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Random skin flaps are commonly used in plastic and reconstructive surgery for patients suffering from severe or large scale wounds or in facial reconstruction. However, skin flaps are sometimes susceptible to partial or complete necrosis at the distal parts of the flaps due to insufficient blood perfusion in the defected area. In order to improve neovascularization in skin flaps, we developed an exogenous growth factor (GF) delivery platform comprised of coacervate-coated poly(lactic-co-glycolic acid) (PLGA) nanofibers. We used a coacervate that is a self-assembled complex of poly(ethylene argininyl aspartate diglyceride) (PEAD) polycation, heparin, and cargo GFs (i.e., vascular endothelial growth factor (VEGF) and/or transforming growth factor beta 3 (TGF-β3)). The coacervate was coated onto a nanofibrous PLGA membrane for co-administration of dual GFs. In vitro proliferation of human dermal fibroblasts and endothelial tube formation using human umbilical vein endothelial cells indicated an enhanced bioactivity of released GFs when both VEGF and TGF-β3 were incorporated into coacervate-coated PLGA nanofibers (Coa-Dual NFs). Moreover, an in vivo study using a mouse skin flap model demonstrated that implantation of Coa-Dual NF reduced necrosis and enhanced blood perfusion in skin flap areas after 10 days, as compared to any single GF-loaded coacervate/PLGA fiber (Coa-Single NF) along with direct administration of the other GF onto the defect site. Moreover, Coa-Dual NFs exhibited a well-composed skin appendage and a significantly higher number of blood vessels. Based upon these results, we conclude that Coa-Dual NFs may stimulate cellular activity by enhancing the bioactivity of the released GF, leading to a synergetic effect of dual GFs for reducing necrosis in the random skin flaps. Therefore, Coa-Dual NFs could be a valuable drug delivery platform for a variety of potential clinical applications for skin tissue regeneration applications., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Treatment and prognosis of diffuse large B-cell lymphoma (DLBCL) depends on the stage of lymphoma. We conducted this study to examine unstaged DLBCL in the United States., Materials and Methods: We used Surveillance Epidemiology and End Result (SEER) 18 registries to select patients with DLBCL diagnosed during January 2000 to December 2012. Limited regional distant Summary stage 2000 was used to determine stage of the disease as localized, regional, distant or unstaged. We used logistic regression to investigate factors associated with unstaged DLBCL. Cox proportional hazards model was used to compare survival outcomes., Results: Among 67,765 patients, disease in 3,194 (4.71%) was unstaged. Age (60+years), non-African American, not married marital status, metropolitan residence, median household income >$50,000, lymph node as the primary site and those with other primary malignancies before diagnosis of DLBCL were the factors associated with cases being unstaged. The 5-year relative survival rate for patients with unstaged DLBCL was inferior to that of those with localized and regional disease, and superior to that of those with distant disease (hazard ratios of 0.58, 0.66 and 1.24 for localized, regional and distant disease, respectively, when compared to unstaged cases)., Conclusion: Several factors are associated with higher risk of unstaged DLBCL. Patients with unstaged DLBC had significantly inferior survival rates compared to patients with localized and regional stage., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Drug efflux pumps confer multidrug resistance to dangerous pathogens which makes these pumps important drug targets. We have synthesised a novel series of compounds based on a 2-naphthamide pharmacore aimed at inhibiting the efflux pumps from Gram-negative bacteria. The archeatypical transporter AcrB from Escherichia coli was used as model efflux pump as AcrB is widely conserved throughout Gram-negative organisms. The compounds were tested for their antibacterial action, ability to potentiate the action of antibiotics and for their ability to inhibit Nile Red efflux by AcrB. None of the compounds were antimicrobial against E. coli wild type cells. Most of the compounds were able to inhibit Nile Red efflux indicating that they are substrates of the AcrB efflux pump. Three compounds were able to synergise with antibiotics and reverse resistance