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Retinol saturase (RetSat) is an endoplasmic reticulum-localized oxidoreductase highly expressed in organs involved in lipid metabolism such as white (WAT) and brown adipose tissue (BAT). Cold exposure was shown to increase RETSAT protein in BAT but its relevance for non-shivering thermogenesis, a process with beneficial effects on metabolic health, is unknown. We analyzed the regulation of RetSat expression in white and brown adipocytes and different murine adipose tissue depots upon β-adrenergic stimulation and cold exposure. RetSat function during the differentiation and β-adrenergic stimulation of brown adipocytes was dissected by loss-of-function experiments. Mice with BAT-specific deletion of RetSat were generated and exposed to cold. Gene expression in human WAT was analyzed and the effect of RetSat depletion on adipocyte lipolysis investigated. We show that cold exposure induces RetSat expression in both WAT and BAT of mice via β-adrenergic signaling. In brown adipocytes, RetSat has minor effects on differentiation but is required for maximal thermogenic gene and protein expression upon β-adrenergic stimulation and mitochondrial respiration. In mice, BAT-specific deletion of RetSat impaired acute but not long-term adaptation to cold exposure. RetSat expression in subcutaneous WAT of humans correlates with the expression of genes related to mitochondrial function. Mechanistically, we found that RetSat depletion impaired β-agonist-induced lipolysis, a major regulator of thermogenic gene expression in adipocytes. Thus, RetSat expression is under β-adrenergic control and determines thermogenic capacity of brown adipocytes and acute cold tolerance in mice. Modulating RetSat activity may allow for therapeutic interventions towards pathologies with inadequate metabolic activity. • Adipose RetSat expression is induced by β-adrenergic signaling and cold exposure. • Loss of RetSat in brown adipocytes reduces thermogenic gene expression. • Mice lacking RetSat in BAT have impaired acute cold tolerance. • RetSat depletion in brown and white adipocytes interferes with lipolysis. [ABSTRACT FROM AUTHOR]

Abstract Hypoxia-inducible factors (HIFs) play a key role in the adaptation to low oxygen by interacting with hypoxia response elements (HREs) in the genome. Cellular levels of the HIF-2α transcription factor subunit influence the histopathology and clinical outcome of neuroblastoma, a malignant childhood tumor of the sympathetic ganglia. Expression of the Wilms tumor gene, WT1 , marks a group of high-risk neuroblastoma. Here, we identify WT1 as a downstream target of HIF-2α in Kelly neuroblastoma cells. In chromatin immunoprecipitation assays, HIF-2α bound to a HRE in intron 3 of the WT1 gene, but not to another predicted HIF binding site (HBS) in the first intron. The identified element conferred oxygen sensitivity to otherwise hypoxia-resistant WT1 and SV40 promoter constructs. Deletion of the HBS in the intronic HRE by genome editing abolished WT1 expression in hypoxic neuroblastoma cells. Physical interaction between the HRE and the WT1 promoter in normoxic and hypoxic Kelly cells was shown by chromosome conformation capture assays. These findings demonstrate that binding of HIF-2α to an oxygen-sensitive enhancer in intron 3 stimulates transcription of the WT1 gene in neuroblastoma cells by hypoxia-independent chromatin looping. This novel regulatory mechanism may have implications for the biology and prognosis of neuroblastoma. Highlights • We identify the first genomic element that is required for WT1 expression, specifically in hypoxia. • We describe a novel mechanism of WT1 gene regulation by hypoxia-independent chromatin looping. • This regulatory mechanism may have implications for the biology and progression neuroblastoma. [ABSTRACT FROM AUTHOR]

Francisella tularensis is the causative agent of tularemia, a zoonotic disease with a wide host range. F. tularensis ssp. holarctica (Fth) is of clinical relevance for European countries, including Germany. Whole genome sequencing methods, including canonical Single Nucleotide Polymorphism (canSNP) typing and whole genome SNP typing, have revealed that European Fth strains belong to a few monophyletic populations. The majority of German Fth isolates belong to two basal phylogenetic clades B.6 (biovar I) and B.12 (biovar II). Strains of B.6 and B.12 seem to differ in their pathogenicity, and it has been shown that strains of biovar II are resistant against erythromycin. In this study, we present data corroborating our previous data demonstrating that basal clade B.12 can be divided into clades B.71 and B.72. By applying phylogenetic whole genome analysis as well as proteome analysis, we could verify that strains of these two clades are distinct from one another. This was confirmed by measuring the intensity of backscatter light on bacteria grown in liquid media. Strains belonging to clades B.6, B.71 or B.72 showed clade-specific backscatter growth curves. Furthermore, we present the whole genome sequence of strain A-1341, as a reference genome of clade B.71, and whole proteomes comparison of Fth strains belonging to clades B.6, B.71 and B.72. Further research is necessary to investigate phenotypes and putative differences in pathogenicity of the investigated different clades of Fth to better understand the relationship between observed phenotypes, pathogenicity and distribution of Fth strains. [ABSTRACT FROM AUTHOR]

