Plac8 is an inducer of C/EBPβ required for brown fat differentiation, thermoregulation, and control of body weight.

Brown adipocytes oxidize fatty acids to produce heat in response to cold or to excessive energy intake; stimulation of brown fat development and function may thus counteract obesity. Brown adipogenesis requires activation of the transcription factor C/EBPβ and recruitment of the zinc finger protein Prdm16, but upstream inducers of these proteins are incompletely defined. Here, we show that genetic inactivation of Plac8, a gene encoding an evolutionarily conserved protein, induces cold intolerance, and late-onset obesity, as well as abnormal morphology and impaired function of brown adipocytes. Using brown preadipocyte lines we show that Plac8 is required for brown fat differentiation, that its overexpression induces C/EBPβ and Prdm16, and that upon induction of differentiation Plac8 associates with C/EBPβ and binds to the C/EBPβ promoter to induce its transcription. Thus, Plac8 is a critical upstream regulator of brown fat differentiation and function that acts, at least in part, by inducing C/EBPβ expression.

Kinematically complete analysis of the CLAS data on the proton structure Function F2 in a Regge-Dual model

The recently measured inclusive electron-proton cross section in the nucleon resonance region, performed with the CLAS detector at the Thomas Jefferson Laboratory, has provided new data for the nucleon structure function F2 with previously unavailable precision. In this paper we propose a description of these experimental data based on a Regge-dual model for F2. The basic inputs in the model are nonlinear complex Regge trajectories producing both isobar resonances and a smooth background. The model is tested against the experimental data, and the Q2 dependence of the moments is calculated. The fitted model for the structure function (inclusive cross section) is a limiting case of the more general scattering amplitude equally applicable to deeply virtual Compton scattering. The connection between the two is discussed.

A Heterozygous Deletion Mutation in the Cardiac Sodium Channel Gene SCN5A with Loss- and Gain-of-Function Characteristics Manifests as Isolated Conduction Disease, without Signs of Brugada or Long QT Syndrome.

BACKGROUND: The SCN5A gene encodes for the α-subunit of the cardiac sodium channel NaV1.5, which is responsible for the rapid upstroke of the cardiac action potential. Mutations in this gene may lead to multiple life-threatening disorders of cardiac rhythm or are linked to structural cardiac defects. Here, we characterized a large family with a mutation in SCN5A presenting with an atrioventricular conduction disease and absence of Brugada syndrome. METHOD AND RESULTS: In a large family with a high incidence of sudden cardiac deaths, a heterozygous SCN5A mutation (p.1493delK) with an autosomal dominant inheritance has been identified. Mutation carriers were devoid of any cardiac structural changes. Typical ECG findings were an increased P-wave duration, an AV-block I° and a prolonged QRS duration with an intraventricular conduction delay and no signs for Brugada syndrome. HEK293 cells transfected with 1493delK showed strongly (5-fold) reduced Na(+) currents with altered inactivation kinetics compared to wild-type channels. Immunocytochemical staining demonstrated strongly decreased expression of SCN5A 1493delK in the sarcolemma consistent with an intracellular trafficking defect and thereby a loss-of-function. In addition, SCN5A 1493delK channels that reached cell membrane showed gain-of-function aspects (slowing of the fast inactivation, reduction in the relative fraction of channels that fast inactivate, hastening of the recovery from inactivation). CONCLUSION: In a large family, congregation of a heterozygous SCN5A gene mutation (p.1493delK) predisposes for conduction slowing without evidence for Brugada syndrome due to a predominantly trafficking defect that reduces Na(+) current and depolarization force.

