Neuregulin 4 Downregulation Induces Insulin Resistance in 3T3-L1 Adipocytes through Inflammation and Autophagic Degradation of GLUT4 Vesicles.

The adipokine Neuregulin 4 (Nrg4) protects against obesity-induced insulin resistance. Here, we analyze how the downregulation of Nrg4 influences insulin action and the underlying mechanisms in adipocytes. Validated shRNA lentiviral vectors were used to generate scramble (Scr) and Nrg4 knockdown (KD) 3T3-L1 adipocytes. Adipogenesis was unaffected in Nrg4 KD adipocytes, but there was a complete impairment of the insulin-induced 2-deoxyglucose uptake, which was likely the result of reduced insulin receptor and Glut4 protein. Downregulation of Nrg4 enhanced the expression of proinflammatory cytokines. Anti-inflammatory agents recovered the insulin receptor, but not Glut4, content. Proteins enriched in Glut4 storage vesicles such as the insulin-responsive aminopeptidase (IRAP) and Syntaxin-6 as well as TBC1D4, a protein involved in the intracellular retention of Glut4 vesicles, also decreased by Nrg4 KD. Insulin failed to reduce autophagy in Nrg4 KD adipocytes, observed by a minor effect on mTOR phosphorylation, at the time that proteins involved in autophagy such as LC3-II, Rab11, and Clathrin were markedly upregulated. The lysosomal activity inhibitor bafilomycin A1 restored Glut4, IRAP, Syntaxin-6, and TBC1D4 content to those found in control adipocytes. Our study reveals that Nrg4 preserves the insulin responsiveness by preventing inflammation and, in turn, benefits the insulin regulation of autophagy.

The timing and spatiotemporal patterning of Neanderthal disappearance.

The timing of Neanderthal disappearance and the extent to which they overlapped with the earliest incoming anatomically modern humans (AMHs) in Eurasia are key questions in palaeoanthropology. Determining the spatiotemporal relationship between the two populations is crucial if we are to understand the processes, timing and reasons leading to the disappearance of Neanderthals and the likelihood of cultural and genetic exchange. Serious technical challenges, however, have hindered reliable dating of the period, as the radiocarbon method reaches its limit at ∼50,000 years ago. Here we apply improved accelerator mass spectrometry (14)C techniques to construct robust chronologies from 40 key Mousterian and Neanderthal archaeological sites, ranging from Russia to Spain. Bayesian age modelling was used to generate probability distribution functions to determine the latest appearance date. We show that the Mousterian ended by 41,030-39,260 calibrated years bp (at 95.4% probability) across Europe. We also demonstrate that succeeding 'transitional' archaeological industries, one of which has been linked with Neanderthals (Châtelperronian), end at a similar time. Our data indicate that the disappearance of Neanderthals occurred at different times in different regions. Comparing the data with results obtained from the earliest dated AMH sites in Europe, associated with the Uluzzian technocomplex, allows us to quantify the temporal overlap between the two human groups. The results reveal a significant overlap of 2,600-5,400 years (at 95.4% probability). This has important implications for models seeking to explain the cultural, technological and biological elements involved in the replacement of Neanderthals by AMHs. A mosaic of populations in Europe during the Middle to Upper Palaeolithic transition suggests that there was ample time for the transmission of cultural and symbolic behaviours, as well as possible genetic exchanges, between the two groups.

Aquaglyceroporins Are Differentially Expressed in Beige and White Adipocytes.