Information about the prevalence and geographical distribution of tick-borne pathogens Anaplasma phagocytophilum , Candidatus Neoehrlichia mikurensis, and Babesia spp. is still rare in Mongolia. We tested 275 Ixodes persulcatus ticks for A. phagocytophilum , Cand . N. mikurensis and Babesia spp. and 125 Dermacentor nuttalli ticks especially for Babesia spp. using different PCR methods. Ticks were collected from three provinces (Selenge, Arkhangai, Khentii) in Mongolia. DNA of A. phagocytophilum , Cand . N. mikurensis and Babesia spp. were found with a prevalence of 6.2%, 1.5% and 3.3% in each case in I. persulcatus ticks. This is the first time Cand . N. mikurensis was found in ticks from Mongolia. Sequence analysis of Babesia spp.-positive amplicons showed exclusively B. venatorum , which had also not been mentioned in Mongolia before. On the contrary, all D. nuttalli ticks tested negatively for Babesia spp. This study demonstrates that all three zoonotic pathogens are present in I. persulcatus ticks in Mongolia, and justify the need for further investigations of a more detailed genetic characterization of these pathogens. [ABSTRACT FROM AUTHOR]

Abstract: The genus Yersinia contains three species pathogenic for humans, one of which is the enteropathogen Yersinia pseudotuberculosis. A recent analysis by Multi Locus Sequence Typing (MLST) of the ‘Y. pseudotuberculosis complex’ revealed that this complex comprises three distinct populations: the Y. pestis/Y. pseudotuberculosis group, the recently described species Yersinia similis, and a third not yet characterized population designated ‘Korean Group’, because most strains were isolated in Korea. The aim of this study was to perform an in depth phenotypic and genetic characterization of the three populations composing the Y. pseudotuberculosis complex (excluding Y. pestis, which belonged to the Y. pseudotuberculosis cluster in the MLST analysis). Using a set of strains representative of each group, we found that the three populations had close metabolic properties, but were nonetheless distinguishable based on D-raffinose and D-melibiose fermentation, and on pyrazinamidase activity. Moreover, high-resolution electrospray mass spectrometry highlighted protein peaks characteristic of each population. Their 16S rRNA gene sequences shared high identity (≥99.5%), but specific nucleotide signatures for each group were identified. Multi-Locus Sequence Analysis also identified three genetically closely related but distinct populations. Finally, an Average Nucleotide Identity (ANI) analysis performed after sequencing the genomes of a subset of strains of each group also showed that intragroup identity (average for each group ≥99%) was higher than intergroup diversity (94.6–97.4%). Therefore, all phenotypic and genotypic traits studied concurred with the initial MLST data indicating that the Y. pseudotuberculosis complex comprises a third and clearly distinct population of strains forming a novel Yersinia species that we propose to designate Yersinia wautersii sp. nov. The isolation of some strains from humans, the detection of virulence genes (on the pYV and pVM82 plasmids, or encoding the superantigen ypmA) in some isolates, and the absence of pyrazinamidase activity (a hallmark of pathogenicity in the genus Yersinia) argue for the pathogenic potential of Y. wautersii. [Copyright &y& Elsevier]

Abstract: Clostridium botulinum is the etiological agent of botulism. Due to food-borne poisoning and the potential use of the extremely toxic botulinum neurotoxin (BoNT) from C. botulinum in bioterror or biocrime related actions, reliable high resolution typing methods for discriminating C. botulinum strains are needed. Partial sequencing of the adk, atpH, gyrB, proC, rpoD and spo0A genes from 51 various C. botulinum/sporogenes isolates was performed, resulting in 37 different sequence types (STs). Analysis of the sequence data revealed a genetic distribution in five larger clusters with a loose correlation to the BoNT serotypes. The developed MLST assay had a slightly lower resolution ability when compared to the MLVA (multilocus variable number of tandem repeat analysis), but the two methods resulted in similar subclusters of the strains possessing the BoNT serotypes A, B and F. The current work presents the development of a novel MLST assay useful for genotyping C. botulinum related to basic phylogenetic research and trace-back analysis in microbial forensic studies. [Copyright &y& Elsevier]

Abstract: In Mongolia, Lyme borreliosis was first reported in 2003. To determine which Borrelia species may contribute to the occurrence of Lyme borreliosis in Mongolia, real-time PCR was conducted on 372 adult Ixodes persulcatus ticks collected in Selenge Aimag, the province with the highest incidence of human Lyme borreliosis. 24.5% of ticks were identified to be positive for Borrelia burgdorferi sensu lato DNA. Species differentiation using an SNP-based real-time PCR and multi-locus sequence analysis revealed that strains phylogenetically closely related to B. bavariensis (previously known as B. garinii OspA serotype 4) is the most prevalent species, showing an unexpectedly high genetic diversity. [Copyright &y& Elsevier]

