Closed-state inactivation of cardiac, skeletal, and neuronal sodium channels is isoform specific.

Voltage-gated sodium (Nav) channels produce the upstroke of action potentials in excitable tissues throughout the body. The gating of these channels is determined by the asynchronous movements of four voltage-sensing domains (VSDs). Past studies on the skeletal muscle Nav1.4 channel have indicated that VSD-I, -II, and -III are sufficient for pore opening, whereas VSD-IV movement is sufficient for channel inactivation. Here, we studied the cardiac sodium channel, Nav1.5, using charge-neutralizing mutations and voltage-clamp fluorometry. Our results reveal that both VSD-III and -IV are necessary for Nav1.5 inactivation, and that steady-state inactivation can be modulated by all VSDs. We also demonstrate that channel activation is partially determined by VSD-IV movement. Kinetic modeling suggests that these observations can be explained from the cardiac channel's propensity to enter closed-state inactivation (CSI), which is significantly higher than that of other Nav channels. We show that skeletal muscle Nav1.4, cardiac Nav1.5, and neuronal Nav1.6 all have different propensities for CSI and postulate that these differences produce isoform-dependent roles for the four VSDs.

Spliced isoforms of the cardiac Nav1.5 channel modify channel activation by distinct structural mechanisms.

Alternative splicing is an important cellular mechanism that fine tunes the gating properties of both voltage- and ligand-gated ion-channels. The cardiac voltage-gated sodium channel, Nav1.5, is subject to alternative splicing of the DI S3-S4 linker, which generates two types of channels with different activation properties. Here, we show that the gating differences between the adult (mH1) and neonatal (Nav1.5e) isoforms of Nav1.5 are mediated by two amino acid residues: Thr/Ser at position 207 and Asp/Lys at position 211. Electrophysiological experiments, in conjunction with molecular dynamics simulations, revealed that each residue contributes equally to the overall gating shifts in activation, but that the underlying structural mechanisms are different. Asp/Lys at position 211 acts through electrostatic interactions, whereas Thr/Ser at position 207 is predicted to alter the hydrogen bond network at the top of the S3 helix. These distinct structural mechanisms work together to modify movement of the voltage-sensitive S4 helix to bring about channel activation. Interestingly, mutation of the homologous Asp and Thr residues of the skeletal muscle isoform, Nav1.4, to Lys and Ser, respectively, confers a similar gating shift in channel activation, suggesting that these residues may fulfill a conserved role across other Nav channel family members.

Successful treatment of SAPHO syndrome with apremilast.

SAPHO (synovitis, acne, pustulosis, hyperostosis and osteitis) syndrome is a rare disease with inflammatory osteoarticular and skin involvement. The pathogenesis of SAPHO syndrome remains unclear, but evidence suggests it may be an autoinflammatory disease triggered upon exposure to infectious agents in genetically predisposed individuals. Induction of the interleukin (IL)-23/T helper 17 axis in addition to neutrophil activation seem to play a key role, and therapies targeting these immunological pathways, including tumour necrosis factor (TNF) inhibitors, ustekinumab, secukinumab and the IL-1 inhibitor anakinra, are potential treatment options that need further investigation. Here we report a case of a 24-year-old woman with SAPHO syndrome who presented at our clinic with palmoplantar pustulosis and sternoclavicular joint involvement. Previous treatments with topical steroids and keratolytics combined with nonsteroidal anti-inflammatory drugs, intravenous methylprednisolone, methotrexate and sulfasalazine had all failed to improve symptoms. Therapy with etanercept was not tolerated, and because of a previous demyelinating peripheral neuropathy, further treatment with TNF inhibitors was avoided. We initiated ustekinumab 45 mg, which improved skin manifestations but not joint pain. Dose escalation to 90 mg initially improved joint pain, but the dose had to be reduced to 45 mg again because of increased infections. During subsequent 45-mg ustekinumab treatment, joint pain exacerbated so we switched to adalimumab which caused an exacerbation of the disease, so we switched to secukinumab, which improved skin and joint symptoms significantly but was associated with a pustular hypersensitivity reaction. Finally, we began treatment with apremilast, a pan-cytokine approach, resulting in stabilization of the skin and joint symptoms without side-effects. To our knowledge, this is the first case report of apremilast as a treatment for SAPHO syndrome.

