Hepatitis B Virus e Antigen Physically Associates With Receptor-Interacting Serine/Threonine Protein Kinase 2 and Regulates IL-6 Gene Expression.

We previously reported that hepatitis B virus (HBV) e antigen (HBeAg) inhibits production of interleukin 6 by suppressing NF-κB activation. NF-κB is known to be activated through receptor-interacting serine/threonine protein kinase 2 (RIPK2), and we examined the mechanisms of interleukin 6 regulation by HBeAg. HBeAg inhibits RIPK2 expression and interacts with RIPK2, which may represent 2 mechanisms through which HBeAg blocks nucleotide-binding oligomerization domain-containing protein 1 ligand-induced NF-κB activation in HepG2 cells. Our findings identified novel molecular mechanisms whereby HBeAg modulates intracellular signaling pathways by targeting RIPK2, supporting the concept that HBeAg could impair both innate and adaptive immune responses to promote chronic HBV infection.

High expression of the HMG box factor sox-13 in arterial walls during embryonic development.

Members of the Sox gene family of transcription factors are defined by the presence of an 80 amino acid homology domain, the High Mobility Group (HMG) box. Here we report the cloning and initial analysis of murine Sox-13 . The 984 amino acids Sox-13 protein contains a single HMG box, a leucine zipper motif and a glutamine-rich stretch. These characteristics are shared with another member of the Sox gene family, Sox-6. High level embryonic expression of Sox-13 occurs uniquely in the arterial walls of 13.5 days post coitum (dpc) mice and later. Low level expression was observed in the inner ear of 13.5 dpc mice and in a limited number of cells in the thymus of 16.5 dpc mice, from which Sox-13 was originally cloned. At 18.5 dpc, Sox-13 is expressed in the tracheal epithelium below the vocal cord and in the hair follicles. The Sox-13 protein binds to the consensus HMG box motif, AACAAAG, but does not transactivate transcription through a concatamer of this motif. Sox-13, like other members of the Sox family likely plays an important role in development.

Maternally inherited Leigh syndrome.

A 6 1/2-year-old girl had developmental regression, and Leigh syndrome was diagnosed. A second girl born to the same mother after heterologous artificial insemination also lost acquired skills and died at 2 1/2 years of age; neuropathologic examination confirmed the diagnosis of Leigh syndrome. Tissues from both children and from the mother had a point mutation at nucleotide 8993 in the adenosinetriphosphatase 6-gene of mitochondrial DNA. This family illustrates that Leigh syndrome can be transmitted by maternal inheritance.

Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery.

BACKGROUND: Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice. RESULTS: Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependent manner. More than 90% of transduced cells were small and medium sized neurons (< 700 microm 2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (approximately 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell types. CONCLUSION: We have found that rAAV2/6 is an efficient vector to deliver transgenes to nociceptive neurons in mice. Furthermore, the characterization of the transduction profile may facilitate gene transfer studies to dissect mechanisms behind neuropathic pain.

Differentially expressed gene profile in the 6-hydroxy-dopamine-induced cell culture model of Parkinson's disease.

Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of dopaminergic (DA) neurons of the substantia nigra pars compacta with unknown aetiology. 6-Hydroxydopamine (6-OHDA) treatment of neuronal cells is an established in vivo model for mimicking the effect of oxidative stress found in PD brains. We examined the effects of 6-OHDA treatment on human neuroblastoma cells (SH-SY5Y) and primary mesencephalic cultures. Using a reverse arbitrarily primed polymerase chain reaction (RAP-PCR) approach we generated reproducible genetic fingerprints of differential expression levels in cell cultures treated with 6-OHDA. Of the resulting sequences, 23 showed considerable homology to known human coding sequences. The results of the RAP-PCR were validated by reverse transcription PCR, real-time PCR and, for selected genes, by Western blot analysis and immunofluorescence. In four cases, [tomoregulin-1 (TMEFF-1), collapsin response mediator protein 1 (CRMP-1), neurexin-1, and phosphoribosylaminoimidazole synthetase (GART)], a down-regulation of mRNA and protein levels was detected. Further studies will be necessary on the physiological role of the identified proteins and their impact on pathways leading to neurodegeneration in PD.

Heparan sulfate 6-O-sulfotransferase 1, a gene involved in extracellular sugar modifications, is mutated in patients with idiopathic hypogonadotrophic hypogonadism.