Browning of white adipocytes has been proposed as a powerful strategy to overcome metabolic complications, since brown adipocytes are more catabolic, expending energy as a heat form. However, the biological pathways involved in the browning process are still unclear. Aquaglyceroporins are a sub-class of aquaporin water channels that also permeate glycerol and are involved in body energy homeostasis. In the adipose tissue, aquaporin-7 (AQP7) is the most representative isoform, being crucial for white adipocyte fully differentiation and glycerol metabolism. The altered expression of AQP7 is involved in the onset of obesity and metabolic disorders. Herein, we investigated if aquaglyceroporins are implicated in beige adipocyte differentiation, similar to white cells. Thus, we optimized a protocol of murine 3T3-L1 preadipocytes browning that displayed increased beige and decreased white adipose tissue features at both gene and protein levels and evaluated aquaporin expression patterns along the differentiation process together with cellular lipid content. Our results revealed that AQP7 and aquaporin-9 (AQP9) expression was downregulated throughout beige adipocyte differentiation compared to white differentiation, which may be related to the beige physiological role of heat production from oxidative metabolism, contrasting with the anabolic/catabolic lipid metabolism requiring glycerol gateways occurring in white adipose cells.

Caveolar targeting links Kv1.3 with the insulin-dependent adipocyte physiology.

The voltage-dependent potassium channel Kv1.3 participates in peripheral insulin sensitivity. Genetic ablation of Kv1.3 triggers resistance to diet-induced weight gain, thereby pointing to this protein as a pharmacological target for obesity and associated type II diabetes. However, this role is under intense debate because Kv1.3 expression in adipose tissue raises controversy. We demonstrated that Kv1.3 is expressed in white adipose tissue from humans and rodents. Moreover, other channels, such as Kv1.1, Kv1.2, Kv1.4 and especially Kv1.5, from the same Shaker family are also present. Although elevated insulin levels and adipogenesis remodel the Kv phenotype, which could lead to multiple heteromeric complexes, Kv1.3 markedly participates in the insulin-dependent regulation of glucose uptake in mature adipocytes. Adipocyte differentiation increased the expression of Kv1.3, which is targeted to caveolae by molecular interactions with caveolin 1. Using a caveolin 1-deficient 3T3-L1 adipocyte cell line, we demonstrated that the localization of Kv1.3 in caveolar raft structures is important for proper insulin signaling. Insulin-dependent phosphorylation of the channel occurs at the onset of insulin-mediated signaling. However, when Kv1.3 was spatially outside of these lipid microdomains, impaired phosphorylation was exhibited. Our data shed light on the putative role of Kv1.3 in weight gain and insulin-dependent responses contributing to knowledge about adipocyte physiology.

Neuregulin improves response to glucose tolerance test in control and diabetic rats.

Neuregulin (NRG) is an EGF-related growth factor that binds to the tyrosine kinase receptors ErbB3 and ErbB4, thus inducing tissue development and muscle glucose utilization during contraction. Here, we analyzed whether NRG has systemic effects regulating glycemia in control and type 2 diabetic rats. To this end, recombinant NRG (rNRG) was injected into Zucker diabetic fatty (ZDF) rats and their respective lean littermates 15 min before a glucose tolerance test (GTT) was performed. rNRG enhanced glucose tolerance without promoting the activation of the insulin receptor (IR) or insulin receptor substrates (IRS) in muscle and liver. However, in control rats, rNRG induced the phosphorylation of protein kinase B (PKB) and glycogen synthase kinase-3 (GSK-3) in liver but not in muscle. In liver, rNRG increased ErbB3 tyrosine phosphorylation and its binding to phosphatidylinositol 3-kinase (PI3K), thus indicating that rNRG activates the ErbB3/PI3K/PKB signaling pathway. rNRG increased glycogen content in liver but not in muscle. rNRG also increased the content of fructose-2,6-bisphosphate (Fru-2,6-P2), an activator of hepatic glycolysis, and lactate in liver but not in muscle. Increases in lactate were abrogated by wortmannin, a PI3K inhibitor, in incubated hepatocytes. The liver of ZDF rats showed a reduced content of ErbB3 receptors, entailing a minor stimulation of the rNRG-induced PKB/GSK-3 cascade and resulting in unaltered hepatic glycogen content. Nonetheless, rNRG increased hepatic Fru-2,6-P2 and augmented lactate both in liver and in plasma of diabetic rats. As a whole, rNRG improved response to the GTT in both control and diabetic rats by enhancing hepatic glucose utilization.

