Bigh3 is upregulated in regenerating zebrafish fin.

Zebrafish is a good model for studying regeneration because of the rapidity with which it occurs. Better understanding of this process may lead in the future to improvement of the regenerating capacity of humans. Signaling factors are the second largest category of genes, regulated during regeneration after the regulators of wound healing. Major developmental signaling pathways play a role in this multistep process, such as Bmp, Fgf, Notch, retinoic acid, Shh, and Wnt. In the present study, we focus on TGF-β-induced genes, bigh3 and bambia. Bigh3 encodes keratoepithelin, a protein first identified as an extracellular matrix protein reported to play a role in cell adhesion, as well as in cornea formation and osteogenesis. The expression of bigh3 in zebrafish fins has previously been reported. Here we demonstrate that tgf-b1 and tgf-b3 mRNA reacted with delay, first showing no regulation at 3âeuro0/00dpa, followed by upregulation at 4 and 5âeuro0/00dpa. Tgf-b1, tgf-2, and tgf-brII mRNA were back to normal levels at 10âeuro0/00dpa. Only tgf-b3 mRNA was still upregulated at that time. Bigh3 mRNA followed the upregulation of tgf-b1, while bambia mRNA behaved similarly to tgf-b2 mRNA. We show that upregulation of bigh3 and bambia mRNA correlated with the process of fin regeneration and regulation of TGF-b signaling, suggesting a new role for these proteins.

Transmission disequilibrium of small CNVs in simplex autism.

We searched for disruptive, genic rare copy-number variants (CNVs) among 411 families affected by sporadic autism spectrum disorder (ASD) from the Simons Simplex Collection by using available exome sequence data and CoNIFER (Copy Number Inference from Exome Reads). Compared to high-density SNP microarrays, our approach yielded ∼2× more smaller genic rare CNVs. We found that affected probands inherited more CNVs than did their siblings (453 versus 394, p = 0.004; odds ratio [OR] = 1.19) and that the probands' CNVs affected more genes (921 versus 726, p = 0.02; OR = 1.30). These smaller CNVs (median size 18 kb) were transmitted preferentially from the mother (136 maternal versus 100 paternal, p = 0.02), although this bias occurred irrespective of affected status. The excess burden of inherited CNVs among probands was driven primarily by sibling pairs with discordant social-behavior phenotypes (p < 0.0002, measured by Social Responsiveness Scale [SRS] score), which contrasts with families where the phenotypes were more closely matched or less extreme (p > 0.5). Finally, we found enrichment of brain-expressed genes unique to probands, especially in the SRS-discordant group (p = 0.0035). In a combined model, our inherited CNVs, de novo CNVs, and de novo single-nucleotide variants all independently contributed to the risk of autism (p < 0.05). Taken together, these results suggest that small transmitted rare CNVs play a role in the etiology of simplex autism. Importantly, the small size of these variants aids in the identification of specific genes as additional risk factors associated with ASD.

Lipoxin A4 is a novel estrogen receptor modulator.

Inflammation is intimately linked with naturally occurring remodeling events in the endometrium. Lipoxins comprise a group of short-lived, nonclassic eicosanoids possessing potent anti-inflammatory and proresolution properties. In the present study, we investigated the role of lipoxin A(4) (LXA(4)) in the endometrium and demonstrated that 15-LOX-2, an enzyme necessary for LX biosynthesis, is expressed in this tissue. Our results establish that LXA(4) possesses robust estrogenic activity through its capacity to alter ERE transcriptional activity, as well as expression of estrogen-regulated genes, alkaline phosphatase activity, and proliferation in human endometrial epithelial cells. Interestingly, LXA(4) also demonstrated antiestrogenic potential, significantly attenuating E2-induced activity. This estrogenic activity was directly mediated through estrogen receptors (ERs). Subsequent investigations determined that the actions of LXA(4) are exclusively mediated through ERα and closely mimic those of the potent estrogen 17β-estradiol (E2). In binding assays, LXA(4) competed with E2 for ER binding, with an IC(50) of 46 nM. Furthermore, LXA(4) exhibited estrogenic activity in vivo, increasing uterine wet weight and modulating E2-regulated gene expression. These findings reveal a previously unappreciated facet of LXA(4) bioactions, implicating this lipid mediator in novel immunoendocrine crosstalk mechanisms.

