Transcriptional regulation of CD4 gene expression by T cell factor-1/beta-catenin pathway.

By interacting with MHC class II molecules, CD4 facilitates lineage development as well as activation of Th cells. Expression of physiological levels of CD4 requires a proximal CD4 enhancer to stimulate basic CD4 promoter activity. T cell factor (TCF)-1/beta-catenin pathway has previously been shown to regulate thymocyte survival via up-regulating antiapoptotic molecule Bcl-xL. By both loss and gain of function studies, in this study we show additional function of TCF-1/beta-catenin pathway in the regulation of CD4 expression in vivo. Mice deficient in TCF-1 displayed significantly reduced protein and mRNA levels of CD4 in CD4+ CD8+ double-positive (DP) thymocytes. A transgene encoding Bcl-2 restored survival but not CD4 levels of TCF-1(-/-) DP cells. Thus, TCF-1-regulated survival and CD4 expression are two separate events. In contrast, CD4 levels were restored on DP TCF-1(-/-) cells by transgenic expression of a wild-type TCF-1, but not a truncated TCF-1 that lacks a domain required for interacting with beta-catenin. Furthermore, forced expression of a stabilized beta-catenin, a coactivator of TCF-1, resulted in up-regulation of CD4. TCF-1 or stabilized beta-catenin greatly stimulated activity of a CD4 reporter gene driven by a basic CD4 promoter and the CD4 enhancer. However, mutation of a potential TCF binding site located within the enhancer abrogated TCF-1 and beta-catenin-mediated activation of CD4 reporter. Finally, recruitment of TCF-1 to CD4 enhancer was detected in wild-type but not TCF-1 null mice by chromatin-immunoprecipitation analysis. Thus, our results demonstrated that TCF/beta-catenin pathway enhances CD4 expression in vivo by recruiting TCF-1 to stimulate CD4 enhancer activity.

Endothelial Cell-Derived Nitric Oxide Enhances Aerobic Glycolysis in Astrocytes via HIF-1α-Mediated Target Gene Activation.

Astrocytes exhibit a prominent glycolytic activity, but whether such a metabolic profile is influenced by intercellular communication is unknown. Treatment of primary cultures of mouse cortical astrocytes with the nitric oxide (NO) donor DetaNONOate induced a time-dependent enhancement in the expression of genes encoding various glycolytic enzymes as well as transporters for glucose and lactate. Such an effect was shown to be dependent on the hypoxia-inducible factor HIF-1α, which is stabilized and translocated to the nucleus to exert its transcriptional regulation. NO action was dependent on both the PI3K/Akt/mTOR and MEK signaling pathways and required the activation of COX, but was independent of the soluble guanylate cyclase pathway. Furthermore, as a consequence of NO treatment, an enhanced lactate production and release by astrocytes was evidenced, which was prevented by downregulating HIF-1α. Several brain cell types represent possible sources of NO. It was found that endothelial cells, which express the endothelial NO synthase (eNOS) isoform, constitutively produced the largest amount of NO in culture. When astrocytes were cocultured with primary cultures of brain vascular endothelial cells, stabilization of HIF-1α and an enhancement in glucose transporter-1, hexokinase-2, and monocarboxylate transporter-4 expression as well as increased lactate production was found in astrocytes. This effect was inhibited by the NOS inhibitor l-NAME and was not seen when astrocytes were cocultured with primary cultures of cortical neurons. Our findings suggest that endothelial cell-derived NO participates to the maintenance of a high glycolytic activity in astrocytes mediated by astrocytic HIF-1α activation.

Urinary oxalate and urate to creatinine ratios in a healthy pediatric population.