Lipopolysaccharide binding protein is an adipokine involved in the resilience of the mouse adipocyte to inflammation.

AIMS/HYPOTHESIS: Lipopolysaccharide (LPS) binding protein (LBP) is a novel 65 kDa adipokine, linked to adipose tissue (AT) inflammation, obesity and insulin resistance, that inhibits adipocyte differentiation. Here, we investigated the molecular mechanisms behind these detrimental effects on adipogenesis through whole-genome transcriptomics and in vitro experiments. METHODS: Permanent and transient knockdown (KD) and co-culture experiments were performed in 3T3-L1 and 3T3-F442A cell lines during adipocyte differentiation. Microarray gene expression was performed using Genechip Affymetrix technology and validated by real-time PCR. RESULTS: LBP KD of 3T3-L1 cells led to a potentiated adipocyte differentiation with a dose-response relationship; genes involved in mitochondrial biogenesis, fatty acid metabolism and peroxisome proliferator-activated receptor γ (PPAR-γ) action were dramatically upregulated in parallel to increased insulin signalling. Cells with LBP KD became refractory to proinflammatory cytokines and other inflammatory stimuli (LPS and palmitate). This phenotype, mediated through disrupted nuclear factor κB (NFκB) signalling, was reversed by a soluble factor present in a co-culture with native cells and by exogenous LBP. Double-silencing of LBP and toll-like receptor 4 (TLR4) again rendered these cells insensitive to co-culture, LBP and inflammatory factors. CONCLUSIONS/INTERPRETATION: In summary, LBP is a proinflammatory soluble adipokine that acts as a brake for adipogenesis, strengthening the negative effects of palmitate and LPS on adipocyte differentiation.

A gold coordination compound as a chemical probe to unravel aquaporin-7 function.

Aquaporins (AQPs) are membrane water/glycerol channels that are involved in many physiological functions. Aquaporin-based modulators are predicted to have potential utility in the treatment of several diseases, as well as chemical tools to assess AQPs function in biological systems. We recently reported gold(III) compounds as human AQP3 inhibitors, with Auphen as the most potent of the series. In this work, we assessed the modulation of aquaporin-7 (AQP7) expressed in an adipocyte cell model and show that Auphen significantly inhibits mouse and human AQP7. By homology modeling and molecular docking it was possible to identify the thioether groups of methionine residues, in particular Met47, as likely candidates for binding to the gold(III) complex. Our data point to Auphen as a useful chemical tool to detect AQP7 function. It might constitute a basis to develop inhibitors with improved affinity towards different aquaglyceroporin isoforms.

A role for adipocyte-derived lipopolysaccharide-binding protein in inflammation- and obesity-associated adipose tissue dysfunction.

AIMS/HYPOTHESIS: Circulating lipopolysaccharide-binding protein (LBP) is an acute-phase reactant known to be increased in obesity. We hypothesised that LBP is produced by adipose tissue (AT) in association with obesity. METHODS: LBP mRNA and LBP protein levels were analysed in AT from three cross-sectional (n = 210, n = 144 and n = 28) and three longitudinal (n = 8, n = 25, n = 20) human cohorts; in AT from genetically manipulated mice; in isolated adipocytes; and in human and murine cell lines. The effects of a high-fat diet and exposure to lipopolysaccharide (LPS) and peroxisome proliferator-activated receptor (PPAR)γ agonist were explored. Functional in vitro and ex vivo experiments were also performed. RESULTS: LBP synthesis and release was demonstrated to increase with adipocyte differentiation in human and mouse AT, isolated adipocytes and human and mouse cell lines (Simpson-Golabi-Behmel syndrome [SGBS], human multipotent adipose-derived stem [hMAD] and 3T3-L1 cells). AT LBP expression was robustly associated with inflammatory markers and increased with metabolic deterioration and insulin resistance in two independent cross-sectional human cohorts. AT LBP also increased longitudinally with weight gain and excessive fat accretion in both humans and mice, and decreased with weight loss (in two other independent cohorts), in humans with acquired lipodystrophy, and after ex vivo exposure to PPARγ agonist. Inflammatory agents such as LPS and TNF-α led to increased AT LBP expression in vivo in mice and in vitro, while this effect was prevented in Cd14-knockout mice. Functionally, LBP knockdown using short hairpin (sh)RNA or anti-LBP antibody led to increases in markers of adipogenesis and decreased adipocyte inflammation in human adipocytes. CONCLUSIONS/INTERPRETATION: Collectively, these findings suggest that LBP might have an essential role in inflammation- and obesity-associated AT dysfunction.