Thirty new loci for age at menarche identified by a meta-analysis of genome-wide association studies.

To identify loci for age at menarche, we performed a meta-analysis of 32 genome-wide association studies in 87,802 women of European descent, with replication in up to 14,731 women. In addition to the known loci at LIN28B (P = 5.4 × 10⁻⁶⁰) and 9q31.2 (P = 2.2 × 10⁻³³), we identified 30 new menarche loci (all P < 5 × 10⁻⁸) and found suggestive evidence for a further 10 loci (P < 1.9 × 10⁻⁶). The new loci included four previously associated with body mass index (in or near FTO, SEC16B, TRA2B and TMEM18), three in or near other genes implicated in energy homeostasis (BSX, CRTC1 and MCHR2) and three in or near genes implicated in hormonal regulation (INHBA, PCSK2 and RXRG). Ingenuity and gene-set enrichment pathway analyses identified coenzyme A and fatty acid biosynthesis as biological processes related to menarche timing.

Treatment of localised resectable neuroblastoma. Results of the LNESG1 study by the SIOP Europe Neuroblastoma Group.

Main objective of this study was to confirm that surgery alone is an effective and safe treatment for localised resectable neuroblastoma except stage 2 with amplified MYCN gene (MYCNA). Of 427 eligible stages 1-2 patients, 411 had normal MYCN and 16 had MYCNA. Of the 288 stage 1 patients with normal MYCN, 1 died of complications and 16 relapsed, 2 of whom died; 5-year relapse-free survival (RFS) and overall survival (OS) rates were 94.3% (95% confidence interval (CI): 91.6-97) and 98.9% (95% CI: 97.7-100), respectively. Of the 123 stage 2 patients with normal MYCN, 1 died of sepsis and 22 relapsed, 8 of whom died (RFS 82.8%, 95% CI: 76.2-89.5; OS 93.2%, 95% CI: 88.7-97.8). In stage 2, OS and RFS were worse for patients with elevated LDH and unfavourable histopathology. Of 16 children with MYCNA, 7 were stage 1 (5 relapses and 4 deaths) and 9 were stage 2 (3 relapses and 2 deaths) patients. In conclusion, surgery alone yielded excellent OS for both stage 1 and 2 neuroblastoma without MYCNA, although stage 2 patients with unfavourable histopathology and elevated LDH suffered a high number of relapses. Both stage 1 and 2 patients with MYCNA were at greater risk of relapse.

Bioinformática: identificar genes en una interfaz gráfica vía web para la comparación de genomas

Este trabajo desarrolla el proceso de diseño e implementación de una interfaz web que permite la exploración en detalle de las relaciones entre genomas completos. La interfaz permite la comparación simultánea de nueve genomas, representando en cada gráfica las relaciones entre cada par de genomas junto los genes identificados de cada uno de ellos. Es capaz de trabajar con genomas del dominio Eukaryota y se adapta a la capacidad de cómputo de la máquina cliente. La información representada son MUMs (Maximal Unique Matching, secuencia máxima y única encontrada en ambos genomas) y SuperMUMs (agrupación de MUMs mediante Approximate String Matching). Los datos son previamente calculados y accesibles desde un servidor web.

Conjugative Transfer of Chromosomal Genes between Fluorescent Pseudomonads in the Rhizosphere of Wheat.