Abstract: Since the year 2005, clinical patterns resembling tick-borne rickettsioses have been noticed in Mongolia. Epidemiological data regarding species of the aetiological agent, tick vector, prevalence, and distribution as well as incidence of human cases throughout Mongolia are still sparse to date. In order to identify Rickettsia species occurring in Mongolia, we investigated Dermacentor nuttalli (n =179) and Ixodes persulcatus (n =374) collected in 4 selected provinces. Rickettsia raoultii was the predominant Rickettsia (82% prevalence) found in D. nuttalli and was also detected in I. persulcatus (0.8%). The Rickettsia prevalence in D. nuttalli from different provinces varied between 70% and 97%. In addition, R. sibirica was identified in approximately 4% of D. nuttalli, but solely from Arkhanghai province. The results of this study extend the common knowledge about the geographic distribution of R. raoultii and its high prevalence in D. nuttalli. Although the pathogenicity of this Rickettsia is still unclear, it should be considered in Mongolian patients suspected of having tick-borne rickettsiosis. [Copyright &y& Elsevier]

Abstract: In the course of this study, examinations were carried out to develop a PCR-based test which allows discrimination of Brucella species and biovars not targeted by the currently established gel-based PCR assays. Appropriate primers were designed based on specific deletions and insertions in the different Brucella genomes as determined by RAPD-PCR and whole-genome comparisons. After testing the specificity of the primers with a set of 22 Brucella reference strains of all species and biovars, they were used to supplement the existing PCR assays resulting in a 19-primer multiplex PCR. In addition to the commonly used PCR assays, the developed assay specifically identified B. neotomae, B. pinnipedialis, B. ceti, and B. microti. Furthermore, it differentiated B. abortus biovars 1, 2, 4 from biovars 3, 5, 6, 9, as well as between B. suis biovar 1, biovars 3, 4, and biovars 2 and 5. When tested in the multiplex assay, all Brucella type and reference strains and the majority of 118 field strains examined could be accurately identified by their respective banding patterns according to their previous typing. B. canis strains were subdivided into 2 groups, one exhibiting a unique pattern and the other one a banding pattern shared with B. suis biovars 3 and 4. Species of the closely related genus Ochrobactrum and several other clinically relevant bacteria showed no amplification product. Hence, the developed PCR assay is useful for rapid identification of Brucella at the species and at the biovar level. [Copyright &y& Elsevier]

Abstract: Ochrobactrum (O.) anthropi is an opportunistic emerging pathogen closely related to the genus Brucella. Identification and differentiation from brucellae and other Ochrobactrum spp. using routine biochemical test systems is not reliable due to the high phenotypic similarity. In this study, antibiotic susceptibilities of 103 Ochrobactrum isolates were determined using Etest™ for 19 clinically relevant antimicrobial agents. Ochrobactrum strains were highly resistant to β-lactam antibiotics, susceptible to ciprofloxacin, and 97.1% were susceptible to trimethoprim/sulfamethoxazole. It was also demonstrated that biochemical reaction profiles of the API® and BD Phoenix™ 100 systems for identifying Ochrobactrum isolates can only be used on the genus level. Our in vitro data suggest that combinations of antimicrobial agents including ciprofloxacin and/or trimethoprim/sulfamethoxazole may be useful for empirical treatment of Ochrobactrum infections. [Copyright &y& Elsevier]

Abstract: The genetic diversity and phylogenetic interrelationships among 106 Ochrobactrum strains (O. anthropi: 72, O. intermedium: 22, O. tritici: 5, O. oryzae: 2, O. grignonense: 2, O. gallinifaecis: 1, O. lupini: 2), the type strains of the eight Brucella species and other closely related taxa were studied by recA and rrs gene (16S rRNA) comparative sequence analysis. Both markers correctly delineated the various Ochrobactrum species; however, resolution at the subspecies level was considerably higher in the recA gene-based approach. Phylogenetic analyses using neighbor-joining, parsimony, and maximum likelihood algorithms generated trees with similar topologies but the overall branching order, and also the order of the subclades, were not stable in either assay, which could be explained by generally high recA and rrs sequence similarities. Ochrobactrum and Pseudochrobactrum formed separate clades distinct from other Alphaproteobacteria with Bartonella, Agrobacterium, and Rhizobium as the closest relatives. O. gallinifaecis was the most distinct member, when compared to the type species O. anthropi, with rrs and recA similarities of 96.2% and 81.4%. Brucella species were indistinguishable, exhibiting high rrs and recA gene similarities of 98.6% and 85.5% compared with Ochrobactrum intermedium. At the protein level, all RecA sequences among the various Ochrobactrum species and between Ochrobactrum and Brucella were highly similar with only a few amino acid substitutions. O. anthropi and O. tritici were indistinguishable by means of their RecA proteins. A set of initially biochemically classified strains did not cluster within their assigned species and they either grouped within other known species or grouped as potential novel Ochrobactrum species. In further investigations, these strains were reclassified and described as novel species. In summary, Ochrobactrum is a highly diverse genus comprising several novel species. We recommend recA- in addition to rrs gene-analysis for correct species allocation and subtyping of novel Ochrobactrum isolates. [Copyright &y& Elsevier]