The purpose of the study was to determine reference percentiles for the urinary (U) oxalate (Ox) and urate (Ura) to creatinine (Cr) concentration ratios in the second morning urine of healthy infants, children, and adolescents. The urinary oxalate and urate to creatinine ratios were determined in the spontaneously voided second morning urine sample. To test reproducibility, two urine samples were analyzed on 2 consecutive weeks in 63% of the subjects. Three hundred eighty-four healthy children (181 girls, 203 boys), aged 1 month to 17 years, from nurseries, kindergartens, and schools of Lausanne, Switzerland, were studied. The 5th and 95th percentiles were determined from the total number of urine samples (627) after confirmation that there was no order effect between repeated measurements and there were no significant sex differences. A nonlinear regression analysis in terms of age was used to smooth the calculated percentiles. In this manner, curves were obtained from which the reference values can be read at any given age. The 95th percentiles decreased with age: for UOx/Cr from 0.175 mg/mg (0.22 mol/mol) at 1 to 6 months to 0.048 mg/mg (0.06 mol/mol) from 7 years and beyond; and UUra/Cr from 2.378 mg/mg (1.6 mol/mol) at 1 to 6 months to 0.594 mg/mg (0.4 mol/mol) in adolescence. We provide 5th and 95th percentile curves for the UOx/Cr and UUra/Cr ratios determined from the second morning urine samples in a large cohort of healthy infants, children, and adolescents. Values were determined by standard analytical chemical techniques and were analyzed by powerful statistical methods. The calculated 95th percentile for the UOx/Cr values fell rather rapidly and reached normal adult values by the age of 7 years, whereas for UUra/Cr, the 95th percentile decreased slowly and stabilized in adolescence.

Identification of MAGI1 as a tumor-suppressor protein induced by cyclooxygenase-2 inhibitors in colorectal cancer cells.

Cyclooxyganase-2 (COX-2), a rate-limiting enzyme in the prostaglandin synthesis pathway, is overexpressed in many cancers and contributes to cancer progression through tumor cell-autonomous and paracrine effects. Regular use of non-steroidal anti-inflammatory drugs or selective COX-2 inhibitors (COXIBs) reduces the risk of cancer development and progression, in particular of the colon. The COXIB celecoxib is approved for adjunct therapy in patients with Familial adenomatous polyposis at high risk for colorectal cancer (CRC) formation. Long-term use of COXIBs, however, is associated with potentially severe cardiovascular complications, which hampers their broader use as preventive anticancer agents. In an effort to better understand the tumor-suppressive mechanisms of COXIBs, we identified MAGUK with Inverted domain structure-1 (MAGI1), a scaffolding protein implicated in the stabilization of adherens junctions, as a gene upregulated by COXIB in CRC cells and acting as tumor suppressor. Overexpression of MAGI1 in CRC cell lines SW480 and HCT116 induced an epithelial-like morphology; stabilized E-cadherin and β-catenin localization at cell-cell junctions; enhanced actin stress fiber and focal adhesion formation; increased cell adhesion to matrix proteins and suppressed Wnt signaling, anchorage-independent growth, migration and invasion in vitro. Conversely, MAGI1 silencing decreased E-cadherin and β-catenin localization at cell-cell junctions; disrupted actin stress fiber and focal adhesion formation; and enhanced Wnt signaling, anchorage-independent growth, migration and invasion in vitro. MAGI1 overexpression suppressed SW480 and HCT116 subcutaneous primary tumor growth, attenuated primary tumor growth and spontaneous lung metastasis in an orthotopic model of CRC, and decreased the number and size of metastatic nodules in an experimental model of lung metastasis. Collectively, these results identify MAG1 as a COXIB-induced inhibitor of the Wnt/β-catenin signaling pathway, with tumor-suppressive and anti-metastatic activity in experimental colon cancer.

Analysis of a finite volume element method for the Stokes problem

In this paper we propose a stabilized conforming finite volume element method for the Stokes equations. On stating the convergence of the method, optimal a priori error estimates in different norms are obtained by establishing the adequate connection between the finite volume and stabilized finite element formulations. A superconvergence result is also derived by using a postprocessing projection method. In particular, the stabilization of the continuous lowest equal order pair finite volume element discretization is achieved by enriching the velocity space with local functions that do not necessarily vanish on the element boundaries. Finally, some numerical experiments that confirm the predicted behavior of the method are provided.