Chronology of the Middle to Upper Palaeolithic transition at Abric Romaní, Catalunya.

This paper presents new data from Abric Romaní, a key site in the region of Catalunya, northeastern Iberia, which is central to discussions of the transition between the Middle and Upper Palaeolithic in Europe. Until now, the Mid-Upper Paleolithic transition had been dated at the site through samples from the remaining baulk sections of levels A and B (typologically classified as 'earliest Aurignacian' and Mousterian, respectively) at the rear of the rockshelter, which were left from excavations in the late 1900s and early 1910s. We dated samples of bone and charcoal from these remnant sections with accelerator mass spectrometry (AMS) methods. We also analysed several humanly-modified artefacts (bone points and perforated shells) excavated from other areas of the same layers. From the initial series, we obtained ages of c. 20 ka BP (thousands of years before present); much younger than expected if they indeed dated to the early Upper Palaeolithic. We sampled additional material to test the robustness of these initial ages, and older determinations that were more comparable with the chronology outlined by Bischoff et al. (1988, 1994) resulted. All of the old and new results have been compared in a Bayesian model using the new INTCAL09 (14)C calibration dataset. The results appear to confirm the suggestion of some researchers (e.g., Zilhão and d'Errico, 1999) that there was no Aurignacian in the north of Iberia until c. 36,500 BP. The chronometric model shows a good level of agreement between the radiocarbon and U-series chronologies previously obtained, and the new results published in this paper.

The relevance of EGFR, ErbB receptors and neuregulins in human adipocytes and adipose tissue in obesity.

OBJECTIVE: To investigate the potential role of EGFR, ErbBs receptors and neuregulins in human adipose tissue physiology in obesity. METHODS: Gene expression analysis in human subcutaneous (SAT) and visceral (VAT) adipose tissue in three independent cohorts [two cross-sectional (N = 150, N = 87) and one longitudinal (n = 25)], and in vitro gene knockdown and overexpression experiments were performed. RESULTS: While both SAT and VAT ERBB2 and ERBB4 mRNA increased in obesity, SAT EGFR mRNA was negatively correlated with insulin resistance, but did not change in obesity. Of note, both SAT and VAT EGFR mRNA were significantly associated with adipogenesis and increased during human adipocyte differentiation. In vitro experiments revealed that EGFR, but not ERBB2 and ERBB4, gene knockdown in preadipocytes and in fully differentiated human adipocytes resulted in decreased expression of adipogenic-related genes. ERBB2 gene knockdown also reduced gene expression of fatty acid synthase in fully differentiated adipocytes. In addition, neuregulin 2 (NRG2) mRNA was associated with expression of adipogenic genes in human adipose tissue and adipocytes, and its overexpression increased expression of EGFR and relevant adipogenic genes. CONCLUSIONS: This study demonstrates the association between adipose tissue ERBB2 and obesity, confirms the relevance of EGFR on human adipogenesis, and suggests a possible adipogenic role of NRG2.