Abstract: A polymerase chain reaction (PCR) assay targeting the flagellin P (fliP)-I S407A genomic region of Burkholderia mallei was developed for the specific detection of this organism in pure cultures and clinical samples from a recent outbreak of equine glanders. Primers deduced from the known fliP-IS407A sequence of B. mallei American Type Culture Collection (ATCC) 23344T allowed the specific amplification of a 989-bp fragment from each of the 20 B. mallei strains investigated, whereas other closely related organisms tested negative. The detection limit of the assay was 10 fg for purified DNA of B. mallei ATCC 23344T. B. mallei DNA was also amplified from various tissues of horses with a generalized B. mallei infection. The developed PCR assay can be used as a simple and rapid tool for the specific and sensitive detection of B. mallei in clinical samples. [Copyright &y& Elsevier]

Abstract: Based on the recently completed genomic sequence of Chromobacterium violaceum American Type Culture Collection (ATCC) 12472 a multiplex PCR assay targeting the prgI, spaO, invG, and sipB genes of the Salmonella SPI-1 homologue type-III secretion system was developed. PCR products of 255bp (prgI), 749bp (spaO), 1685bp (invG), and 1752bp (sipB) were successfully amplified simultaneously in a single reaction with all Chr. violaceum strains investigated whereas other bacteria tested negative. The detection limit for pure cultures in multiplex PCR analysis was 100CFU. The developed assay significantly improves rapid identification of Chr. violaceum and allows its differentiation from closely related organisms. [Copyright &y& Elsevier]

Abstract: Anthrax Blood Agar® (ABA) and Cereus Ident Agar® (CEI) were evaluated as selective growth media for the isolation of Bacillus anthracis using 92 B. anthracis and 132 other Bacillus strains from 30 species. The positive predictive values for the identification of B. anthracis on ABA, CEI, and the combination of both were 72%, 71%, and 90%, respectively. Thus, less than 10% of all species were misidentified using both nutrient media. Species which might be misidentified as B. anthracis were B. cereus, B. mycoides, and B. thuringiensis. Particularly, 30% of B. weihenstephanensis strains were misidentified as B. anthracis. [Copyright &y& Elsevier]

The homeoboxB9 (HOXB9) gene is necessary for specification of the anterior-posterior body axis during embryonic development and expressed in various types of cancer. Here we show that the Wilms tumor transcription factor WT1 regulates the HOXB9 gene in a bidirectional manner. Silencing of WT1 activates HOXB9 in Wt1 expressing renal cell adenocarcinoma-derived 786-0 cells, mesonephric M15 cells and ex vivo cultured murine embryonic kidneys. In contrast, HOXB9 expression in U2OS osteosarcoma and human embryonic kidney (HEK) 293 cells, which lack endogenous WT1, is enhanced by overexpression of WT1. Consistently, Hoxb9 promoter activity is stimulated by WT1 in transiently transfected U2OS and HEK293 cells, but inhibited in M15 cells with CRISPR/Cas9-mediated Wt1 deletion. Electrophoretic mobility shift assay and chromatin immunoprecipitation demonstrate binding of WT1 to the HOXB9 promoter in WT1-overexpressing U2OS cells and M15 cells. BASP1, a transcriptional co-repressor of WT1, is associated with the HOXB9 promoter in the chromatin of these cell lines. Co-transfection of U2OS and HEK293 cells with BASP1 plus WT1 prevents the stimulatory effect of WT1 on the HOXB9 promoter. Our findings identify HOXB9 as a novel downstream target gene of WT1. Depending on the endogenous expression of WT1 , forced changes in WT1 can either stimulate or repress HOXB9 , and the inhibitory effect of WT1 on transcription of HOXB9 involves BASP1. Consistent with inhibition of Hoxb9 expression by WT1, both transcripts are distributed in an almost non-overlapping pattern in embryonic mouse kidneys. Regulation of HOXB9 expression by WT1 might become relevant during kidney development and cancer progression. Bidirectional regulation of Hoxb9 by WT1. Endogenous WT1 suppresses transcription of the Hoxb9 gene in M15 cells, 786–0 cells and embryonic kidney organ culture involving the co-repressor BASP1 (A). Knockdown of WT1 in these cells increases Hoxb9 expression (B). Wt1 overexpression in cells lacking endogenous WT1 (U2OS, HEK293) stimulates Hoxb9 expression presumably due to a loss of Basp1 at the Hoxb9 promoter (C). Combined overexpression of WT1 plus BASP1 represses Hoxb9 (D). [Display omitted] • We identify the homeobox gene HOXB9 as a novel downstream target of the Wilms tumor protein WT1. • Depending on the cell-type, WT1 can either stimulate or inhibit transcription of the HOXB9 gene. • Transcriptional inhibition of HOXB9 by WT1 involves the co-repressor BASP1. • We propose that HOXB9 gene regulation by WT1 is important in disease and development, particularly of the kidney. [ABSTRACT FROM AUTHOR]