Rapidly fatal poisoning with an insecticide containing rotenone.

Introduction: Rotenone is a botanical pesticide derived from extracts of Derris roots, which is traditionally used as piscicide, but also as an industrial insecticide for home gardens. Its mechanism of action is potent inhibition of mitochondrial respiratory chain by uncoupling oxidative phosphorylation by blocking electron transport at complex-I. Despite its classification as mild to moderately toxic to humans (estimated LD50, 300-500 mg/kg), there is a striking variety of acute toxicity of rotenone depending on the formulation (solvents). Human fatalities with rotenone-containing insecticides have been rarely reported, and a rapid deterioration within a few hours of the ingestion has been described previously in one case. Case report: A 49-year-old Tamil man with a history of asthma, ingested 250 mL of an insecticide containing 1.24% of rotenone (3.125 g, 52.1-62.5 mg/kg) in a suicide attempt at home. The product was not labeled as toxic. One hour later, he vomited repeatedly and emergency services were alerted. He was found unconscious with irregular respiration and was intubated. On arrival at the emergency department, he was comatose (GCS 3) with fixed and dilated pupils, and absent corneal reflexes. Physical examination revealed hemodynamic instability with hypotension (55/30 mmHg) and bradycardia (52 bpm). Significant laboratory findings were lactic acidosis (pH 6.97, lactate 17 mmol/L) and hypokalemia (2 mmol/L). Cranial computed tomography (CT) showed early cerebral edema. A single dose of activated charcoal was given. Intravenous hydration, ephedrine, repeated boli of dobutamine, and a perfusor with 90 micrograms/h norepinephine stabilized blood pressure temporarily. Atropine had a minimal effect on heart rate (58 bpm). Intravenous lipid emulsion was considered (log Pow 4.1), but there was a rapid deterioration with refractory hypotension and acute circulatory failure. The patient died 5h after ingestion of the insecticide. No autopsy was performed. Quantitative analysis of serum performed by high-resolution/accurate mass-mass spectrometry and liquid chromatography (LC-HR/AM-MS): 560 ng/mL rotenone. Other substances were excluded by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS/MS). Conclusion: The clinical course was characterized by early severe symptoms and a rapidly fatal evolution, compatible with inhibition of mitochondrial energy supply. Although rotenone is classified as mild to moderately toxic, physicians must be aware that suicidal ingestion of emulsified concentrates may be rapidly fatal. (n=3): stridor, cyanosis, cough (one each). Local swelling after chewing or swallowing soap developed at the earliest after 20 minutes and persisted beyond 24 hours in some cases. Treatment with antihistamines and/or steroids relieved the symptoms in 9 cases. Conclusion: Bar soap ingestion by seniors carries a risk of severe local reactions. Half the patients developed symptoms, predominantly swellings of tongue and/or lips (38%). Cognitive impairment, particularly in the cases of dementia (37%), may increase the risk of unintentional ingestion. Chewing and intraoral retention of soap leads to prolonged contact with the mucosal membranes. Age-associated physiological changes of oral mucosa probably promote the irritant effects of the surfactants. Medical treatment with antihistamines and corticosteroids usually leads to rapid decline of symptoms. Without treatment, there may be a risk of airway obstruction.

Physicians' estimates of the 10 year cardiovascular risk in hypertensive patients: an evaluation in primary care physicians in training.