Urban growth and water access in sub-Saharan Africa: Progress, challenges, and emerging research directions.

For the next decade, the global water crisis remains the risk of highest concern, and ranks ahead of climate change, extreme weather events, food crises and social instability. Across the globe, nearly one in ten people is without access to an improved drinking water source. Least Developed Countries (LDCs) especially in sub-Saharan Africa (SSA) are the most affected, having disproportionately more of the global population without access to clean water than other major regions. Population growth, changing lifestyles, increasing pollution and accelerating urbanization will continue to widen the gap between the demand for water and available supply especially in urban areas, and disproportionately affect informal settlements, where the majority of SSA's urban population resides. Distribution and allocation of water will be affected by climate-induced water stresses, poor institutions, ineffective governance, and weak political will to address scarcity and mediate uncertainties in future supply. While attempts have been made by many scientists to examine different dimensions of water scarcity and urban population dynamics, there are few comprehensive reviews, especially focused on the particular situation in Sub-Saharan Africa. This paper contributes to interdisciplinary understanding of urban water supply by distilling and integrating relevant empirical knowledge on urban dynamics and water issues in SSA, focusing on progress made and associated challenges. It then points out future research directions including the need to understand how alternatives to centralized water policies may help deliver sustainable water supply to cities and informal settlements in the region.

Hydroxytyrosol modulates the levels of microRNA-9 and its target sirtuin-1 thereby counteracting oxidative stress-induced chondrocyte death.

OBJECTIVE: Nutraceutical compounds, such as hydroxytyrosol (HT), have been found to exert protective effects in osteoarthritis (OA) by affecting a variety of key molecular and cellular processes in chondrocytes. However, to our knowledge, no relationship has been reported between nutraceuticals and microRNA (miR) network in OA models. Here, we identified a miR that is implicated in HT-mediated chondroprotection following oxidative stress condition by targeting sirtuin-1 (SIRT-1). METHODS: Human primary and C-28/I2 chondrocytes were pre-treated with 100 µM HT 30 min before 100 µM H2O2 addition. In silico analyses were exploited to select putative candidate miRs able to target SIRT-1 mRNA. Luciferase-based gene reporter assay was employed to demonstrate the direct link between miR-9 and its putative mRNA target. Transient transfection approach was performed to examine the effects of miR-9 levels on caspase activity, cell viability and expression of OA-related genes. RESULTS: MiR-9 was identified and confirmed as a post-transcriptional regulator of SIRT-1. MiR-9 and SIRT-1 levels showed opposite changes in chondrocytes following H2O2 and HT treatment. Moreover mir-9 silencing inhibited cell death induced by H2O2 partly through down-regulation of SIRT-1, whereas miR-9 overexpression markedly reduced the protective effect of HT. The manipulation of miR-9 levels also resulted in the modulation of OA-related gene expression, including MMP-13, VEGF and RUNX-2. CONCLUSIONS: These results show that miR-9 is a critical mediator of the deleterious and OA-related effects of oxidative stress in chondrocytes and that modulation of miR expression may be a crucial mechanism underlying the protective action of HT.

MicroRNA-155 suppresses autophagy in chondrocytes by modulating expression of autophagy proteins.

OBJECTIVE: Autophagy dysfunction has been reported in osteoarthritis (OA) cartilage. The objective of this study was to investigate the role of microRNA-155 (miR-155), which is overexpressed in OA, in the regulation of autophagy in human chondrocytes. DESIGN: Rapamycin (50 nM) and 2-deoxyglucose (2-DG) (5 mM) were used to stimulate autophagy in primary human articular chondrocytes and in the T/C28a2 human chondrocyte cell line. Cells were transfected with LNA GapmeR or mimic specific for miR-155 and autophagy flux was assessed by LC3 western blotting and by Cyto-ID(®) dye quantification in autophagic vacuoles. Expression of predicted miR-155 targets in the autophagy pathway were analyzed by real-time PCR and western blotting. RESULTS: Autophagy flux induced by rapamycin and 2-DG was significantly increased by miR-155 LNA, and significantly decreased after miR-155 mimic transfection in T/C28a2 cells and in human primary chondrocytes. These effects of miR-155 on autophagy were related to suppression of gene and protein expression of key autophagy regulators including Ulk1, FoxO3, Atg14, Atg5, Atg3, Gabarapl1, and Map1lc3. CONCLUSION: MiR-155 is an inhibitor of autophagy in chondrocytes and contributes to the autophagy defects in OA.