Characterization of the expression, regulation and function of Sorting Nexin 9 and Sorting Nexin 18

Summary : Sorting nexin (SNX) family members play important roles in intracellular protein and membrane trafficking, The membrane-tubulating SNX9 protein has been shown to interact with multiple components of the endocytic machinery and to participate in clathrin-mediated endocytosis of cell surface receptors. It has not been investigated if SNX9 may also participate in other protein sorting pathways that involve vesicular transport, specifically the biogenesis of lysosome-related organelles (LROs). Closely related to SNX9 is SNXl8, whose function is largely unknown. In this work, we have characterized the expression of SNX9 and SNXl8 in LRO-containing cells and investigated their role in protein trafficking during the formation of LROs. Our results indicate that SNX9 and SNXl8 are not essential for the formation of LROs, nor for the sorting of melanosomal proteins. We investigated how the level of intracellular SNX9 protein is regulated and found that it is a substrate of the ubiquitin ligase Itch, a member of the NEDD4 family of E3 ubiquitin ligases. Itch ubiquitylates SNX9 and regulates SNX9 levels by enhancing its degradation. Using ? truncated proteins we found that the interaction with SNX9 is mediated by the proline-rich domain of Itch, a domain distinct from the conventional WW recognition domain, and the SH3 domain of SNX9. Interaction with the PRD of Itch is essential for SNX9 ubiquitylation and degradation. We further showed that Itch binding is not affected by tyrosine phosphorylation of SNX9. Using lentivector-mediated siRNA techniques, we found that Itch regulates the level of melanosomal proteins, while knock-down of SNX9 does not alter their level. Interestingly, we revealed that silencing of SNXIS affects the amount of the melanosomal protein Melan-A, but also of SNX9, and that SNXl8 can interact with SNX9. Taken together, our results highlight that the pool of substrates of NEDD4 family E3 ligases extends to proteins containing SH3 domains and provide insight into the potential functions of SNXI8. Résumé : Les membres de la famille des Sorting Nexins (SNX) jouent des rôles importants dans le trafic intracellulaire de protéines et membranes. Il a été démontré que la protéine SNX9, qui génère les tubules membranaires, interagit avec plusieurs composants de la machinerie d'endocytose et participe à l'endocytose des récepteurs de surface mediée par la clathrine. Aucune étude n'a investigué si SNX9 pourrait aussi participer à d'autres voies de trafic de protéines tel que le transport vésiculaire, et plus particulièrement la biogenèse des organites lysosomaux ("lysosome-related organelles", LR©s). SNXl8 est similaire à SNX9, mais sa fonction est largement inconnue. Dans ce travail, nous avons caractérisé l'expression de SNX9 et SNX18 dans des cellules contenants des LROs et investigué leur rôle dans le trafic de protéines pendant la formation des LROS. Nos résultats indiquent que SNX9 et SNXI8 ne sont essentiels ni pour la formation des LR©s, ni pour le trafic de protéines mélanosomales. Nous avons examiné la régulation du niveau intracellulaire de la protéine SNX9 et avons trouvé qu'elle est un substrat de l'ubiquitine ligase Itch, un membre de la famille NEDD4 des ubiquitine ligases E3. Itch ubiquitine SNX9 et régule les niveaux de SNX9 en augmentant sa dégradation. En utilisant des protéines mutées nous avons découvert que l'interaction avec SNX9 est médiée par le domaine riche en proline de Itch, qui est différent du domaine conventionnel de reconnaissance WW, et par le domaine SH3 de SNX9. L'interaction avec le domaine riche en proline de Itch est essentielle pour l'ubiquitination et la dégradation de SNX9. De plus, nous avons montré que cette liaison n'est pas affectée par la phosphorylation des résidus tyrosine de SNX9. En utilisant des vecteurs lentiviraux exprimant des siARN, nous avons trouvé que Itch régule les niveaux de protéines mélanosomales, alors que l'extinction de l'expression de SNX9 ne change pas leurs niveaux. En autre, nous avons révélé que la diminution de SNXl8 affecte le niveau de la protéine mélanosomale Melan-A et de SNX9, et aussi que SNXl8 peut interagir avec SNX9. En résumé, nos résultats démontrent que l'ensemble des substrats de la famille NEDD4 des ubiquitine ligases E3 s'élargit aux protéines contenant des domaines SH3 et ouvrent des perspectives sur les fonctions potentielles de SNXl8.