To evaluate how young physicians in training perceive their patients' cardiovascular risk based on the medical charts and their clinical judgment. Cross sectional observational study. University outpatient clinic, Lausanne, Switzerland. Two hundred hypertensive patients and 50 non-hypertensive patients with at least one cardiovascular risk factor. Comparison of the absolute 10-year cardiovascular risk calculated by a computer program based on the Framingham score and adapted for physicians by the WHO/ISH with the perceived risk as assessed clinically by the physicians. Physicians underestimated the 10-year cardiovascular risk of their patients compared to that calculated with the Framingham score. Concordance between methods was 39% for hypertensive patients and 30% for non-hypertensive patients. Underestimation of cardiovascular risks for hypertensive patients was related to the fact they had a stabilized systolic blood pressure under 140 mm Hg (OR = 2.1 [1.1; 4.1]). These data show that young physicians in training often have an incorrect perception of the cardiovascular risk of their patients with a tendency to underestimate the risk. However, the calculated risk could also be slightly overestimated when applying the Framingham Heart Study model to a Swiss population. To implement a systematic evaluation of risk factors in primary care a greater emphasis should be placed on the teaching of cardiovascular risk evaluation and on the implementation of quality improvement programs.

Gastric banding induces negative bone remodelling in the absence of secondary hyperparathyroidism: potential role of serum C telopeptides for follow-up.

OBJECTIVE: Data about the consequences of laparoscopic adjustable gastric banding (LAGB) on phospho-calcic and bone metabolism remain scarce. SUBJECTS: We studied a group of 37 obese premenopausal women (age: 24-52 y; mean BMI = 43.7 kg/m2) who underwent LAGB. METHODS: Serum calcium, phosphate, alkaline phosphatase, parathormone (PTH), vitamin D3, serum C-telopeptides, IGFBP-3 and IGF-1 were measured at baseline, 6, 12, 18 and 24 months after surgery. Body composition, bone mineral content (BMC) and density (BMD) were measured using dual-X-ray absorptiometry (DXA) at baseline, 6, 12 and 24 months after surgery. RESULTS: There was no clinically significant decrease of calcemia; PTH remained stable. Serum telopeptides increased by 100% (P < 0.001) and serum IGFBP-3 decreased by 16% (P < 0.001) during the first 6 months, and then stabilized, whereas IGF-1 remained stable over the 2 y. BMC and BMD decreased, especially at the femoral neck; this decrease was significantly correlated with the decrease of waist and hip circumference. CONCLUSIONS: We concluded that there was no evidence of secondary hyperparathyroidism 24 months after LAGB. The observed bone resorption could be linked to the decrease of IGFBP-3, although this decrease could be attributable to other confounding factors. Serum telopeptides seem to be a reliable marker of bone metabolism after gastric banding. DXA must be interpreted cautiously during major weight loss, because of the artefacts caused by the important variation of fat tissue after LAGB.

Functional and transcriptional induction of aquaporin-1 gene by hypoxia; analysis of promoter and role of Hif-1α.

Aquaporin-1 (AQP1) is a water channel that is highly expressed in tissues with rapid O(2) transport. It has been reported that this protein contributes to gas permeation (CO(2), NO and O(2)) through the plasma membrane. We show that hypoxia increases Aqp1 mRNA and protein levels in tissues, namely mouse brain and lung, and in cultured cells, the 9L glioma cell line. Stopped-flow light-scattering experiments confirmed an increase in the water permeability of 9L cells exposed to hypoxia, supporting the view that hypoxic Aqp1 up-regulation has a functional role. To investigate the molecular mechanisms underlying this regulatory process, transcriptional regulation was studied by transient transfections of mouse endothelial cells with a 1297 bp 5' proximal Aqp1 promoter-luciferase construct. Incubation in hypoxia produced a dose- and time-dependent induction of luciferase activity that was also obtained after treatments with hypoxia mimetics (DMOG and CoCl(2)) and by overexpressing stabilized mutated forms of HIF-1α. Single mutations or full deletions of the three putative HIF binding domains present in the Aqp1 promoter partially reduced its responsiveness to hypoxia, and transfection with Hif-1α siRNA decreased the in vitro hypoxia induction of Aqp1 mRNA and protein levels. Our results indicate that HIF-1α participates in the hypoxic induction of AQP1. However, we also demonstrate that the activation of Aqp1 promoter by hypoxia is complex and multifactorial and suggest that besides HIF-1α other transcription factors might contribute to this regulatory process. These data provide a conceptual framework to support future research on the involvement of AQP1 in a range of pathophysiological conditions, including edema, tumor growth, and respiratory diseases.