Serum neuregulin 4 is negatively correlated with insulin sensitivity in humans and impairs mitochondrial respiration in HepG2 cells.

Neuregulin 4 (NRG4) has been described to improve metabolic disturbances linked to obesity status in rodent models. The findings in humans are controversial. We aimed to investigate circulating NRG4 in association with insulin action in humans and the possible mechanisms involved. Insulin sensitivity (euglycemic hyperinsulinemic clamp) and serum NRG4 concentration (ELISA) were analysed in subjects with a wide range of adiposity (n = 89). In vitro experiments with human HepG2 cell line were also performed. Serum NRG4 was negatively correlated with insulin sensitivity (r = -0.25, p = 0.02) and positively with the inflammatory marker high-sensitivity C reative protein (hsCRP). In fact, multivariant linear regression analyses showed that insulin sensitivity contributed to BMI-, age-, sex-, and hsCRP-adjusted 7.2% of the variance in serum NRG4 (p = 0.01). No significant associations were found with adiposity measures (BMI, waist circumference or fat mass), plasma lipids (HDL-, LDL-cholesterol, or fasting triglycerides) or markers of liver injury. Cultured hepatocyte HepG2 treated with human recombinant NRG4 had an impact on hepatocyte metabolism, leading to decreased gluconeogenic- and mitochondrial biogenesis-related gene expression, and reduced mitochondrial respiration, without effects on expression of lipid metabolism-related genes. Similar but more pronounced effects were found after neuregulin 1 administration. In conclusion, sustained higher serum levels of neuregulin-4, observed in insulin resistant patients may have deleterious effects on metabolic and mitochondrial function in hepatocytes. However, findings from in vitro experiments should be confirmed in human primary hepatocytes.

Crosstalk Between LXR and Caveolin-1 Signaling Supports Cholesterol Efflux and Anti-Inflammatory Pathways in Macrophages.

Macrophages are immune cells that play crucial roles in host defense against pathogens by triggering their exceptional phagocytic and inflammatory functions. Macrophages that reside in healthy tissues also accomplish important tasks to preserve organ homeostasis, including lipid uptake/efflux or apoptotic-cell clearance. Both homeostatic and inflammatory functions of macrophages require the precise stability of lipid-rich microdomains located at the cell membrane for the initiation of downstream signaling cascades. Caveolin-1 (Cav-1) is the main protein responsible for the biogenesis of caveolae and plays an important role in vascular inflammation and atherosclerosis. The Liver X receptors (LXRs) are key transcription factors for cholesterol efflux and inflammatory gene responses in macrophages. Although the role of Cav-1 in cellular cholesterol homeostasis and vascular inflammation has been reported, the connection between LXR transcriptional activity and Cav-1 expression and function in macrophages has not been investigated. Here, using gain and loss of function approaches, we demonstrate that LXR-dependent transcriptional pathways modulate Cav-1 expression and compartmentation within the membrane during macrophage activation. As a result, Cav-1 participates in LXR-dependent cholesterol efflux and the control of inflammatory responses. Together, our data show modulation of the LXR-Cav-1 axis could be exploited to control exacerbated inflammation and cholesterol overload in the macrophage during the pathogenesis of lipid and immune disorders, such as atherosclerosis.

Neuregulin, an Effector on Mitochondria Metabolism That Preserves Insulin Sensitivity.

Various external factors modulate the metabolic efficiency of mitochondria. This review focuses on the impact of the growth factor neuregulin and its ErbB receptors on mitochondria and their relationship with several physiopathological alterations. Neuregulin is involved in the differentiation of heart, skeletal muscle, and the neuronal system, among others; and its deficiency is deleterious for the health. Information gathered over the last two decades suggests that neuregulin plays a key role in regulating the mitochondrial oxidative machinery, which sustains cell survival and insulin sensitivity.

Neuregulin 4 Is a Novel Marker of Beige Adipocyte Precursor Cells in Human Adipose Tissue.