What to Do, and What Not to Do, When Diagnosing and Treating Breakthrough Cancer Pain (BTcP): Expert Opinion.

Clinical management of breakthrough cancer pain (BTcP) is still not satisfactory despite the availability of effective pharmacological agents. This is in part linked to the lack of clarity regarding certain essential aspects of BTcP, including terminology, definition, epidemiology and assessment. Other barriers to effective management include a widespread prejudice among doctors and patients concerning the use of opioids, and inadequate assessment of pain severity, resulting in the prescription of ineffective drugs or doses. This review presents an overview of the appropriate and inappropriate actions to take in the diagnosis and treatment of BTcP, as determined by a panel of experts in the field. The ultimate aim is to provide a practical contribution to the unresolved issues in the management of BTcP. Five 'things to do' and five 'things not to do' in the diagnosis and treatment of BTcP are proposed, and evidence supporting said recommendations are described. It is the duty of all healthcare workers involved in managing cancer patients to be mindful of the possibility of BTcP occurrence and not to underestimate its severity. It is vital that all the necessary steps are carried out to establish an accurate and timely diagnosis, principally by establishing effective communication with the patient, the main information source. It is crucial that BTcP is treated with an effective pharmacological regimen and drug(s), dose and administration route prescribed are designed to suit the particular type of pain and importantly the individual needs of the patient.

Context dependency and generality of fever in insects.

Fever can reduce mortality in infected animals. Yet, despite its fitness-enhancing qualities, fever often varies among animals. We used several approaches to examine this variation in insects. Texas field crickets (Gryllus texensis) exhibited a modest fever (1 °C increase in preferred body temperature, T pref) after injection of prostaglandin, which putatively mediates fever in both vertebrates and invertebrates, but they did not exhibit fever during chronic exposure to heat-killed bacteria. Further, chronic food limitation and mating status did not affect T pref or the expression of behavioural fever, suggesting limited context dependency of fever in G. texensis. Our meta-analysis of behavioural fever studies indicated that behavioural fever occurs in many insects, but it is not ubiquitous. Thus, both empirical and meta-analytical results suggest that the fever response in insects 'is widespread, although certainly not inevitable' (Moore 2002). We highlight the need for future work focusing on standardizing an experimental protocol to measure behavioural fever, understanding the specific mechanism(s) underlying fever in insects, and examining whether ecological or physiological costs often outweigh the benefits of fever and can explain the sporadic nature of fever in insects.

Counseling in primary care improves depression and quality of life.

INTRODUCTION: To measure the effectiveness on Quality of Life of adjunctive cognitive behavioral counseling in the setting of General Practitioners (GPs) along with the treatment as usual (TAU;) for the treatment of depression. METHODS: Six month-controlled trial of patients who were referred to randomly assigned GPs (four for experimental group of patients and ten for the control) was done. Experimental sample had 34 patients with DSM-IV diagnosis of Depression (Depressed Episode, Dysthymia, or Adjustment Disorder with Depressed Mood) receiving the TAU supplemented with counseling. Control group had 30 patients with diagnosis of Depression receiving only the TAU. RESULTS: The Beck Depression Inventory (BDI) score improved in both groups. Patients in the experimental group showed greater improvement compared to the control group at T2. The World Health Organization Quality OF Life Questionnaire (WHOQOL) score also improved in the experimental group but not in the control group. The improvement in the experimental group was statistically significant in terms of both BDI and WHOQOL scores. CONCLUSIONS: Adding counseling to TAU in general medical practice settings is more effective in controlling the symptoms of depression and improving the quality of life as measured over a period of six months, than TAU alone. These results while encouraging, also calls for a larger study involving a largersample size and a longer period of time.