Bacteria released in large numbers for biocontrol or bioremediation purposes might exchange genes with other microorganisms. Two model systems were designed to investigate the likelihood of such an exchange and some factors which govern the conjugative exchange of chromosomal genes between root-colonizing pseudomonads in the rhizosphere of wheat. The first model consisted of the biocontrol strain CHA0 of Pseudomonas fluorescens and transposon-facilitated recombination (Tfr). A conjugative IncP plasmid loaded with transposon Tn5, in a CHA0 derivative carrying a chromosomal Tn5 insertion, promoted chromosome transfer to auxotrophic CHA0 recipients in vitro. A chromosomal marker (pro) was transferred at a frequency of about 10(sup-6) per donor on wheat roots under gnotobiotic conditions, provided that the Tfr donor and recipient populations each contained 10(sup6) to 10(sup7) CFU per g of root. In contrast, no conjugative gene transfer was detected in soil, illustrating that the root surface stimulates conjugation. The second model system was based on the genetically well-characterized strain PAO of Pseudomonas aeruginosa and the chromosome mobilizing IncP plasmid R68.45. Although originally isolated from a human wound, strain PAO1 was found to be an excellent root colonizer, even under natural, nonsterile conditions. Matings between an auxotrophic R68.45 donor and auxotrophic recipients produced prototrophic chromosomal recombinants at 10(sup-4) to 10(sup-5) per donor on wheat roots in artificial soil under gnotobiotic conditions and at about 10(sup-6) per donor on wheat roots in natural, nonsterile soil microcosms after 2 weeks of incubation. The frequencies of chromosomal recombinants were as high as or higher than the frequencies of R68.45 transconjugants, reflecting mainly the selective growth advantage of the prototrophic recombinants over the auxotrophic parental strains in the rhizosphere. Although under field conditions the formation of chromosomal recombinants is expected to be reduced by several factors, we conclude that chromosomal genes, whether present naturally or introduced by genetic modification, may be transmissible between rhizosphere bacteria.

Genome-wide association and genetic functional studies identify autism susceptibility candidate 2 gene (AUTS2) in the regulation of alcohol consumption.

Alcohol consumption is a moderately heritable trait, but the genetic basis in humans is largely unknown, despite its clinical and societal importance. We report a genome-wide association study meta-analysis of ∼2.5 million directly genotyped or imputed SNPs with alcohol consumption (gram per day per kilogram body weight) among 12 population-based samples of European ancestry, comprising 26,316 individuals, with replication genotyping in an additional 21,185 individuals. SNP rs6943555 in autism susceptibility candidate 2 gene (AUTS2) was associated with alcohol consumption at genome-wide significance (P = 4 × 10(-8) to P = 4 × 10(-9)). We found a genotype-specific expression of AUTS2 in 96 human prefrontal cortex samples (P = 0.026) and significant (P < 0.017) differences in expression of AUTS2 in whole-brain extracts of mice selected for differences in voluntary alcohol consumption. Down-regulation of an AUTS2 homolog caused reduced alcohol sensitivity in Drosophila (P < 0.001). Our finding of a regulator of alcohol consumption adds knowledge to our understanding of genetic mechanisms influencing alcohol drinking behavior.

Structural prediction of peptides bound to MHC class I.

An ab initio structure prediction approach adapted to the peptide-major histocompatibility complex (MHC) class I system is presented. Based on structure comparisons of a large set of peptide-MHC class I complexes, a molecular dynamics protocol is proposed using simulated annealing (SA) cycles to sample the conformational space of the peptide in its fixed MHC environment. A set of 14 peptide-human leukocyte antigen (HLA) A0201 and 27 peptide-non-HLA A0201 complexes for which X-ray structures are available is used to test the accuracy of the prediction method. For each complex, 1000 peptide conformers are obtained from the SA sampling. A graph theory clustering algorithm based on heavy atom root-mean-square deviation (RMSD) values is applied to the sampled conformers. The clusters are ranked using cluster size, mean effective or conformational free energies, with solvation free energies computed using Generalized Born MV 2 (GB-MV2) and Poisson-Boltzmann (PB) continuum models. The final conformation is chosen as the center of the best-ranked cluster. With conformational free energies, the overall prediction success is 83% using a 1.00 Angstroms crystal RMSD criterion for main-chain atoms, and 76% using a 1.50 Angstroms RMSD criterion for heavy atoms. The prediction success is even higher for the set of 14 peptide-HLA A0201 complexes: 100% of the peptides have main-chain RMSD values &lt; or =1.00 Angstroms and 93% of the peptides have heavy atom RMSD values &lt; or =1.50 Angstroms. This structure prediction method can be applied to complexes of natural or modified antigenic peptides in their MHC environment with the aim to perform rational structure-based optimizations of tumor vaccines.

Dihydroaeruginoic acid synthetase and pyochelin synthetase, products of the pchEF genes, are induced by extracellular pyochelin in Pseudomonas aeruginosa.