ADAMTS16 (a disintegrin and metalloproteinase with thrombospondin motifs) is a secreted mammalian metalloproteinase with unknown function. We report here that murine Adamts16 is co-expressed with the Wilms tumor protein, Wt1, in the developing glomeruli of embryonic kidneys. Adamts16 mRNA levels were significantly reduced upon transfection of embryonic murine kidney explants with Wt1 antisense vivomorpholinos. Antisense knockdown of Adamts16 inhibited branching morphogenesis in kidney organ cultures. Adamts16 was detected by in situ mRNA hybridization and/or immunohistochemistry also in embryonic gonads and in spermatids and granulosa cells of adult testes and ovaries, respectively. Silencing of Wt1 by transfection with antisense vivo-morpholinos significantly increased Adamts16 mRNA in cultured embryonic XY gonads (11.5 and 12.5 days postconception), and reduced Adamts16 transcripts inXXgonads (12.5 and 13.5 days postconception). Three predicted Wt1 consensus motifs could be identified in the promoter and the 5'-untranslated region of the murine Adamts16 gene. Binding of Wt1 protein to these elements was verified by EMSA and ChIP. A firefly luciferase reporter gene under control of the Adamts16 promoter was activated ~8-fold by transient co-transfection of human granulosa cells with a Wt1 expression construct. Gradual shortening of the 5'-flanking sequence successively reduced and eventually abrogated Adamts16 promoter activation by Wt1. These findings demonstrate that Wt1 differentially regulates the Adamts16 gene in XX and XY embryonic gonads. It is suggested that Adamts16 acts immediately downstream of Wt1 during murine urogenital development. We propose that Adamts16 is involved in branching morphogenesis of the kidneys in mice. [ABSTRACT FROM AUTHOR]

Abstract: The diagnosis of brucellosis is mainly based on the detection of anti-LPS antibodies. Due to substantial similarity of the O-polysaccharide of Brucella LPS to that of various other Gram-negative bacteria, serological tests of samples containing high amounts of LPS lack specificity. Hence, the development of assays based on more specific protein antigens is an essential subject in brucellosis research. The aim of this study was proteomic characterization of various antigen preparations of the diagnostic reference strain Brucella abortus 1119-3 and the identification of immunogenic proteins suitable for serological assays. Seventeen out of 383 protein spots of B. abortus 1119-3 were identified to be immunogenic by 2-D immunoblotting. These immunogenic spots were assigned to 6 proteins by MALDI-MS and nLC-ESI-MS/MS: Cu–Zn SOD, BCSP31, L7/L12, GroEL, GroES, and DnaK. All immunogenic proteins were present in three different antigen preparations investigated, i.e. native antigen, standard agglutination and commercially available agglutination antigen. 2-D immunoblotting of bacteria cross-reacting with Brucellae in agglutination tests proved that cross-reactivity of proteins is negligible. Surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS) spectra also differentiated B. abortus clearly from cross-reacting bacteria. The combination of SELDI-MS analysis with the specificity of antibody binding will improve the identification of Brucella specific immunogenic proteins. [Copyright &y& Elsevier]

Wilms tumor protein 1 (WT1) has been implicated in the control of several genes in sexual development, but its function in gonad formation is still unclear. Here, we report thatWT1stimulates expression of Kdr, the gene encoding VEGF receptor 2, in murine embryonic gonads. We found that WT1 and KDR are co-expressed in Sertoli cells of the testes and somatic cells of embryonic ovaries. Vivo-morpholino-mediated WT1 knockdown decreased Kdr transcripts in cultured embryonic gonads at multiple developmental stages. Furthermore, WT1 bound to the Kdr promoter in the chromatin of embryonic testes and ovaries. Forced expression of theWT1(- KTS) isoform, which functions as a transcription factor, increased KDR mRNA levels, whereas the WT1(+ KTS) isoform, which acts presumably on the post-transcriptional level, did not. ChIP indicated that WT1(- KTS), but not WT1(+ KTS), binds to the KDR promoter. Treatment with the KDR tyrosine kinase inhibitor SU1498 or the KDR ligand VEGFA revealed that KDR signaling represses the testis-promoting gene Sox9 in embryonic XX gonads. WT1 knockdown abrogated the stimulatory effect of SU1498-mediated KDR inhibition on Sox9 expression. Exposure to 1% O2 to mimic the low-oxygen conditions in the embryo increased Vegfa expression but did not affect Sox9 mRNA levels in gonadal explants. However, incubation in 1% O2 in the presence of SU1498 significantly reduced Sox9 transcripts in cultured testes and increased Sox9 levels in ovaries. These findings demonstrate that both the local oxygen environment and WT1, which enhances KDR expression, contribute to sex-specific Sox9 expression in developing murine gonads. [ABSTRACT FROM AUTHOR]

