Sulfurospirillum Finally, we quantified the expression of a large number of Helicobacter pylori genes and found high expression of genes involved in pH regulation and nickel transport. Our study is the first to dissect the viable microbiota of the human stomach by metatranscriptomic analysis, and it shows that metatranscriptomic analysis of the gastric microbiota is feasible and can provide new insights into how bacteria respond in vivo to variations in the stomach microenvironment and at different stages of disease progression.

Whole Genome Sequence and Phylogenetic Analysis Show Helicobacter pylori Strains from Latin America Have Followed a Unique Evolution Pathway.

Helicobacter pylori (HP) genetics may determine its clinical outcomes. Despite high prevalence of HP infection in Latin America (LA), there have been no phylogenetic studies in the region. We aimed to understand the structure of HP populations in LA mestizo individuals, where gastric cancer incidence remains high. The genome of 107 HP strains from Mexico, Nicaragua and Colombia were analyzed with 59 publicly available worldwide genomes. To study bacterial relationship on whole genome level we propose a virtual hybridization technique using thousands of high-entropy 13 bp DNA probes to generate fingerprints. Phylogenetic virtual genome fingerprint (VGF) was compared with Multi Locus Sequence Analysis (MLST) and with phylogenetic analyses of cagPAI virulence island sequences. With MLST some Nicaraguan and Mexican strains clustered close to Africa isolates, whereas European isolates were spread without clustering and intermingled with LA isolates. VGF analysis resulted in increased resolution of populations, separating European from LA strains. Furthermore, clusters with exclusively Colombian, Mexican, or Nicaraguan strains were observed, where the Colombian cluster separated from Europe, Asia, and Africa, while Nicaraguan and Mexican clades grouped close to Africa. In addition, a mixed large LA cluster including Mexican, Colombian, Nicaraguan, Peruvian, and Salvadorian strains was observed; all LA clusters separated from the Amerind clade. With cagPAI sequence analyses LA clades clearly separated from Europe, Asia and Amerind, and Colombian strains formed a single cluster. A NeighborNet analyses suggested frequent and recent recombination events particularly among LA strains. Results suggests that in the new world, H. pylori has evolved to fit mestizo LA populations, already 500 years after the Spanish colonization. This co-adaption may account for regional variability in gastric cancer risk.

Rapid evolution of distinct Helicobacter pylori subpopulations in the Americas.

For the last 500 years, the Americas have been a melting pot both for genetically diverse humans and for the pathogenic and commensal organisms associated with them. One such organism is the stomach-dwelling bacterium Helicobacter pylori, which is highly prevalent in Latin America where it is a major current public health challenge because of its strong association with gastric cancer. By analyzing the genome sequence of H. pylori isolated in North, Central and South America, we found evidence for admixture between H. pylori of European and African origin throughout the Americas, without substantial input from pre-Columbian (hspAmerind) bacteria. In the US, strains of African and European origin have remained genetically distinct, while in Colombia and Nicaragua, bottlenecks and rampant genetic exchange amongst isolates have led to the formation of national gene pools. We found three outer membrane proteins with atypical levels of Asian ancestry in American strains, as well as alleles that were nearly fixed specifically in South American isolates, suggesting a role for the ethnic makeup of hosts in the colonization of incoming strains. Our results show that new H. pylori subpopulations can rapidly arise, spread and adapt during times of demographic flux, and suggest that differences in transmission ecology between high and low prevalence areas may substantially affect the composition of bacterial populations.

Comparative Analysis of Two Helicobacter pylori Strains using Genomics and Mass Spectrometry-Based Proteomics.

Helicobacter pylori, a gastroenteric pathogen believed to have co-evolved with humans over 100,000 years, shows significant genetic variability. This motivates the study of different H. pylori strains and the diseases they cause in order to identify determinants for disease evolution. In this study, we used proteomics tools to compare two H. pylori strains. Nic25_A was isolated in Nicaragua from a patient with intestinal metaplasia, and P12 was isolated in Europe from a patient with duodenal ulcers. Differences in the abundance of surface proteins between the two strains were determined with two mass spectrometry-based methods, label-free quantification (MaxQuant) or the use of tandem mass tags (TMT). Each approach used a lipid-based protein immobilization (LPITM) technique to enrich peptides of surface proteins. Using the MaxQuant software, we found 52 proteins that differed significantly in abundance between the two strains (up- or downregulated by a factor of 1.5); with TMT, we found 18 proteins that differed in abundance between the strains. Strain P12 had a higher abundance of proteins encoded by the cag pathogenicity island, while levels of the acid response regulator ArsR and its regulatory targets (KatA, AmiE, and proteins involved in urease production) were higher in strain Nic25_A. Our results show that differences in protein abundance between H. pylori strains can be detected with proteomic approaches; this could have important implications for the study of disease progression.