The siderophore pyochelin of Pseudomonas aeruginosa is derived from one molecule of salicylate and two molecules of cysteine. Two cotranscribed genes, pchEF, encoding peptide synthetases have been identified and characterized. pchE was required for the conversion of salicylate to dihydroaeruginoate (Dha), the condensation product of salicylate and one cysteine residue and pchF was essential for the synthesis of pyochelin from Dha. The deduced PchE (156 kDa) and PchF (197 kDa) proteins had adenylation, thiolation and condensation/cyclization motifs arranged as modules which are typical of those peptide synthetases forming thiazoline rings. The pchEF genes were coregulated with the pchDCBA operon, which provides enzymes for the synthesis (PchBA) and activation (PchD) of salicylate as well as a putative thioesterase (PchC). Expression of a translational pchE'-'lacZ fusion was strictly dependent on the PchR regulator and was induced by extracellular pyochelin, the end product of the pathway. Iron replete conditions led to Fur (ferric uptake regulator)-dependent repression of the pchE'-'lacZ fusion. A translational pchD'-'lacZ fusion was also positively regulated by PchR and pyochelin and repressed by Fur and iron. Thus, autoinduction by pyochelin (or ferric pyochelin) and repression by iron ensure a sensitive control of the pyochelin pathway in P. aeruginosa.

An activation-independent role of transcription factors in insulator function.

Chromatin insulators are defined as transcriptionally neutral elements that prevent negative or positive influence from extending across chromatin to a promoter. Here we show that yeast subtelomeric anti-silencing regions behave as boundaries to telomere-driven silencing and also allow discontinuous propagation of silent chromatin. These two facets of insulator activity, boundary and silencing discontinuity, can be recapitulated by tethering various transcription activation domains to tandem sites on DNA. Importantly, we show that these insulator activities do not involve direct transcriptional activation of the reporter promoter. These findings predict that certain promoters behave as insulators and partition genomes in functionally independent domains.

Preferential binding of the methyl-CpG binding domain protein 2 at methylated transcriptional start site regions.

Methyl-CpG Binding Domain (MBD) proteins are thought to be key molecules in the interpretation of DNA methylation signals leading to gene silencing through recruitment of chromatin remodeling complexes. In cancer, the MBD-family member, MBD2, may be primarily involved in the repression of genes exhibiting methylated CpG at their 5' end. Here we ask whether MBD2 randomly associates methylated sequences, producing chance effects on transcription, or exhibits a more specific recognition of some methylated regions. Using chromatin and DNA immunoprecipitation, we analyzed MBD2 and RNA polymerase II deposition and DNA methylation in HeLa cells on arrays representing 25,500 promoter regions. This first whole-genome mapping revealed the preferential localization of MBD2 near transcription start sites (TSSs), within the region analyzed, 7.5 kb upstream through 2.45 kb downstream of 5' transcription start sites. Probe by probe analysis correlated MBD2 deposition and DNA methylation. Motif analysis did not reveal specific sequence motifs; however, CCG and CGC sequences seem to be overrepresented. Nonrandom association (multiple correspondence analysis, p < 0.0001) between silent genes, DNA methylation and MBD2 binding was observed. The association between MBD2 binding and transcriptional repression weakened as the distance between binding site and TSS increased, suggesting that MBD2 represses transcriptional initiation. This hypothesis may represent a functional explanation for the preferential binding of MBD2 at methyl-CpG in TSS regions.

Bioinformática: interfaz web para estudiar el efecto de diferentes condiciones sobre la expresión de los genes

El trabajo realizado se divide en dos bloques bien diferenciados, ambos relacionados con el análisis de microarrays. El primer bloque consiste en agrupar las condiciones muestrales de todos los genes en grupos o clústers. Estas agrupaciones se obtienen al aplicar directamente sobre la microarray los siguientes algoritmos de agrupación: SOM,PAM,SOTA,HC y al aplicar sobre la microarray escalada con PC y MDS los siguientes algoritmos: SOM,PAM,SOTA,HC y K-MEANS. El segundo bloque consiste en realizar una búsqueda de genes basada en los intervalos de confianza de cada clúster de la agrupación activa. Las condiciones de búsqueda ajustadas por el usuario se validan para cada clúster respecto el valor basal 0 y respecto el resto de clústers, para estas validaciones se usan los intervalos de confianza. Estos dos bloques se integran en una aplicación web ya existente, el applet PCOPGene, alojada en el servidor: http://revolutionresearch.uab.es.