in the resistant phenotype. Compound A3, 4-(isopentyloxy)-2-naphthamide, reduced the MICs of erythromycin and chloramphenicol to the MIC levels of the drug sensitive strain that lacks an efflux pump. A3 had no effect on the MIC of the non-substrate rifampicin indicating that this compound acts specifically through the AcrB efflux pump. A3 also does not act through non-specific mechanisms such as outer membrane or inner membrane permeabilisation and is not cytotoxic against mammalian cell lines. Therefore, we have designed and synthesised a novel chemical compound with great potential to further optimisation as inhibitor of drug efflux pumps., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Multi-domain voltage-gated ion channels appear to have evolved through sequential rounds of intragenic duplication from a primordial one-domain precursor. Whereas modularity within one-domain symmetrical channels is established, little is known about the roles of individual regions within more complex asymmetrical channels where the domains have undergone substantial divergence. Here we isolated and characterised both of the divergent pore regions from human TPC2, a two-domain channel that holds a key intermediate position in the evolution of voltage-gated ion channels. In HeLa cells, each pore localised to the ER and caused Ca 2+ depletion, whereas an ER-targeted pore mutated at a residue that inactivates full-length TPC2 did not. Additionally, one of the pores expressed at high levels in E. coli. When purified, it formed a stable, folded tetramer. Liposomes reconstituted with the pore supported Ca 2+ and Na + uptake that was inhibited by known blockers of full-length channels. Computational modelling of the pore corroborated cationic permeability and drug interaction. Therefore, despite divergence, both pores are constitutively active in the absence of their partners and retain several properties of the wild-type pore. Such symmetrical 'pore-only' proteins derived from divergent channel domains may therefore provide tractable tools for probing the functional architecture of complex ion channels.

Rationale: GlycA, an emerging inflammatory biomarker, predicted cardiovascular events in population-based studies. Psoriasis, an inflammatory disease associated with increased cardiovascular risk, provides a model to study inflammatory biomarkers in cardiovascular disease (CVD). Whether GlycA associates with psoriasis and how it predicts subclinical CVD beyond high-sensitivity C-reactive protein in psoriasis is unknown., Objective: To investigate the relationships between GlycA and psoriasis and between GlycA and subclinical CVD., Methods and Results: Patients with psoriasis and controls (n=412) participated in a 2-stage study. We measured GlycA by nuclear magnetic resonance spectroscopy. National Institutes of Health (NIH) participants underwent 18-F Fluorodeoxyglucose Positron Emission Tomography Computed Tomography (18-FDG PET/CT) scans to assess vascular inflammation (VI) and coronary computed tomographic angiography to quantify coronary artery disease burden. Psoriasis cohorts were young (mean age=47.9), with low cardiovascular risk and moderate skin disease. high-sensitivity C-reactive protein and GlycA were increased in psoriasis compared with controls (GlycA: [PENN: 408.8±75.4 versus 289.4±60.2, P<0.0001; NIH: 415.8±63.2 versus 346.2±46, P<0.0001]) and demonstrated a dose-response with psoriasis severity. In stage 2, VI (β=0.36, P<0.001) and coronary artery disease (β=0.29, P=0.004) associated with GlycA beyond CV risk factors in psoriasis. In receiver operating characteristic analysis, GlycA added value in predicting VI (P=0.01) and coronary artery disease (P<0.01). Finally, initiating anti-tumor necrosis factor therapy (n=16) reduced psoriasis severity (P<0.001), GlycA (463.7±92.5 versus 370.1±78.5, P<0.001) and VI (1.93±0.36 versus 1.76±0.19, P<0.001), whereas GlycA remained associated with VI (β=0.56, P<0.001) post treatment., Conclusions: GlycA associated with psoriasis severity and subclinical CVD beyond traditional CV risk and high-sensitivity C-reactive protein. Moreover, psoriasis treatment reduced GlycA and VI. These findings support the potential use of GlycA in subclinical CVD risk assessment in psoriasis and potentially other inflammatory diseases., Competing Interests: AND FINANCIAL DISCLOSURES: All other authors declare no conflicts of interest to disclose., (© 2016 American Heart Association, Inc.)