The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA

The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3'UTR prompted studies to investigate if this transcript associated with HuR. The interaction between HuR and DNMT3b mRNA was studied by immunoprecipitation of endogenous HuR ribonucleoprotein complexes followed by RT-qPCR detection of DNMT3b mRNA, and by in vitro pulldown of biotinylated DNMT3b RNAs followed by western blotting detection of HuR. These studies revealed that binding of HuR stabilized the DNMT3b mRNA and increased DNMT3b expression. Unexpectedly, cisplatin treatment triggered the dissociation of the [HuR-DNMT3b mRNA] complex, in turn promoting DNMT3b mRNA decay, decreasing DNMT3b abundance, and lowering the methylation of repeated sequences and global DNA methylation. In summary, our data identify DNMT3b mRNA as a novel HuR target, present evidence that HuR affects DNMT3b expression levels post-transcriptionally, and reveal the functional consequences of the HuR-regulated DNMT3b upon DNA methylation patterns.

Nanopartícul·les core-shell estabilitzades en polímer amb propietats calítiques i bactericides medioambientalment segure

Aquest projecte de doctorat és un treball interdisciplinari adreçat a l’obtenció de nous nanocompòsits (NCs) funcionals sintetitzats a partir de materials polimèrics bescanviadors d’ions que són modificats amb nanopartícules metàl•liques (NPMs) de diferent composició. Els materials desenvolupats s’avaluen en funció de dues possibles aplicacions: 1) com a catalitzadors de reaccions orgàniques d’interès actual (NCs basats en pal•ladi) i, 2) la seva dedicació a aplicacions bactericides en el tractament d’aigües domèstiques o industrials (NCs basats en plata). El desenvolupament de nanomaterials és de gran interès a l’actualitat donades les seves especials propietats, l’aprofitament de les quals és la força impulsora per a la fabricació de nous NCs. Les nanopartícules metàl•liques estabilitzades en polímer (Polymer Stabilized Metal Nanoparticles, PSNPM) s’han preparat mitjançant la tècnica in-situ de síntesi intermatricial (Inter-matrix synthesis, IMS) que consisteix en la càrrega seqüencial dels grups funcionals de les matrius polimèriques amb ions metàl•lics, i la seva posterior reducció química dins de la matriu polimèrica de bescanvi iònic. L’estabilització en matrius polimèriques evita l’agregació entre elles (self-aggreagtion), un dels principals problemes coneguts de les NPs. Pel desenvolupament d’aquesta metodologia, s’han emprat diferents tipus de matrius polimèriques de bescanvi iònic: membrana Sulfonated PolyEtherEtherKetone, SPEEK, així com fibres sintètiques basades en polypropilè amb diferents tipus de grups funcionals, que ens permeten el seu ús com a filtres en la desinfecció de solucions aquoses o com a material catalitzador. Durant el projecte s’ha anat avançant en l’optimització del material nanocomposite final per a les aplicacions d’interès, en quant activitat i funcionalitat de les nanopartícules i estabilitat del nanocomposite. Així, s’ha optimitzat la síntesi de NPs estabilitzades en resines de bescanvi iònic, realitzant un screening de diferents tipus de resines i la seva avaluació en aplicacions industrials d’interès.

Quantization of energy and writhe in self-repelling knots

Probably the most natural energy functional to be considered for knotted strings is that given by electrostatic repulsion. In the absence of counter-charges, a charged, knotted string evolving along the energy gradient of electrostatic repulsion would progressively tighten its knotted domain into a point on a perfectly circular string. However, in the presence of charge screening self-repelling knotted strings can be stabilized. It is known that energy functionals in which repulsive forces between repelling charges grow inversely proportionally to the third or higher power of their relative distance stabilize self-repelling knots. Especially interesting is the case of the third power since the repulsive energy becomes scale invariant and does not change upon Mobius transformations (reflections in spheres) of knotted trajectories. We observe here that knots minimizing their repulsive Mobius energy show quantization of the energy and writhe (measure of chirality) within several tested families of knots.