Why should an immune response activate the stress response? Insights from the insects (the cricket Gryllus texensis).

Mediators of the stress response (e.g. glucocorticoids and norepinephrine) can be immunosuppressive. Nevertheless, immune challenge leads to the release of these compounds in vertebrates. To resolve this paradox, it has been suggested that stress hormones help restore immune homeostasis, preventing self-damage. A comparative approach may provide additional hypotheses as to why an immune challenge induces the release of stress hormones/neurohormones. Octopamine, a neurohormonal mediator of the stress response in the cricket Gryllus texensis, increased in concentration in the hemolymph during an immune challenge. Therefore, the release of stress hormones during an immune response occurs in animals across phyla. Octopamine induced an increase in lipid concentration in the hemolymph. After an acute stress (flying or running) the total number of hemocytes in the hemolymph increased. Injections of octopamine had the same effect, suggesting that it may enhance hemocyte-dependent immune functions. On the other hand, octopamine decreased lysozyme-like activity in vitro, suggesting that it inhibits some immune functions. However, lysozyme-like activity was increased by the presence of heat-killed bacteria in vitro and this increase was significantly augmented by the presence of octopamine. Therefore, the effect of octopamine on immune function differed depending on the presence of pathogens. Stress hormones may help shift immune function into the most optimal configuration depending on the physiological context.

Small intestine bacterial overgrowth in patients with irritable bowel syndrome.

Recent investigations in patients with irritable bowel syndrome (IBS) undergoing a breath test (BT) with lactulose, have shown inconclusive results on a possible association between IBS and a small intestine bacterial overgrowth (SIBO), as well as on the effective prevalence of SIBO in IBS patients, because of different geographic areas involved and different criteria adopted for the BT positivity. The aim of this study was to estimate the prevalence of SIBO among IBS patients by means a lactulose BT. Between January 2005 and December 2006, all the patients who were sent to our Gastroenterology Unit by general practitioners (GPs) for "functional" gastrointestinal (GI) symptoms, underwent a lactulose BT for diagnosis of SIBO. The test was considered positive if the hydrogen concentrations in the expired air increased more than 20 ppm over basal values within 90 minutes. A total of 127 patients have been selected, 28 males and 99 females, aged between 17 and 76 (mean age: 41.4 years), with an IBS diagnosis based on the Roma II criteria. Fifty-five patients (43%) resulted positive to the lactulose BT. No significant difference was observed between IBS patients with (SIBO+) and without (SIBO-) an intestinal bacteria contamination. In conclusion, our results indicate that SIBO is relatively frequent in IBS patients and that execution of a lactulose BT should be encouraged in all these patients, being the only way to make correct diagnosis of SIBO and establish a valid therapeutic treatment.

Competition between immune function and lipid transport for the protein apolipophorin III leads to stress-induced immunosuppression in crickets.

Intense physical activity results in transient immunosuppression in a wide range of animals. We tested the hypothesis that competition between immune function and lipid transport for the protein apolipophorin III (apoLpIII) can cause transient immunosuppression in crickets. Both flying, an energetically demanding behavior, and an immune challenge reduced the amount of monomeric (free) apoLpIII in the hemolymph of crickets. Because both immune function and flying depleted free apoLpIII, these two phenomena could be in competition for this protein. We showed that immune function was sensitive to the amount of free apoLpIII in the hemolymph. Reducing the amount of free apoLpIII in the hemolymph using adipokinetic hormone produced immunosuppression. Increasing apoLpIII levels after flight by pre-loading animals with trehalose reduced immunosuppression. Increasing post-flight apoLpIII levels by injecting purified apoLpIII also reduced flight-induced immunosuppression. These results show that competition between lipid transport and immune function for the same protein can produce transient immunosuppression after flight-or-fight behavior. Intertwined physiological systems can produce unexpected trade-offs.

Mechanisms behind inter-individual differences in lung diffusing capacity.