Strategies to improve usability and preserve accuracy in biological sequence databases.

Biology is increasingly dependent on large-scale analysis, such as proteomics, creating a requirement for efficient bioinformatics. Bioinformatic predictions of biological functions rely upon correctly annotated database sequences, and the presence of inaccurately annotated or otherwise poorly described sequences introduces noise and bias to biological analyses. Accurate annotations are, for example, pivotal for correct identification of polypeptide fragments. However, standards for how sequence databases are organized and presented are currently insufficient. Here, we propose five strategies to address fundamental issues in the annotation of sequence databases: (i) to clearly separate experimentally verified and unverified sequence entries; (ii) to enable a system for tracing the origins of annotations; (iii) to separate entries with high-quality, informative annotation from less useful ones; (iv) to integrate automated quality-control software whenever such tools exist; and (v) to facilitate postsubmission editing of annotations and metadata associated with sequences. We believe that implementation of these strategies, for example as requirements for publication of database papers, would enable biology to better take advantage of large-scale data.

Identification of a Latin American-specific BabA adhesin variant through whole genome sequencing of Helicobacter pylori patient isolates from Nicaragua.

BACKGROUND: Helicobacter pylori (H. pylori) is one of the most common bacterial infections in humans and this infection can lead to gastric ulcers and gastric cancer. H. pylori is one of the most genetically variable human pathogens and the ability of the bacterium to bind to the host epithelium as well as the presence of different virulence factors and genetic variants within these genes have been associated with disease severity. Nicaragua has particularly high gastric cancer incidence and we therefore studied Nicaraguan clinical H. pylori isolates for factors that could contribute to cancer risk. METHODS: The complete genomes of fifty-two Nicaraguan H. pylori isolates were sequenced and assembled de novo, and phylogenetic and virulence factor analyses were performed. RESULTS: The Nicaraguan isolates showed phylogenetic relationship with West African isolates in whole-genome sequence comparisons and with Western and urban South- and Central American isolates using MLSA (Multi-locus sequence analysis). A majority, 77 % of the isolates carried the cancer-associated virulence gene cagA and also the s1/i1/m1 vacuolating cytotoxin, vacA allele combination, which is linked to increased severity of disease. Specifically, we also found that Nicaraguan isolates have a blood group-binding adhesin (BabA) variant highly similar to previously reported BabA sequences from Latin America, including from isolates belonging to other phylogenetic groups. These BabA sequences were found to be under positive selection at several amino acid positions that differed from the global collection of isolates. CONCLUSION: The discovery of a Latin American BabA variant, independent of overall phylogenetic background, suggests hitherto unknown host or environmental factors within the Latin American population giving H. pylori isolates carrying this adhesin variant a selective advantage, which could affect pathogenesis and risk for sequelae through specific adherence properties.

Transcriptome signatures in Helicobacter pylori-infected mucosa identifies acidic mammalian chitinase loss as a corpus atrophy marker.

BACKGROUND: The majority of gastric cancer cases are believed to be caused by chronic infection with the bacterium Helicobacter pylori, and atrophic corpus gastritis is a predisposing condition to gastric cancer development. We aimed to increase understanding of the molecular details of atrophy by performing a global transcriptome analysis of stomach tissue. METHODS: Biopsies from patients with different stages of H. pylori infection were taken from both the antrum and corpus mucosa and analyzed on microarrays. The stages included patients without current H. pylori infection, H. pylori-infected without corpus atrophy and patients with current or past H. pylori-infection with corpus-predominant atrophic gastritis. RESULTS: Using clustering and integrated analysis, we found firm evidence for antralization of the corpus mucosa of atrophy patients. This antralization harbored gain of gastrin expression, as well as loss of expression of corpus-related genes, such as genes associated with acid production, energy metabolism and blood clotting. The analyses provided detailed molecular evidence for simultaneous intestinal metaplasia (IM) and spasmolytic polypeptide expressing metaplasia (SPEM) in atrophic corpus tissue. Finally, acidic mammalian chitinase, a chitin-degrading enzyme produced by chief cells, was shown to be strongly down-regulated in corpus atrophy. CONCLUSIONS: Transcriptome analysis revealed several gene groups which are related to development of corpus atrophy, some of which were increased also in H. pylori-infected non-atrophic patients. Furthermore, loss of acidic chitinase expression is a promising marker for corpus atrophy.