RANKL expression, function, and therapeutic targeting in multiple myeloma and chronic lymphocytic leukemia.

Bone destruction is a prominent feature of multiple myeloma, but conflicting data exist on the expression and pathophysiologic involvement of the bone remodeling ligand RANKL in this disease and the potential therapeutic benefits of its targeted inhibition. Here, we show that RANKL is expressed by primary multiple myeloma and chronic lymphocytic leukemia (CLL) cells, whereas release of soluble RANKL was observed exclusively with multiple myeloma cells and was strongly influenced by posttranscriptional/posttranslational regulation. Signaling via RANKL into multiple myeloma and CLL cells induced release of cytokines involved in disease pathophysiology. Both the effects of RANKL on osteoclastogenesis and cytokine production by malignant cells could be blocked by disruption of RANK-RANKL interaction with denosumab. As we aimed to combine neutralization of RANKL with induction of antibody-dependent cellular cytotoxicity of natural killer (NK) cells against RANKL-expressing malignant cells and as denosumab does not stimulate NK reactivity, we generated RANK-Fc fusion proteins with modified Fc moieties. The latter displayed similar capacity compared with denosumab to neutralize the effects of RANKL on osteoclastogenesis in vitro, but also potently stimulated NK cell reactivity against primary RANKL-expressing malignant B cells, which was dependent on their engineered affinity to CD16. Our findings introduce Fc-optimized RANK-Ig fusion proteins as attractive tools to neutralize the detrimental function of RANKL while at the same time potently stimulating NK cell antitumor immunity.

A homozygous missense mutation in SCNN1A is responsible for a transient neonatal form of pseudohypoaldosteronism type 1.

Pseudohypoaldosteronism type 1 (PHA1) is a monogenic disorder of mineralocorticoid resistance characterized by salt wasting, hyperkalemia, high aldosterone levels, and failure to thrive. An autosomal recessive form (AR-PHA1) is caused by mutations in the epithelial sodium channel ENaC with usually severe and persisting multiorgan symptoms. The autosomal dominant form of PHA1 (AD-PHA1) is due to mutations in the mineralocorticoid receptor causing milder and transient symptoms restricted to the kidney. We identified a homozygous missense mutation in the SCNN1A gene (c.727T>C/p.Ser(243)Pro), encoding α-subunit of ENaC (α-ENaC) in a prematurely born boy with a severe salt-losing syndrome. The patient improved rapidly under treatment, and dietary salt supplementation could be stopped after 6 mo. Interestingly, the patient's sibling born at term and harboring the same homozygous Ser(243)Pro mutation showed no symptom of salt-losing nephropathy. In vitro expression of the αSer(243)Pro ENaC mutant revealed a slight but significant decrease in ENaC activity that is exacerbated in the presence of high Na(+) load. Our study provides the first evidence that ENaC activity is critical for the maintenance of salt balance in the immature kidney of preterm babies. Together with previous studies, it shows that, when the kidney is fully mature, the severity of the symptoms of AR-PHA1 is related to the degree of the ENaC loss of function. Finally, this study identifies a novel functional domain in the extracellular loop of ENaC.

Peripheral neuropathy is linked to a severe form of myotonic dystrophy in transgenic mice.

Myotonic dystrophy type 1 (DM1) is a multisystem disorder with a variable phenotype. The involvement of peripheral nerves in DM1 disease is controversial. The DM1 animal model DM300 transgenic mice that carry 350 to 500 CTG repeats express a mild DM1 phenotype but do not exhibit motor or sensory pathology. Here, we investigated the presence or absence of peripheral neuropathy in transgenic mice (DMSXL) that carry more than 1,300 CTG repeats and display a severe form of DM1. Electrophysiologic, histologic, and morphometric methods were used to investigate the structure and function of peripheral nerves. We observed lower compound muscle action potentials recorded from hind limb muscles and slowing of sciatic nerve conduction velocity in DMSXL versus control mice. Morphometric analyses showed an axonopathy and neuronopathy in the DMSXL mice characterized by a decrease in numbers of myelinatedmotor axons in sciatic nerve and in spinal cord motor neurons. Pathologic alterations in the structure of hind limb neuromuscular junctions were also detected in the DMSXL mice. These results suggest that peripheral neuropathy can be linked to a large CTG expansion and a severe form of DM1.