We measured diffusing capacity (DLCO), alveolar membrane properties (D (m)), capillary lung volume (V (c)), and alveolar volume (V (A) ) in 20 healthy subjects (12 males; age 32.4 +/- 13 (SD); BMI 21.7 +/- 3; non smokers) at total lung capacity (TLC) and at approximately 80, 60, and 40% TLC. In all subjects, D (m) increased with lung volume, the increase being significantly greater for higher values of D (m)(TLC): the inter-individual differences can be interpreted by a greater number of alveolar units coupled to a lower thickness of the air-blood barrier (thus a higher alveolar surface to thickness ratio S (A)/tau). On the average, the volume-dependent increase of D (m) from approximately 40 to 100% TLC is less than expected based on geometrical increase of S (A) /tau. In fact, up to approximately 80% TLC, the increase in D (m) closely reflects only the increase of S (A), suggesting "unfolding" of the septa with no appreciable decrease in tau. Conversely, above 80% TLC, the decrease in tau due to parenchymal stretching becomes the main factor affecting D (m). In all subjects, V (c) decreased with increasing lung volume, in line with an increase in parenchymal stretching; the decrease was significantly larger for higher values of V (c) (40% TLC). Possibly reflecting differences in alveolar capillary density. No correlation was found between D (m)(TLC) and V (c)(40%TLC). The individual specificity in the lung volume dependence of V (c) and D (m) can be reasonably described by evaluating the V (c)/D (m) ratio at TLC and at approximately 40%TLC.

Stem cell-mediated muscle regeneration and repair in aging and neuromuscular diseases.

One of the most exciting aspirations of current medical science is the regeneration of damaged body parts. The capacity of adult tissues to regenerate in response to injury stimuli represents an important homeostatic process that until recently was thought to be limited in mammals to tissues with high turnover such as blood and skin. However, it is now generally accepted that each tissue type, even those considered post-mitotic, such as nerve or muscle, contains a reserve of undifferentiated progenitor cells, loosely termed stem cells, participating in tissue regeneration and repair. Skeletal muscle regeneration is a coordinate process in which several factors are sequentially activated to maintain and preserve muscle structure and function upon injury stimuli. In this review, we will discuss the role of stem cells in muscle regeneration and repair and the critical role of specific factors, such as IGF-1, vasopressin and TNF-alpha, in the modulation of the myogenic program and in the regulation of muscle regeneration and homeostasis.

[PCBs cause necrosis of L6C5 myoblasts]. / I PCB causano necrosi dei mioblasti L6C5.

Polychlorinated biphenyls (PCBs) are structurally related to dioxins, widely used in the past in various industrial applications and daily used products. Although PCBs production was discontinued more than twenty years ago, their chemical stability and high lipophilicity make them persistent pollutants and dangerous occupational contaminants. Skeletal muscle is an important site of PCB accumulation. Our previous results about the effects of PCBs on L6C5 myoblasts, showed that "low concentrations" (< 10 microg/ml) of these compounds inhibit in vitro myogenic differentiation in a concentration-dependent fashion, while toxic effects only begin to be evident at PCB concentrations > or = 10 microg/ml. In the present paper we wondered if the observed cell mortality is due to necrosis or if it depends on the activation of programmed cell death mechanisms (apoptosis). Using different methods of analysis, we have observed that PCBs cause necrosis of myogenic cells and that such effect is related to the employed concentrations and to the time of exposure (EC50 approximately = 50 microg/ml). Our results may help to explain the creatin kinase elevation, observed in the blood of patients acutely exposed to high concentrations of PCBs, as the consequence of a necrotic damage of the skeletal muscle. It will be therefore interesting to evaluate the presence of muscular damages in the chronic exposures to PCBs.

Hypertrophy and transcriptional regulation induced in myogenic cell line L6-C5 by an increase of extracellular calcium.