Orosomucoid system: 17 additional orosomucoid variants and proposal for a new nomenclature.

There are two forms of orosomucoid (ORM) in the sera of most individuals. They are encoded by two separate but closely linked loci, ORM1 and ORM2. A number of variants have been identified in various populations. Duplication and nonexpression are also observed in some populations. Thus, the ORM system is very complicated and its nomenclature is very confusing. In order to propose a new nomenclature, ORM variants detected by several laboratories have been compared and characterized by isoelectric focusing (IEF) followed by immunoprinting. A total of 57 different alleles including 17 new ones were identified. The 27 alleles were assigned to the ORM1 locus, and the others to the ORM2 locus. The designations ORM*F1, ORM1*F2, ORM1*S and ORM2*M were adopted for the four common alleles instead of ORM1*1, ORM1*3, ORM1*2 and ORM2*1 (ORM2*A), respectively. The variants were designated alpha numerically according to their relative mobilities after IEF in a pH gradient of 4.5-5.4 with Triton X-100 and glycerol. For the duplicated genes a prefix is added to a combined name of two alleles, e.g. ORM1*dB9S. Silent alleles were named ORM1*Q0 and ORM2*Q0 conventionally. In addition, the effects of diseases to ORM band patterns after IEF are also discussed.

Molecular genetics of "PRPF31" dominant mutations linked to retinitis pigmentosa

ABSTRACT : The retina is one of the most important human sensory tissues since it detects and transmits all visual information from the outside world to the brain. Retinitis pigmentosa (RP) is the name given to a group of inherited diseases that affect specifically the photoreceptors present in the retina and in many instances lead to blindness. Dominant mutations in PRPF31, a gene that encodes for a pre-mRNA splicing factor, cause retinitis pigmentosa with reduced penetrance. We functionally investigated a novel mutation, identified in a large family with autosomal dominant RP, and 7 other mutations, substitutions and microdeletions, in 12 patients from 7 families with PRPF31-linked RP. Seven mutations lead to PRPF31 mRNA with premature stop codons and one to mRNA lacking the exon containing the initiation codon. Quantification of PRPF31 mRNA and protein levels revealed a significant reduction in cell lines derived from patients, compared to non carriers of mutations in PRPF31. Allelic quantification of PRPF31 mRNA indicated that the level of mutated mRNA is very low compared to wild-type mRNA. No mutant protein was detected and the subnuclear localization of wild-type PRPF31 remains the same in cell lines from patients and controls. Blocking nonsense-mediated mRNA decay in cell lines derived from patients partially restored PRPF31 mutated mRNA but derived proteins were still undetectable, even when protein degradation pathways were inhibited. Our results demonstrated that the vast majority of PRPF31 mutations result in null alleles, since they are subject to surveillance mechanisms that degrade mutated mRNA and possibly block its translation. Altogether, these data indicate that the likely cause of PRPF31-linked RP is haploinsufficiency, rather than a dominant negative effect. Penetrance of PRPF31 mutations has been previously demonstrated to be inversely correlated with the level of PRPF31 mRNA, since high expression of wild-type PRPF31 mRNA protects from the disease. Consequently, we have investigated the genetic modifiers that control the expression of PRPF31 by quantifying PRPF31 mRNA levels in cell lines derived from 200 individuals from 15 families representative of the general population. By linkage analyses we identified a 8.2Mb-region on chromosome 14q21-23 that contains a gene involved in the modulation of PRPF31 expression. We also assessed apreviously-mapped penetrance factor invariably located on the wild-type allele and linked to the PRPF31 locus in asymptomatic patients from different families with RP. We demonstrated that this modifier increases the expression of both PRPF31 alleles already at the pre-mRNA level. Finally, our data suggest that PRPF31 mRNA expression and consequently the penetrance of PRPF31 mutations is modulated by at least 2 diffusible compounds, which act on both PRPF31 alleles during their transcription.