Background: Nrg4 expression has been linked to brown adipose tissue activity and browning of white adipocytes in mice. Here, we aimed to investigate whether these observations could be translated to humans by investigating NRG4 mRNA and markers of brown/beige adipocytes in human visceral (VAT) and subcutaneous adipose tissue (SAT). We also studied the possible association of NRG4 with insulin action. Methods: SAT and VAT NRG4 and markers of brown/beige (UCP1, UCP3, and TMEM26)-related gene expression were analyzed in two independent cohorts (n = 331 and n = 59). Insulin resistance/sensitivity was measured using HOMAIR and glucose infusion rate during euglycemic hyperinsulinemic clamp. Results: In both cohort 1 and cohort 2, NRG4 and thermogenic/beige-related gene expression were significantly increased in VAT compared to SAT. Adipogenic-related genes followed an opposite pattern. In cohort 1, VAT NRG4 gene expression was positively correlated with BMI and expression of UCP1, UCP3, TMEM26, and negatively with adipogenic (FASN, PPARG, and SLC2A4)- and inflammatory (IL6 and IL8)-related genes. In SAT, NRG4 gene expression was negatively correlated with HOMAIR and positively with UCP1 and TMEM26 gene expression. Multiple linear regression analysis revealed that expression of TMEM26 gene was the best predictor of NRG4 gene expression in both VAT and SAT. Specifically, NRG4 and TMEM26 gene expression was significantly increased in VAT, but not in SAT stromal vascular fraction cells (p < 0.001). In cohort 2, the significant association between NRG4 and TMEM26 gene expression in both VAT and SAT was confirmed, and SAT NRG4 gene expression also was positively correlated with insulin action and the expression of UCP1. Conclusion: Current findings suggest NRG4 gene expression as a novel marker of beige adipocytes in human adipose tissue.

Oral insulin-mimetic compounds that act independently of insulin.

The hallmarks of insulin action are the stimulation and suppression of anabolic and catabolic responses, respectively. These responses are orchestrated by the insulin pathway and are initiated by the binding of insulin to the insulin receptor, which leads to activation of the receptor's intrinsic tyrosine kinase. Severe defects in the insulin pathway, such as in types A and B and advanced type 1 and 2 diabetes lead to severe insulin resistance, resulting in a partial or complete absence of response to exogenous insulin and other known classes of antidiabetes therapies. We have characterized a novel class of arylalkylamine vanadium salts that exert potent insulin-mimetic effects downstream of the insulin receptor in adipocytes. These compounds trigger insulin signaling, which is characterized by rapid activation of insulin receptor substrate-1, Akt, and glycogen synthase kinase-3 independent of insulin receptor phosphorylation. Administration of these compounds to animal models of diabetes lowered glycemia and normalized the plasma lipid profile. Arylalkylamine vanadium compounds also showed antidiabetic effects in severely diabetic rats with undetectable circulating insulin. These results demonstrate the feasibility of insulin-like regulation in the complete absence of insulin and downstream of the insulin receptor. This represents a novel therapeutic approach for diabetic patients with severe insulin resistance.

Prevalence of human metapneumovirus among hospitalized children younger than 1 year in Catalonia, Spain.