Insulin-like growth factor (IGF) binding proteins (IGFBPs) constitute a family of six secreted proteins that regulate the signaling of insulin-like growth factors (IGFs). IGFBP5 is the most conserved family member in vertebrates and the major IGF binding protein in bone. IGFBP5 is required for normal development of the musculoskeletal system, and various types of cancer frequently express high levels of IGFP5. Here we identify the gene encoding IGFBP5 as a novel downstream target of the Wilms tumor protein WT1. IGFBP5 and WT1 are expressed in an overlapping pattern in the condensing metanephric mesenchyme of embryonic murine kidneys. Down-regulation of WT1 by transfection with antisense vivo -morpholino significantly decreased Igfbp5 transcripts in murine embryonic kidney explants. Likewise, silencing of Wt1 in a mouse mesonephros-derived cell line reduced Igfbp5 mRNA levels by approximately 80%. Conversely, induction of the WT1(− KTS) isoform, whose role as transcriptional regulator has been firmly established, significantly increased IGFBP5 mRNA and protein levels in osteosarcoma cells. IGFBP5 expression was not significantly changed by WT1(+ KTS) protein, which exhibits lower DNA binding affinity than the WT1(− KTS) isoform and has a presumed role in post-transcriptional gene regulation. Luciferase reporter constructs harboring 0.8 and 1.6 kilobases of the murine Igfbp5 promoter, respectively, were stimulated approximately 5-fold by co-transfection of WT1(− KTS). The WT1(+ KTS) variant had no significant effect on IGFBP5 promoter activity. Binding of WT1(− KTS), but not of WT1(+ KTS) protein, to the IGFBP5 promoter in human osteosarcoma cells was proven by chromatin immunoprecipitation (ChIP) and confirmed by electrophoretic mobility shift assay. These findings demonstrate that WT1 activates transcription of the IGFBP5 gene with possible implications for kidney development and bone (patho)physiology. [ABSTRACT FROM AUTHOR]

The basic helix-loop-helix transcription factor hASH1, encoded by the ASCL1 gene, plays an important role in neurogenesis and tumor development. Recent findings indicate that local oxygen tension is a critical determinant for the progression of neuroblastomas. Here we investigated the molecular mechanisms underlying the oxygen-dependent expression of hASH1 in neuroblastoma cells. Exposure of human neuroblastoma-de-rived Kelly cells to 1% O2 significantly decreased ASCL1 mRNA and hASH1 protein levels. Using reporter gene assays, we show that the response of hASH1 to hypoxia is mediated mainly by post-transcriptional inhibition via the ASCL1 mRNA 5'- and 3'-UTRs, whereas additional inhibition of the ASCL1 promoter was observed under prolonged hypoxia. By RNA pulldown experiments followed by MALDI/TOF-MS analysis, we identified heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1 and hnRNP-R as interactors binding directly to the ASCL1 mRNA 5'- and 3'-UTRs and influencing its expression. We further demonstrate that hnRNP-A2/B1 is a key positive regulator of ASCL1, findings that were also confirmed by analysis of a large compilation of gene expression data. Our data suggest that a prominent down-regulation of hnRNP-A2/B1 during hypoxia is associated with the post-transcriptional suppression of hASH1 synthesis. This novel post-transcriptional mechanism for regulating hASH1 levels will have important implications in neural cell fate development and disease. [ABSTRACT FROM AUTHOR]

Amine oxidase copper-containing 1 (AOC1; formerly known as amiloride-binding protein 1) is a secreted glycoprotein that catalyzes the degradation of putrescine and histamine. Polyamines and their diamine precursor putrescine are ubiquitous to all organisms and fulfill pivotal functions in cell growth and proliferation. Despite the importance of AOC1 in regulating polyamine breakdown, very little is known about the molecular mechanisms that control its expression.Wereport here that the Wilms tumor protein, WT1, which is necessary for normal kidney development, activates transcription of the AOC1 gene. Expression of a firefly luciferase reporter under control of the proximal AOC1 promoter was significantly enhanced by co-transfection of aWT1expression construct. Binding ofWT1 protein to a cis-regulatory element in the AOC1 promoter was confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Antisense inhibition of WT1 protein translation strongly reduced Aoc1 transcripts in cultured murine embryonic kidneys and gonads. Aoc1 mRNA levels correlated with WT1 protein in several cell lines. Double immunofluorescent staining revealed a co-expression ofWT1andAOC1 proteins in the developing genitourinary system of mice and rats. Strikingly, induced changes in polyamine homeostasis affected branching morphogenesis of cultured murine embryonic kidneys in a developmental stage-specific manner. These findings suggest that WT1-dependent control of polyamine breakdown, which is mediated by changes in AOC1 expression, has a role in kidney organogenesis. [ABSTRACT FROM AUTHOR]