The rate of recovery in renal function when patients with HIV infection discontinue treatment with tenofovir.

OBJECTIVES: Tenofovir is associated with reduced renal function. It is not clear whether patients can be expected to fully recover their renal function if tenofovir is discontinued. METHODS: We calculated the estimated glomerular filtration rate (eGFR) for patients in the Swiss HIV Cohort Study remaining on tenofovir for at least 1 year after starting a first antiretroviral therapy regimen with tenofovir and either efavirenz or the ritonavir-boosted protease inhibitor lopinavir, atazanavir or darunavir. We estimated the difference in eGFR slope between those who discontinued tenofovir after 1 year and those who remained on tenofovir. RESULTS: A total of 1049 patients on tenofovir for at least 1 year were then followed for a median of 26 months, during which time 259 patients (25%) discontinued tenofovir. After 1 year on tenofovir, the difference in eGFR between those starting with efavirenz and those starting with lopinavir, atazanavir and darunavir was - 0.7 [95% confidence interval (CI) -2.3 to 0.8], -1.4 (95% CI -3.2 to 0.3) and 0.0 (95% CI -1.7 to 1.7) mL/min/1.73 m(2) , respectively. The estimated linear rate of decline in eGFR on tenofovir was -1.1 (95% CI -1.5 to -0.8) mL/min/1.73 m(2) per year and its recovery after discontinuing tenofovir was 2.1 (95% CI 1.3 to 2.9) mL/min/1.73 m(2) per year. Patients starting tenofovir with either lopinavir or atazanavir appeared to have the same rates of decline and recovery as those starting tenofovir with efavirenz. CONCLUSIONS: If patients discontinue tenofovir, clinicians can expect renal function to recover more rapidly than it declined.

A natural structural variant of the mouse TCR beta-chain displays intrinsic receptor function and antigen specificity.

The Cbeta0 alternate cassette exon is located between the Jbeta1 and Cbeta1 genes in the mouse TCR beta-locus. In T cells with a VDJbeta1 rearrangement, the Cbeta0 exon may be included in TCRbeta transcripts (herein called TCRbeta-Cbeta0 transcripts), potentially inserting an additional 24 aa between the V and C domains of the TCR beta-chain. These TCRbeta splice isoforms may be differentially regulated after Ag activation, because we detected TCRbeta-Cbeta0 transcripts in a high proportion (>60%) of immature and mature T cells having VDJbeta1 rearrangements but found a substantially reduced frequency (<35%) of TCRbeta-Cbeta0 expression among CD8 T cells selected by Ag in vivo. To study the potential activity of the TCRbeta-Cbeta0 splice variant, we cloned full-length TCR cDNAs by single-cell RT-PCR into retroviral expression vectors. We found that the TCRbeta-Cbeta0 splice isoform can function during an early stage of T cell development normally dependent on TCR beta-chain expression. We also demonstrate that T hybridoma-derived cells expressing a TCRbeta-Cbeta0 isoform together with the clonally associated TCR alpha-chain recognize the same cognate peptide-MHC ligand as the corresponding normal alphabetaTCR. This maintenance of receptor function and specificity upon insertion of the Cbeta0 peptide cassette signifies a remarkable adaptability for the TCR beta-chain, and our findings open the possibility that this splice isoform may function in vivo.

Effects of fou8/fry1 mutation on sulfur metabolism: is decreased internal sulfate the trigger of sulfate starvation response?