Calcium plays a pivotal role in the establishment of the differentiated phenotype in myogenic cells but the involved molecular mechanisms are still matter of debate. Here we studied the effects of exposing L6-C5 myogenic cells to high extracellular Ca2+ concentration ([Ca2+]o), which induces an increase of intracellular calcium ([Ca2+]i) without involving Ca2+ release from the intracellular stores but exclusively due to plasma membrane influx (Naro et al., 2003). Exposure of L6-C5 cells to [Ca2+]o up to 20 mM for 30 min, before shifting them into a differentiative medium, induced the appearance of multinucleated, myosin-positive myotubes, much larger than in control cells with an increased protein/DNA ratio. These large myotubes showed nuclear accumulation of the hypertrophy marker GATA-2. The hypertrophic growth of these cells was blocked by cyclosporin A (CsA), FK506, or overexpression of a calcineurin-dominant negative protein, suggesting the involvement in this process of the Ca2+ responsive phosphatase calcineurin. Furthermore, transient exposure of L6-C5 cells to high [Ca2+]o increased the expression of luciferase reporter driven by myoglobin (Mb) and beta-MHC promoters but not IIB-MHC and MCK promoters. Luciferase transcription driven by CK promoter was, instead, enhanced by mobilizing Ca2+ from the intracellular stores. These data indicate that a transient increase of [Ca2+]i due to plasma-membrane influx is sufficient to induce a hypertrophic phenotype and an increased expression of slow-fiber genes but not fast-fiber genes.

Surgical results and functional outcome after total anorectal reconstruction by double graciloplasty supported by external-source electrostimulation and/or implantable pulse generators: an 8-year experience.

BACKGROUND AND AIMS: Surgical and functional results after abdominoperineal resection and total anorectal reconstruction by electrostimulated gracilis muscle transposition are still poorly documented. This study prospectively evaluated surgical and functional outcome over time in our patients. PATIENTS AND METHODS: Twenty-three patients underwent abdominoperineal resection, coloperineal pullthrough, double graciloplasty, and loop abdominal stoma. Temporary external-source intermittent electrostimulation, biofeedback training, and selective delayed stimulator implantation to improve unsatisfactory results were carried out in the first 13 patients (1st series); thereafter (2nd series) the stimulator was implanted during graciloplasty. Surgical and oncological results were followed up in all patients. Functional results were evaluated in 16 patients who underwent abdominal stoma takedown, eight in each of the two series, by anomanometry (up to 1 year) and our own 0-20 scoring system (up to 8 years from initial surgery). RESULTS: The rate of major and minor postoperative complications was 21.7% and 65%, respectively. Continuous electrostimulation proved effective on resting anal pressure. Early clinical assessments showed satisfactory functional results (considered as having a score < or =8) in all first-group patients, including five who had stimulator support, and in one-half of second-group patients. After impairment (at least 2 points) at 1 year in five patients, four of whom were from the first group, all functional results improved and became satisfactory from 5 years on (1st series) and from 4 years on (2nd series). CONCLUSION: Despite marked morbidity the high rate of good results, which improved over time, suggests that total anorectal reconstruction is worth being performed as part of abdominoperineal resection in well-selected patients with a strong motivation to avoid a permanent colostomy.

Oral beclometasone dipropionate in the treatment of extensive and left-sided active ulcerative colitis: a multicentre randomised study.

AIM: To explore the efficacy and safety of the topically acting steroid beclometasone dipropionate (BDP) in an oral controlled release formulation in the treatment of extensive or left-sided ulcerative colitis. METHODS: In a multicentre, randomised, parallel-group, single-blind study, patients with active mild to moderate ulcerative colitis were randomised to a 4-week treatment with BDP 5 mg/day o.d. vs. 5-ASA 0.8 g t.d.s. The primary efficacy variable was the decrease of Disease Activity Index (DAI) (clinical symptoms and endoscopic appearance of mucosa). Safety was evaluated by monitoring adverse events, vital signs, haematochemical parameters and adrenal function. RESULTS: One hundred and seventy-seven patients were enrolled and randomly treated with BDP (n = 90) or 5-ASA (n = 87). Mean DAI score decreased in both treatments groups (P < 0.0001 vs. baseline for both groups). Clinical remission was achieved in 63.0% of patients in the BDP group vs. 62.5% in the 5-ASA group. A significant DAI score improvement (P < 0.05) in favour of BDP was observed in patients with extensive disease. Both treatments were well tolerated. Mean plasma cortisol levels were significantly reduced vs. baseline in BDP recipients, but without signs of pituitary-adrenal function depletion. CONCLUSION: Oral BDP gave an overall treatment result in patients with active ulcerative colitis without signs of systemic side-effects.