Fragile X syndrome is a common inherited cause of mental retardation that results from loss or mutation of the fragile X mental retardation protein (FMRP). In this study, we identified the mRNA of the basic helix-loop-helix transcription factor human achaetescute homologue-1 (hASH1 or ASCL1), which is required for normal development of the nervous system and has been implicated in the formation of neuroendocrine tumors, as a new FMRP target. Using a double-immunofluorescent staining technique we detected an overlapping pattern of both proteins in the hippocampus, temporal cortex, subventricular zone, and cerebellum of newborn rats. Forced expression of FMRP and gene silencing by small interference RNA transfection revealed a positive correlation between the cellular protein levels of FMRP and hASH1. A luciferase reporter construct containing the 5′-untranslated region of hASH1 mRNA was activated by the full-length FMRP, but not by naturally occurring truncated FMR proteins, in transient co-transfections. The responsible cis-element was mapped by UV-cross-linking experiments and reporter mutagenesis assays to a (U)10 sequence located in the 5′-untranslated region of the hASH1 mRNA. Sucrose density gradient centrifugation revealed that hASH1 transcripts were transiocated into a translationally active polysomal fraction upon transient transfection of HEK293 cells with FMRP, thus indicating translational activation of hASH1 mRNA. In conclusion, we identified hASH1 as a novel downstream target of FMRP. Improved translation efficiency of hASH1 mRNA by FMRP may represent an important regulatory switch in neuronal differentiation. [ABSTRACT FROM AUTHOR]

Renin plays a crucial role in the control of various physiological processes such as blood pressure and body fluid homeostasis. Here, we show that a splice variant of the Wilms' tumor protein lacking three amino acids WT1(−KTS) suppresses renin gene transcription. Using bioinformatics tools, we initially predicted that a WT1-binding site exists in a regulatory region about 12 kb upstream of the renin promoter; this was confirmed by reporter gene assays and gel shift experiments in heterologous cells. Co-expression of Wt1 and renin proteins was found in rat kidney sections, mouse kidney blood vessels, and a cell line derived from the juxtaglomerular apparatus that produces renin. Knockdown of WT1 protein by siRNA significantly increased the cellular renin mRNA content, while overexpression of WT1(−KTS) reduced renin gene expression in stable and transiently transfected cells. A mutant WT1(−KTS) protein found in Wilms' tumors failed to suppress renin gene reporter activity and endogenous renin expression. Our findings show that renin gene transcription is regulated by the WT1(−KTS) protein and this may explain findings in patients with WT1 gene mutations of increased plasma renin and hypertension.Kidney International (2008) 74, 458–466; doi:10.1038/ki.2008.194; published online 21 May 2008 [ABSTRACT FROM AUTHOR]

Neurotrophins and their cognate receptors play a pivotal role in the development and function of the nervous system. High expression levels of the neurotrophin receptor TrkB and its ligands in neuroblastomas are associated with an unfavorable outcome. We report here that NTRK2, which encodes the TrkB receptor tyrosine kinase, is an oxygen-regulated gene, whose expression is stimulated by the hypoxia-inducible factor-1 (HIF-1). TrkB mRNA and protein levels were elevated nearly 30-fold in neuroblastoma-derived Kelly cells in hypoxia (1% O2) versus normoxia (21% O2). A luciferase reporter construct containing ≈2.1 kilobases of the human TrkB promoter was activated about 6-fold both in hypoxia and after stimulation with the hypoxia mimetic 2,2′-dipyridyl (100 µM) at 21% O2. Luciferase activity in the presence of 2,2′-dipyridyl was reduced significantly upon small interfering RNA knockdown of HIF-1α but not of HIF-2α. Accordingly, hypoxia failed to stimulate the TrkB promoter in mouse embryonic fibroblasts that lacked HIF-1α. The hypoxia-responsive promoter region could be mapped to three HIF-1 binding elements that were located between -923 and -879 bp relative to the transcription start site. The migration of cultured neuroblastoma cells was increased ~2-fold upon incubation at 1 versus 21% °2! This effect of hypoxia was abrogated with the tyrosine kinase inhibitor K252a (200 nM). Our findings indicate that transcription of the NTRK2 gene is stimulated at low oxygen tension through a HIF-1-dependent mechanism. In conclusion, enhanced expression of TrkB could represent a critical switch for the previously reported dedifferentiation of neuroblastoma cells under hypoxic conditions. [ABSTRACT FROM AUTHOR]

Abstract: Human brucellosis is still the most common bacterial zoonosis worldwide. Neither well-known molecular tools nor the classical biotyping methods are satisfactory for subtyping of Brucella spp. Loci containing Variable Number of Tandem Repeats (VNTRs) have recently proved their usefulness in typing strains from animal origin despite the high genetic homogeneity within the genus Brucella (DNA–DNA homology >90%). The aim of this study was to evaluate MLVA (Multiple Locus VNTR Analysis) for diagnostic and epidemiological use in human brucellosis. One hundred and twenty-eight B. melitensis isolates of all three biovars were typed using eight minisatellite (panel 1) and eight microsatellite (panel 2) markers. One hundred and ten different genotypes were identified. The MLVA clustering pattern correlated with the geographic origin of the strains. Brucella strains isolated from different patients within the same outbreak or from the same patient before first-line therapy and after relapse showed identical genotypes. Fuchsin sensitive B. melitensis strains were found in closely related clusters giving evidence for an association between VNTRs and some phenotypic characteristics. However, the validity of biovars established by classical microbiological methods could not be confirmed by MLVA clustering. The original data can be queried on the genotyping web page at http://bacterial-genotyping.igmors.u-psud.fr. The MLVA assay is rapid, highly discriminatory, and reproducible within human Brucella isolates. MLVA can significantly contribute to epidemiological trace-back analysis of Brucella infections and may advance surveillance and control of human brucellosis. [Copyright &y& Elsevier]