Human metapneumovirus was discovered recently respiratory virus implicated in both upper and lower respiratory tract infection. In children, the clinical symptoms of human metapneumovirus are similar to those produced by respiratory syncytial virus, ranging from mild to severe diseases such as bronchiolitis and pneumonia. The aim of the present study was to describe the prevalence of human metapneumovirus and other common respiratory viruses among admitted to hospital infants. From January 2006 to June 2006, 99 nasopharyngeal aspirates were collected from hospitalized children younger than 12 months in order to study respiratory viruses. Human metapneumovirus detection was performed by cell culture and two RT-PCR targeting on polymerase and fusion genes. The latter gene was used for phylogenetic analysis. In 67/99 children (67%) at least one viral pathogen was identified, the viruses detected most frequently were respiratory syncytial virus (35%), human metapneumovirus (25%) and rhinovirus (19%). The results obtained in this study, show that: (1) human metapneumovirus is one of the most important viruses among children less than 12 months; (2) children infected with human metapneumovirus were significantly older than those infected by respiratory syncytial virus; (3) human metapneumovirus was associated more frequently with pneumonia whereas respiratory syncytial virus was only detected in patients with bronchiolitis; (4) there was a clear epidemiological succession pattern with only a small overlap among the viruses detected most frequently; (5) all human metapneumovirus samples were clustered within sublineage A2.

The role of viruses in the aetiology of community-acquired pneumonia in adults.

BACKGROUND: The role of viruses in community-acquired pneumonia may have been previously underestimated. We aimed to study the incidence and clinical characteristics of community-acquired pneumonia (CAP) due to respiratory viruses in adults adding PCR to routine conventional laboratory tests. METHODS: Consecutive adult patients diagnosed of CAP from January 2003 to March 2004 were included. Conventional tests including cultures of blood, sputum, urine antigen detection of Streptococcus pneumoniae and Legionella pneumophila, and paired serologies were routinely performed. Nasopharyngeal swabs were processed for study of respiratory viruses through antigen detection by indirect immunofluorescence assay, isolation of viruses in cell culture and detection of nucleic acids by two independent multiplex RT-PCR assays. According to the aetiology, patients were categorized in 4 groups: group 1, only virus detected; group 2, only bacteria detected; group 3, viral and bacterial; and group 4, unkown aetiology. RESULTS: Of 340 patients diagnosed with CAP, 198 had nasopharyngeal swabs available and were included in this study. Aetiology was established in 112 (57%) patients: group 1, n=26 (13%); group 2, n=66 (33%); group 3, n=20 (10%). The most common aetiological agent was S. neumoniae (58 patients, 29%), followed by respiratory viruses (46 patients, 23%). Forty-eight respiratory viruses were identified: influenza virus A (n=16), respiratory syncytial virus A (n=5), adenovirus (n=8), parainfluenza viruses (n=5), enteroviruses (n=1), rhinoviruses (n=8) and coronavirus (n=5). There were two patients coinfected by two respiratory viruses. Serology detected 6 viruses, immunofluorescence 8, viral culture 12, and PCR 45. For the viruses that could be diagnosed with conventional methods, the sensitivity and specificity of RT-PCR was 85% and 92%, respectively. The only clinical characteristic that significantly distinguished viral from bacterial aetiology was a lower number of leukocytes (P=0.004). CONCLUSION: PCR revealed that viruses represent a common aetiology of CAP. There is an urgent need to reconsider routine laboratory tests for an adequate diagnosis of respiratory viruses, as clinical characteristics are unable to reliably distinguish viral from bacterial aetiology.

Teaching research methods in nursing using Aronson's Jigsaw Technique. A cross-sectional survey of student satisfaction.

BACKGROUND: To adapt nursing studies to the European Higher Education Area, new teaching methods have been included that assign maximum importance to student-centered learning and collaborative work. The Jigsaw Technique is based on collaborative learning and everyone in the group must play their part because each student's mark depends on the other students. Home group members are given the responsibility to become experts in a specific area of knowledge. Experts meet together to reach an agreement and improve skills. Finally, experts return to their home groups to share all their findings. OBJECTIVE: The aim of this study was to evaluate nursing student satisfaction with the Jigsaw Technique used in the context of a compulsory course in research methods for nursing. METHODS: A cross-sectional study was conducted using a self-administered anonymous questionnaire administered to students who completed the Research Methods course during the 2012-13 and 2013-14 academic years. The questionnaire was developed taking into account the learning objectives, competencies and skills that should be acquired by students, as described in the course syllabus. The responses were compared by age group (younger or older than 22years). RESULTS: A total of 89.6% of nursing students under 22years believed that this methodology helped them to develop teamwork, while this figure was 79.6% in older students. Nursing students also believed it helped them to work independently, with differences according to age, 79.7% and 58% respectively (p=0.010). Students disagreed with the statement "The Jigsaw Technique involves little workload", with percentages of 88.5% in the group under 22years and 80% in older students. Most believed that this method should not be employed in upcoming courses, although there were differences by age, with 44.3% of the younger group being against and 62% of the older group (p=0.037). CONCLUSION: The method was not highly valued by students, mainly by those older than 22years, who concluded that they did not learn more with it than with other traditional techniques. The results of this study question whether this form of learning meets students' learning needs and its compatibility with individual and group realities.