Bicarbonate and functional CFTR channel are required for proper mucin secretion and link cystic fibrosis with its mucus phenotype.

Cystic fibrosis (CF) is caused by a nonfunctional chloride and bicarbonate ion channel (CF transmembrane regulator [CFTR]), but the link to the phenomenon of stagnant mucus is not well understood. Mice lacking functional CFTR (CftrΔ508) have no lung phenotype but show similar ileal problems to humans. We show that the ileal mucosa in CF have a mucus that adhered to the epithelium, was denser, and was less penetrable than that of wild-type mice. The properties of the ileal mucus of CF mice were normalized by secretion into a high concentration sodium bicarbonate buffer (~100 mM). In addition, bicarbonate added to already formed CF mucus almost completely restored the mucus properties. This knowledge may provide novel therapeutic options for CF.

Verification of genes differentially expressed in neuroblastoma tumours: a study of potential tumour suppressor genes.

BACKGROUND: One of the most striking features of the childhood malignancy neuroblastoma (NB) is its clinical heterogeneity. Although there is a great need for better clinical and biological markers to distinguish between tumours with different severity and to improve treatment, no clear-cut prognostic factors have been found. Also, no major NB tumour suppressor genes have been identified. METHODS: In this study we performed expression analysis by quantitative real-time PCR (QPCR) on primary NB tumours divided into two groups, of favourable and unfavourable outcome respectively. Candidate genes were selected on basis of lower expression in unfavourable tumour types compared to favourables in our microarray expression analysis. Selected genes were studied in two steps: (1) using TaqMan Low Density Arrays (TLDA) targeting 89 genes on a set of 12 NB tumour samples, and (2) 12 genes were selected from the TLDA analysis for verification using individual TaqMan assays in a new set of 13 NB tumour samples. RESULTS: By TLDA analysis, 81 out of 87 genes were found to be significantly differentially expressed between groups, of which 14 have previously been reported as having an altered gene expression in NB. In the second verification round, seven out of 12 transcripts showed significantly lower expression in unfavourable NB tumours, ATBF1, CACNA2D3, CNTNAP2, FUSIP1, GNB1, SLC35E2, and TFAP2B. The gene that showed the highest fold change in the TLDA analysis, POU4F2, was investigated for epigenetic changes (CpG methylation) and mutations in order to explore the cause of the differential expression. Moreover, the fragile site gene CNTNAP2 that showed the largest fold change in verification group 2 was investigated for structural aberrations by copy number analysis. However, the analyses of POU4F2 and CNTNAP2 showed no genetic alterations that could explain a lower expression in unfavourable NB tumours. CONCLUSION: Through two steps of verification, seven transcripts were found to significantly discriminate between favourable and unfavourable NB tumours. Four of the transcripts, CACNA2D3, GNB1, SLC35E2, and TFAP2B, have been observed in previous microarray studies, and are in this study independently verified. Our results suggest these transcripts to be markers of malignancy, which could have a potential usefulness in the clinic.

SPI-C and STAT6 can cooperate to stimulate IgE germline transcription.

SPI-C is a novel ETS protein that is expressed in B lymphocytes. No target gene for SPI-C has so far been defined. We have performed a yeast two-hybrid screen using SPI-C as bait in order to further analyze the functional role of this orphan transcription factor. We found that SPI-C interacted specifically with the C-terminus of STAT6 in yeast. By co-immunoprecipitation in transfected COS7 cells the physical interaction between SPI-C and STAT6 was confirmed. Furthermore, this protein-protein interaction is functional since we could demonstrate that SPI-C and STAT6 stimulated IL4 induced Iepsilon transcription synergistically but only when both proteins bound to DNA. Thus, a protein interaction between SPI-C and STAT6 is the basis for a novel mechanism for regulation of IL4 induced gene expression.