The fou8 loss of function allele of adenosine bisphosphate phosphatase FIERY1 results in numerous phenotypes including the increased enzymatic oxygenation of fatty acids and increased jasmonate synthesis. Here we show that the mutation causes also profound alterations of sulfur metabolism. The fou8 mutants possess lower levels of sulfated secondary compounds, glucosinolates, and accumulate the desulfo-precursors similar to previously described mutants in adenosine 5'phosphosulfate kinase. Transcript levels of genes involved in sulfate assimilation differ in fou8 compared to wild type Col-0 plants and are similar to plants subjected to sulfate deficiency. Indeed, independent microarray analyses of various alleles of mutants in FIERY1 showed similar patterns of gene expression as in sulfate deficient plants. This was not caused by alterations in signalling, as the fou8 mutants contained significantly lower levels of sulfate and glutathione and, consequently, of total elemental sulfur. Analysis of mutants with altered levels of sulfate and glutathione confirmed the correlation of sulfate deficiency-like gene expression pattern with low internal sulfate but not low glutathione. The changes in sulfur metabolism in fou8 correlated with massive increases in 3'-phosphoadenosine 5'-phosphate levels. The analysis of fou8 thus revealed that sulfate starvation response is triggered by a decrease in internal sulfate as opposed to external sulfate availability and that the presence of desulfo-glucosinolates does not induce the glucosinolate synthesis network. However, as well as resolving these important questions on the regulation of sulfate assimilation in plants, fou8 has also opened an array of new questions on the links between jasmonate synthesis and sulfur metabolism.

Functional glycosylation of dystroglycan is crucial for thymocyte development in the mouse.

BACKGROUND: Alpha-dystroglycan (alpha-DG) is a cell surface receptor providing a molecular link between the extracellular matrix (ECM) and the actin-based cytoskeleton. During its biosynthesis, alpha-DG undergoes specific and unusual O-glycosylation crucial for its function as a high-affinity cellular receptor for ECM proteins. METHODOLOGY/PRINCIPAL FINDINGS: We report that expression of functionally glycosylated alpha-DG during thymic development is tightly regulated in developing T cells and largely confined to CD4(-)CD8(-) double negative (DN) thymocytes. Ablation of DG in T cells had no effect on proliferation, migration or effector function but did reduce the size of the thymus due to a significant loss in absolute numbers of thymocytes. While numbers of DN thymocytes appeared normal, a marked reduction in CD4(+)CD8(+) double positive (DP) thymocytes occurred. In the periphery mature naïve T cells deficient in DG showed both normal proliferation in response to allogeneic cells and normal migration, effector and memory T cell function when tested in acute infection of mice with either lymphocytic choriomeningitis virus (LCMV) or influenza virus. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that DG function is modulated by glycosylation during T cell development in vivo and that DG is essential for normal development and differentiation of T cells.

Induction of macrophage nitric oxide production by interferon-gamma and tumor necrosis factor-alpha is enhanced by interleukin-10.

Interleukin-10 (IL-10) has been reported to inhibit nitric oxide (NO) synthesis and microbicidal activity of interferon-gamma (IFN-gamma)-stimulated macrophages (M phi) by preventing the secretion of tumor necrosis factor-alpha (TNF-alpha) which serves as an autocrine activating signal. We have examined the effects of recombinant IL-10 on the capacity of IFN-gamma together with exogenous TNF-alpha to induce NO synthesis by bone marrow-derived M phi. Under these conditions and in contrast to its reported deactivating potential, IL-10 strongly enhanced NO synthesis measured as nitrite (NO2-) release (half maximal stimulation at approximately 10 U/ml). IL-10 further increased NO2- production by M phi stimulated in the presence of optimal concentrations of prostaglandin E2, a positive modulator of M phi activation by IFN-gamma/TNF-alpha. Increased steady state levels of NO synthase mRNA were observed in 4-h IFN-gamma/TNF-alpha cultures and enhanced NO2(-)-release was evident 24 h but not 48 h after stimulation. These results suggest that the effects of IL-10 on M phi function are more complex than previously recognized.