Cell-matrix interaction through specific adhesion molecules is a critical step during organ development. In addition, down-regulation of cell adhesion receptors may promote tumor invasion and metastasis. We show here that the Wilms tumor suppressor Wt1, which is necessary for normal development of the epicardium, coronary vessels, genitourinary system, and other tissues, activates transcription of the α4integrin gene. Binding of the Wt1(-KTS) form, which is transcriptionally active, to the proximal α4integrin promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. A reporter construct harboring ~1.9 kb of the human α4integrin gene promoter was activated significantly by transient co-transfection of a Wt1(-KTS) expression plasmid. Introducing mutations in two identified Wt1(-KTS) binding motifs in the proximal promoter of the α4integrin gene abrogated this stimulatory effect. Endogenous α4integrin transcripts were increased more than 3-fold in human embryonic kidney 293 cells with stable expression of the Wt1(-KTS) protein. Wt1-overexpressing cells showed augmented adhesion to the α4integrin ligand vascular cell adhesion molecule-1 that was abolished upon incubation with an inhibitory α4integrin anti-body. Double immunofluorescent staining revealed co-localization of Wt1 and α4integrin in the developing epicardium of mouse embryos. Cardiac expression of α4integrin was reduced significantly in embryos with a homozygous Wt1 defect (Wt1-/-). These findings demonstrate that Wt1 can support cell adhesion through enhanced expression of α4integrin. This transcriptional activation of the α4integrin gene by Wt1(-KTS) might contribute to normal formation of the epicardium and other tissues in the developing embryo. [ABSTRACT FROM AUTHOR]

Cardiac arrhythmia is a serious clinical condition, which is frequently associated with abnormalities of mechanical loading and changes in wall tension of the heart. Recent novel findings suggest that fibroblasts may function as mechano-electric transducers in healthy and diseased hearts. Cardiac fibroblasts are electrically non-excitable cells that respond to spontaneous contractions of the myocardium with rhythmical changes of their resting membrane potential. This phenomenon is referred to as mechanically induced potential (MIP) and has been implicated in the mechano-electric feedback mechanism of the heart. Mechano-electric feedback is thought to adjust the frequency of spontaneous myocardial contractions to changes in wall tension, which may result from variable filling pressure. Electrophysiological recordings of single atrial fibroblasts indicate that mechanical compression of the cells may activate a non-selective cation conductance leading to depolarisation of the membrane potential. Reduced amplitudes of MIPs due to pharmacological disruption of F-actin and tubulin suggest a role for the cytoskeleton in the mechano-electric signal transduction process. Enhanced sensitivity of the membrane potential of the fibroblasts to mechanical stretch after myocardial infarction correlates with depression of heart rates. It is assumed that altered electrical function of cardiac fibroblasts may contribute to the increased risk of post-infarct arrhythmia. [Copyright &y& Elsevier]

The Wilms’ tumor gene Wt1 encodes a zinc finger protein, which is required for normal formation of the genitourinary system and mesothelial tissues. Our recent findings indicate that Wt1 also plays a critical role in the development of ganglion cells in the vertebrate retina. Here we show that the POU-domain factor Pou4f2 (formerly Brn-3b), which is necessary for retinal ganglion cell survival, is up-regulated in human embryonic kidney (HEK)293 cells with stable Wt1 expression. Consistent with our previous observations of increased Pou4f2 mRNA in stably Wt1-transfeced HEK293 cells [EMBO J. 21 (2002) 1398], endogenous Pou4f2 was also elevated at the protein level in the HEK293 transfectants as well as in U2OS osteosarcoma cells that expressed an inducible Wt1 isoform. Transient co-transfection of a Wt1 expression construct activated a Pou4f2 promoter-reporter construct approximately 4-fold. Stimulation of the Pou4f2 promoter required a Wt1 binding element that was similar to a degenerative consensus site previously identified in other Wt1 responsive genes. Double-immunofluorescent labeling revealed co-expression of Pou4f2 and Wt1 in glomerular podocytes of adult kidney and in developing retinal ganglion cells of mouse embryos. Pou4f2 immunoreactivity was absent from the retinas of Wt1−/− embryos. In conclusion, we identified Pou4f2 as a novel downstream target gene of Wt1. Co-localization of both proteins in glomerular podocytes of the kidney and in developing retinal ganglion cells suggests a role for Wt1-Pou4f2 interaction in these tissues. [Copyright &y& Elsevier]

Abstract: To facilitate routine laboratories in the effective diagnosis of brucellosis, we report a robust and rapid multiplex PCR assay, which allows for the differentiation of all nine currently recognised Brucella species. This includes the recently described species B. microti, B. inopinata, B. ceti and B. pinnipedialis. [Copyright &y& Elsevier]