Neuronal development is the result of a multitude of neural migrations, which require extensive cell-cell communication. These processes are modulated by extracellular matrix components, such as heparan sulfate (HS) polysaccharides. HS is molecularly complex as a result of nonrandom modifications of the sugar moieties, including sulfations in specific positions. We report here mutations in HS 6-O-sulfotransferase 1 (HS6ST1) in families with idiopathic hypogonadotropic hypogonadism (IHH). IHH manifests as incomplete or absent puberty and infertility as a result of defects in gonadotropin-releasing hormone neuron development or function. IHH-associated HS6ST1 mutations display reduced activity in vitro and in vivo, suggesting that HS6ST1 and the complex modifications of extracellular sugars are critical for normal development in humans. Genetic experiments in Caenorhabditis elegans reveal that HS cell-specifically regulates neural branching in vivo in concert with other IHH-associated genes, including kal-1, the FGF receptor, and FGF. These findings are consistent with a model in which KAL1 can act as a modulatory coligand with FGF to activate the FGF receptor in an HS-dependent manner.

Growth arrest-specific gene 6 (GAS6) as an intra-hospital mortality predictor for patients in septic shock

Aim: Gas6 is known to be elevated in sepsis, correlating with the severity of infection and organ failure. We aimed to investigate the performance of Gas6 plasma levels at admission to predict the risk of mortality in a cohort of septic patients.Methods: We used prospectively collected data and plasma samples from the 'Sepsis Cohorte Romande'. Gas6 level was measured by ELISA at admission and expressed in percentage relative to its level in a pool of normal plasma.Results: Non-survivors (n = 19) presented higher Gas6 levels than survivors (n = 78; median 287% vs. 158%, IQR 182 and 119 respectively; P = 0.0003). Gas6 correlated positively with different cytokine and was the best mortality predictor, as shown by the ROC curves area (Fig. 1). In patients with septic shock (n = 67), using 249% as a cut-off value, Gas6 measurement had a specificity of 81% and a sensitivity of 68% for predicting mortality. ROC curve area was 0.76. Positive and negative predictive values were 59% and 87%, respectively.Conclusion: Thus, Gas6 plasma level at admission might be a useful tool to predict mortality in patients with septic shock. Nevertheless, independent association of Gas6 level with mortality still needs to be assessed. Although Gas6 hold promise as an early sepsis marker, its precise implication in sepsis remains to be elucidated.

Growth arrest-specific gene 6 » (Gas6) as an intra-hospital mortality predictor for patients in septic shock

Aim: Gas6 is known to be elevated in sepsis, correlating with the severity of infection and¦organ failure. We aimed to investigate the performance of Gas6 plasma levels at¦admission to predict the risk of mortality in a cohort of septic patients.¦Methods: We used prospectively collected data and plasma samples from the "Sepsis¦Cohorte Romande". Gas6 level was measured by ELISA at admission and expressed in¦percentage relative to its level in a pool of normal plasma.¦Results: Non-survivors (n=21) presented higher Gas6 levels than survivors (n=73) (median¦258% vs 164%, IQR 194 and 117 respectively) (p=0.0027). Gas6 correlated positively with¦different cytokines and was the best mortality predictor, as shown by the ROC curves area.¦In patients with septic shock (n=66), using 249% as a cut-off value, Gas6 measurement¦had a specificity of 67% and a sensitivity of 81% for predicting mortality. ROC curve area¦was 0.75. Positive and negative predictive values were 57% and 87%, respectively.¦Conclusion: Thus, Gas6 plasma level at admission might be a useful tool to predict¦mortality in patients with septic shock. Although Gas6 hold promise as an early sepsis¦marker, its precise implication in sepsis remains to be elucidated. Our observation should¦be further investigated in larger prospective clinical trials.