Evaluation of ammonia volatilization losses by adjusted parameters of a logistic function

The dynamics of N losses in fertilizer by ammonia volatilization is affected by several factors, making investigation of these dynamics more complex. Moreover, some features of the behavior of the variable can lead to deviation from normal distribution, making the main commonly adopted statistical strategies inadequate for data analysis. Thus, the purpose of this study was to evaluate the patterns of cumulative N losses from urea through ammonia volatilization in order to find a more adequate and detailed way of assessing the behavior of the variable. For that reason, changes in patterns of ammonia volatilization losses as a result of applying different combinations of two soil classes [Planossolo and Chernossolo (Typic Albaqualf and Vertic Argiaquolls)] and different rates of urea (50, 100 and 150 kg ha-1 N), in the presence or absence of a urease inhibitor, were evaluated, adopting a 2 × 3 × 2 factorial design with four replications. Univariate and multivariate analysis of variance were performed using the adjusted parameter values of a logistic function as a response variable. The results obtained from multivariate analysis indicated a prominent effect of the soil class factor on the set of parameters, indicating greater relevance of soil adsorption potential on ammonia volatilization losses. Univariate analysis showed that the parameters related to total N losses and rate of volatilization were more affected by soil class and the rate of urea applied. The urease inhibitor affected only the rate and inflection point parameters, decreasing the rate of losses and delaying the beginning of the process, but had no effect on total ammonia losses. Patterns of ammonia volatilization losses provide details on behavior of the variable, details which can be used to develop and adopt more accurate techniques for more efficient use of urea.

In defense of the "tunneling" wave function of the universe

The tunneling approach to the wave function of the Universe has been recently criticized by Bousso and Hawking who claim that it predicts a catastrophic instability of de Sitter space with respect to pair production of black holes. We show that this claim is unfounded. First, we argue that different horizon size regions in de Sitter space cannot be treated as independently created, as they contend. And second, the WKB tunneling wave function is not simply the inverse of the Hartle-Hawking one, except in very special cases. Applied to the related problem of pair production of massive particles, we argue that the tunneling wave function leads to a small constant production rate, and not to a catastrophe as the argument of Bousso and Hawking would suggest.

Preoperative assessment of neurovesical function in children with anorectal malformation: association with vertebral and spinal malformations.

PURPOSE: We preoperatively assessed neurovesical function and spinal cord function in children with anorectal malformations. In cases of neurovesical dysfunction we looked for an association with vertebral malformation or myelodysplasia. MATERIALS AND METHODS: We prospectively evaluated 80 children with anorectal malformations via preoperative urodynamics and magnetic resonance imaging of the spine. Bladder compliance and volume, detrusor activity and vesicosphincteric synergy during voiding allowed urodynamic evaluation. Results were reported according to Wingspread and Krickenbeck classifications of anorectal malformations. RESULTS: Urodynamic findings were pathological in 14 children (18%). Pathological evaluations did not seem related to type of fistula or level of anorectal malformation. Vertebral anomalies were seen in 34 patients (43%) and myelodysplasia in 16 (20%). Neither vertebral anomaly nor myelodysplasia seemed associated with type of fistula or severity of anorectal malformation. Of 14 children with pathological urodynamics no vertebral anomaly or myelodysplasia was found in 7. Of 66 children with normal urodynamics 40 presented with vertebral or spinal malformation. CONCLUSIONS: Lower urinary tract dysfunction is common in patients with anorectal malformations. Normal spine or spinal cord does not exclude neurovesical dysfunction. Myelodysplasia or vertebral anomaly does not determine lower urinary tract dysfunction. Thus, we recommend preoperative urodynamic assessment of the bladder and magnetic resonance imaging of the spine in children with anorectal malformations.