Caveolin interaction governs Kv1.3 lipid raft targeting.

The spatial localization of ion channels at the cell surface is crucial for their functional role. Many channels localize in lipid raft microdomains, which are enriched in cholesterol and sphingolipids. Caveolae, specific lipid rafts which concentrate caveolins, harbor signaling molecules and their targets becoming signaling platforms crucial in cell physiology. However, the molecular mechanisms involved in such spatial localization are under debate. Kv1.3 localizes in lipid rafts and participates in the immunological response. We sought to elucidate the mechanisms of Kv1.3 surface targeting, which govern leukocyte physiology. Kv1 channels share a putative caveolin-binding domain located at the intracellular N-terminal of the channel. This motif, lying close to the S1 transmembrane segment, is situated near the T1 tetramerization domain and the determinants involved in the Kvß subunit association. The highly hydrophobic domain (FQRQVWLLF) interacts with caveolin 1 targeting Kv1.3 to caveolar rafts. However, subtle variations of this cluster, putative ancillary associations and different structural conformations can impair the caveolin recognition, thereby altering channel's spatial localization. Our results identify a caveolin-binding domain in Kv1 channels and highlight the mechanisms that govern the regulation of channel surface localization during cellular processes.

Semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 activity exerts an antidiabetic action in Goto-Kakizaki rats.

In this study we have explored whether the bifunctional protein semicarbazide-sensitive amine oxidase (SSAO)/vascular adhesion protein-1 (VAP-1) represents a novel target for type 2 diabetes. To this end, Goto-Kakizaki (GK) diabetic rats were treated with the SSAO substrate benzylamine and with low ineffective doses of vanadate previously shown to have antidiabetic effects in streptozotocin-induced diabetic rats. The administration of benzylamine in combination with vanadate in type 2 diabetic rats acutely stimulated glucose tolerance, and the chronic treatment normalized hyperglycemia, stimulated glucose transport in adipocytes, and reversed muscle insulin resistance. Acute in vivo administration of benzylamine and vanadate stimulated skeletal muscle glucose transport, an effect that was also observed in incubated muscle preparations coincubated with adipose tissue explants or with human recombinant SSAO. Acute administration of benzylamine/vanadate also ameliorated insulin secretion in diabetic GK rats, and this effect was also observed in incubated pancreatic islets. In keeping with these observations, we also demonstrate that pancreatic islets express SSAO/VAP-1. As far as mechanisms of action, we have found that benzylamine/vanadate causes enhanced tyrosine phosphorylation of proteins and reduced protein tyrosine phosphatase activity in adipocytes. In addition, incubation of human recombinant SSAO, benzylamine, and vanadate generates peroxovanadium compounds in vitro. Based on these data, we propose that benzylamine/vanadate administration generates peroxovanadium locally in pancreatic islets, which stimulates insulin secretion and also produces peroxovanadium in adipose tissue, activating glucose metabolism in adipocytes and in neighboring muscle. This opens the possibility of using the SSAO/VAP-1 activity as a local generator of protein tyrosine phosphatase inhibitors in antidiabetic therapy.