Growth Arrest-Specific Gene 6 product modulates innate immunity in sepsis

Background: Growth Arrest-Specific Gene 6 product (Gas6) is, like anticoagulant protein C, a vitamin K-dependent protein. Our aim was to determine whether Gas6 plays a role in sepsis. Materials and methods: We submitted mice lacking Gas6 (Gas6)/)) or one of its receptors (Axl)/), Tyro3)/) or Mertk)/)) to LPS-induced endotoxemia and peritonitis (cecal ligation and puncture (CLP) and inoculation of E. coli). In addition, we measured Gas6 or its soluble receptors in plasma of eight volunteers that received LPS, 13 healthy subjects, 28 patients with severe sepsis, and 18 patients with non-infectious inflammatory diseases. Results: Gas6 and its soluble receptor sAxl raised in mice models and TNF-a was more elevated in Gas6)/) mice than in wild-type (WT). Protein array showed that before and after LPS injection, titers of 62 cytokines were more elevated in plasma of Gas6)/) than WT mice. Endotoxemia-induced mortality was higher in Gas6)/), Axl)/), Tyro3)/) and Mertk)/) compared to WT mice and mortality subsequent to CLP was amplified in Gas6)/) mice. LPS-stimulated Gas6)/) macrophages produced more cytokines than WT macrophages. This production was dampened by recombinant Gas6. Phosphorylation of Akt in Gas6)/) macrophages was reduced, but p38 phosphorylation and NF-jB translocation were increased. In human, Gas6 raised in plasma after LPS (2 ng/kg). Gas6 and sAxl were higher in patients with severe sepsis than in healthy subjects or control patients, and there was a non-significant trend for higher Gas6 in the survival group. Conclusions: Our data point to Gas6 as a major modulator of innate immunity and provide thereby novel insights into the mechanism of sepsis. Thus Gas6 and its receptors might constitute potential therapeutic targets for the development of new immunomodulating drugs.

Changes in reproductive roles are associated with changes in gene expression in fire ant queens.

Abstract In species with social hierarchies, the death of dominant individuals typically upheaves the social hierarchy and provides an opportunity for subordinate individuals to become reproductives. Such a phenomenon occurs in the monogyne form of the fire ant, Solenopsis invicta, where colonies typically contain a single wingless reproductive queen, thousands of workers and hundreds of winged nonreproductive virgin queens. Upon the death of the mother queen, many virgin queens shed their wings and initiate reproductive development instead of departing on a mating flight. Workers progressively execute almost all of them over the following weeks. To identify the molecular changes that occur in virgin queens as they perceive the loss of their mother queen and begin to compete for reproductive dominance, we collected virgin queens before the loss of their mother queen, 6 h after orphaning and 24 h after orphaning. Their RNA was extracted and hybridized against microarrays to examine the expression levels of approximately 10 000 genes. We identified 297 genes that were consistently differentially expressed after orphaning. These include genes that are putatively involved in the signalling and onset of reproductive development, as well as genes underlying major physiological changes in the young queens.

Genome-wide association study of metabolic traits reveals novel gene-metabolite-disease links.

Metabolic traits are molecular phenotypes that can drive clinical phenotypes and may predict disease progression. Here, we report results from a metabolome- and genome-wide association study on (1)H-NMR urine metabolic profiles. The study was conducted within an untargeted approach, employing a novel method for compound identification. From our discovery cohort of 835 Caucasian individuals who participated in the CoLaus study, we identified 139 suggestively significant (P<5×10(-8)) and independent associations between single nucleotide polymorphisms (SNP) and metabolome features. Fifty-six of these associations replicated in the TasteSensomics cohort, comprising 601 individuals from São Paulo of vastly diverse ethnic background. They correspond to eleven gene-metabolite associations, six of which had been previously identified in the urine metabolome and three in the serum metabolome. Our key novel findings are the associations of two SNPs with NMR spectral signatures pointing to fucose (rs492602, P = 6.9×10(-44)) and lysine (rs8101881, P = 1.2×10(-33)), respectively. Fine-mapping of the first locus pinpointed the FUT2 gene, which encodes a fucosyltransferase enzyme and has previously been associated with Crohn's disease. This implicates fucose as a potential prognostic disease marker, for which there is already published evidence from a mouse model. The second SNP lies within the SLC7A9 gene, rare mutations of which have been linked to severe kidney damage. The replication of previous associations and our new discoveries demonstrate the potential of untargeted metabolomics GWAS to robustly identify molecular disease markers.

Identification of C7orf11 (TTDN1) gene mutations and genetic heterogeneity in nonphotosensitive trichothiodystrophy.