Artrodesis de tobillo: resultados comparativos de diferentes técnicas

INTRODUCTION: Ankle arthrodesis in an intervention that may be performed using a variety of surgical approaches. The joint may be stabilized using diferent systems, with or without bone graft. MATERIAL AND METHODS: Retrospective study that collected the results of 40 patients who underwent ankle arthrodesis; the complications that arose were analyzed, results according to the AOFAS clinical rating system, global patient satisfaction and the relationship with the articular approach route, the !xation system or the usage of grafts.RESULTS: 55% of the patients presented one or more complication that motivated a total of 12 re-interventions in 10 patients (excludes removal of osteosynthesis material). The absence of consolidation, which required a new arthrodesis was more frequent in patients where osteosynthesis was not used (two out of three cases) than in those using screws (four out of 24 cases), while there was no register of this in 13 patients in which intramedullary pin. The use of the distal extreme of the fibula or the iliac crest as bone graft has failed to improve the consolidation. Arthroscopic approaches avoid the appearance of soft tissue complications, although the consolidation disorders were more frequent. The average score on the AOFAS clinical rating system was 56.4±21.2 and 55% of the patients were satisfied. CONCLUSIONS: Although the case histories fail to provide significant results, it is a demanding intervention, with an elevated complication rate. Intramedullary pins seem to favor improved consolidation.

Age-Related Macular Degeneration: Clinical Findings following Treatment with Antiangiogenic Drugs.

Purpose. To survey the management of patients with neovascular age-related macular degeneration (nvAMD) in Spain. Methods. An observational retrospective multicenter study was conducted. The variables analyzed were sociodemographic characteristics, foveal and macular thickness, visual acuity (VA), type of treatment, number of injections, and the initial administration of a loading dose of an antiangiogenic drug. Results. 208 patients were followed up during 23.4 months in average. During the first and second years, patients received a mean of 4.5 ± 1.8 and 1.6 ± 2.1 injections of antiangiogenic drugs, and 5.4 ± 2.8 and 3.6 ± 2.2 follow-up visits were performed, respectively. The highest improvement in VA was observed at 3 months of follow-up, followed by a decrease in the response that stabilized above baseline values until the end of the study. Patients who received an initial loading dose presented greater VA gains than those without. Conclusions. Our results suggest the need for a more standardized approach in the management and diagnosis of nvAMD receiving VEGF inhibitors. To achieve the visual outcomes reported in pivotal trials, an early diagnosis, proactive approach (more treating than follow-up visits), and a close monitoring might be the key to successfully manage nvAMD.

The N-terminal DNA-binding 'zinc finger' of the oestrogen and glucocorticoid receptors determines target gene specificity.

Steroid hormone receptors activate specific gene transcription by binding as hormone-receptor complexes to short DNA enhancer-like elements termed hormone response elements (HREs). We have shown previously that a highly conserved 66 amino acid region of the oestrogen (ER) and glucocorticoid (GR) receptors, which corresponds to part of the receptor DNA binding domain (region C) is responsible for determining the specificity of target gene activation. This region contains two sub-regions (CI and CII) analogous to the 'zinc-fingers' of the transcription factor TFIIIA. We show here that CI and CII appear to be separate domains both involved in DNA binding. Furthermore, using chimaeric ERs in which either the first (N-terminal) (CI) or second (CII) 'zinc finger' region has been exchanged with that of the GR, indicates that it is the first 'zinc finger' which largely determines target gene specificity. We suggest that receptor recognition of the HRE is analogous to that of the helix-turn-helix DNA binding motif in that the receptor binds to DNA as a dimer with the first 'zinc finger' lying in the major groove recognizing one half of the palindromic HRE, and that protein-DNA interaction is stabilized through non-specific DNA binding and dimer interactions contributed by the second 'zinc finger'.