We have identified C7orf11, which localizes to the nucleus and is expressed in fetal hair follicles, as the first disease gene for nonphotosensitive trichothiodystrophy (TTD). C7orf11 maps to chromosome 7p14, and the disease locus has been designated "TTDN1" (TTD nonphotosensitive 1). Mutations were found in patients with Amish brittle-hair syndrome and in other nonphotosensititive TTD cases with mental retardation and decreased fertility but not in patients with Sabinas syndrome or Pollitt syndrome. Therefore, genetic heterogeneity in nonphotosensitive TTD is a feature similar to that observed in photosensitive TTD, which is caused by mutations in transcription factor II H (TFIIH) subunit genes. Comparative immunofluorescence analysis, however, suggests that C7orf11 does not influence TFIIH directly. Given the absence of cutaneous photosensitivity in the patients with C7orf11 mutations, together with the protein's nuclear localization, C7orf11 may be involved in transcription but not DNA repair.

CYP3A activity measured by the midazolam test is not related to 3435 C >T polymorphism in the multiple drug resistance transporter gene.

A recent study with 69 Japanese liver transplants treated with tacrolimus found that the MDR13435 C >T polymorphism, but not the MDR12677 G >T polymorphism, was associated with differences in the intestinal expression level of CYP3A4 mRNA. In the present study, over 6 h, we measured the kinetics of a 75 microg oral dose of midazolam, a CYP3A substrate, in 21 healthy subjects genotyped for the MDR13435 C >T and 2677 G >T polymorphism. No statistically significant differences were found in the calculated pharmacokinetic parameters between the three 3435 C >T genotypes (TT, CT and CC group, respectively: Cmax (mean +/- SD: 0.30 +/- 0.08 ng/ml, 0.31 +/- 0.09 ng/ml and 0.31 +/- 0.11 ng/ml; Apparent clearance: 122 +/- 29 l/h, 156 +/- 92 l/h and 111 +/- 35 l/h; t1/2: 1.9 +/- 1.1 h, 1.6 +/- 0.90 h and 1.7 +/- 0.7 h). In addition, the 30-min 1'OH midazolam to midazolam ratio, a marker of CYP3A activity, determined in 74 HIV-positive patients before the introduction of antiretroviral treatment, was not significantly different between the three 3435 C >T genotypes (mean ratio +/- SD: 3.65 +/- 2.24, 4.22 +/- 3.49 and 4.24 +/- 2.03, in the TT, CT and CC groups, respectively). Similarly, no association was found between the MDR12677 G >T polymorphism and CYP3A activity in the healthy subjects or in the HIV-positive patients. The existence of a strong association between the activity of CYP3A and MDR13435 C >T and 2677 G >T polymorphisms appears unlikely, at least in Caucasian populations and/or in the absence of specific environmental factors.

Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs.

Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor betaPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNA-modified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo.

Characterization of pip5k3 fleck corneal dystrophy-linked gene in zebrafish.

PIKfyve is a kinase encoded by pip5k3 involved in phosphatidylinositols (PdtIns) pathways. These lipids building cell membranes have structural functions and are involved in complex intracellular regulations. Mutations in human PIP5K3 are associated with François-Neetens mouchetée fleck corneal dystrophy [Li, S., Tiab, L., Jiao, X., Munier, F.L., Zografos, L., Frueh, B.E., Sergeev, Y., Smith, J., Rubin, B., Meallet, M.A., Forster, R.K., Hejtmancik, J.F., Schorderet, D.F., 2005. Mutations in PIP5K3 are associated with François-Neetens mouchetee fleck corneal dystrophy. Am. J. Hum. Genet. 77, 54-63]. We cloned the zebrafish pip5k3 and report its molecular characterization and expression pattern in adult fish as well as during development. The zebrafish PIKfyve was 70% similar to the human homologue. The gene encompassed 42 exons and presented four alternatively spliced variants. It had a widespread expression in the adult organs and was localized in specific cell types in the eye as the cornea, lens, ganglion cell layer, inner nuclear layer and outer limiting membrane. Pip5k3 transcripts were detected in early cleavage stage embryos. Then it was uniformly expressed at 10 somites, 18 somites and 24 hpf. Its expression was then restricted to the head region at 48 hpf, 72 hpf and 5 dpf and partial expression was found in somites at 72 hpf and 5 dpf. In situ on eye sections at 3 dpf showed a staining mainly in lens, outer limiting membrane, inner nuclear layer and ganglion cell layer. A similar expression pattern was found in the eye at 5 dpf. A temporal regulation of the spliced variants was observed at 1, 3 and 5 dpf and they were also found in the adult eye.