Construction of 3-dimensional (3-D) engineered tissue is increasingly being investigated for use in drug discovery and regenerative medicine. Here, we developed multi-layered 3-D cellular assembly by using magnetic nanoparticles (MNP) isolated from Magnetospirillum sp. AMB-1 magnetotactic bacteria. Magnetized human dermal fibroblasts (HDFBs) were prepared by treatment with the MNP, induced to form 3-D assembly under a magnetic field. Analyses including LIVE/DEAD assay, transmission electron microscopy revealed that the MNP were internalized via clathrin-mediated endocytosis without cytotoxicity. The magnetized HDFBs could build 3-D structure as a function of seeding density. When the highest seeding density (5 × 105 cells/mm2 was used, the thickness of assembly was 41.90 ± 1.69 μm, with approximately 9.3 ± 1.6 cell layers being formed. Immunofluorescence staining confirmed homogeneous distribution of ECM and junction proteins throughout the 3-D assembly. Real-time PCR analysis showed decrease in expression levels of collagen types I and IV but increase in that of connexin 43 in the 3-D assembly compared with the 2-D culture. Finally, we demonstrated that the discernible layers can be formed hierarchically by serial assembly. In conclusion, our study showed that a multi-layered structure can be easily prepared using magnetically-assisted cellular assembly with highlighting cell-cell and cell-ECM communication.

Background: Afghanistan has made considerable gains in improving maternal and child health and survival since 2001. However, socioeconomic and regional inequities may pose a threat to reaching universal coverage of health interventions and further health progress. We explored coverage and socioeconomic inequalities in key life-saving reproductive, maternal, newborn and child health (RMNCH) interventions at the national level and by region in Afghanistan. We also assessed gains in child survival through scaling up effective community-based interventions across wealth groups., Methods: Using data from the Afghanistan Multiple Indicator Cluster Survey (MICS) 2010/11, we explored 11 interventions that spanned all stages of the continuum of care, including indicators of composite coverage. Asset-based wealth quintiles were constructed using standardised methods, and absolute inequalities were explored using wealth quintile (Q) gaps (Q5-Q1) and the slope index of inequality (SII), while relative inequalities were assessed with ratios (Q5/Q1) and the concentration index (CIX). The lives saved tool (LiST) modeling used to estimate neonatal and post-neonatal deaths averted from scaling up essential community-based interventions by 90 % coverage by 2025. Analyses considered the survey design characteristics and were conducted via STATA version 12.0 and SAS version 9.4., Results: Our results underscore significant pro-rich socioeconomic absolute and relative inequalities, and mass population deprivation across most all RMNCH interventions studied. The most inequitable are antenatal care with a skilled attendant (ANCS), skilled birth attendance (SBA), and 4 or more antenatal care visits (ANC4) where the richest have between 3.0 and 5.6 times higher coverage relative to the poor, and Q5-Q1 gaps range from 32 % - 65 %. Treatment of sick children and breastfeeding interventions are the most equitably distributed. Across regions, inequalities were highest in the more urbanised East, West and Central regions of the country, while they were lowest in the South and Southeast. About 7700 newborns and 26,000 post-neonates could be saved by scaling up coverage of community outreach interventions to 90 %, with the most gains in the poorest quintiles., Conclusions: Afghanistan is a pervasively poor and conflict-prone nation that has only recently experienced a decade of relative stability. Though donor investments during this period have been plentiful and have contributed to rebuilding of health infrastructure in the country, glaring inequities remain. A resolution to scaling up health coverage in insecure and isolated regions, and improving accessibility for the poorest and marginalised populations, should be at the forefront of national policy and programming efforts.

Background: Prior studies have not examined which cardiovascular outcomes most frequently develop in participants with atrial fibrillation (AF) from population-based cohorts of the elderly., Methods: This analysis included 4,304 (85% white; 61% women) participants from the Cardiovascular Health Study who were free of baseline cardiovascular disease. AF cases were identified at baseline and as time-updated events during follow-up. Kaplan-Meier estimates were used to compute the 1-, 5-, 10-, and 15-year cumulative incidence rates of the following outcomes: coronary heart disease (CHD), myocardial infarction (MI), heart failure, and ischemic stroke. Cox regression was used to compute hazard ratios (HR) and 95% confidence intervals (CI) for the association between AF and each outcome., Results: For all time periods, the cumulative incidence estimates of CHD, MI, heart failure, and ischemic stroke were higher for those with AF compared with those without AF. Heart failure was the most frequent outcome in those with AF, while CHD events were the most frequently detected outcome in participants without AF. Compared with persons who did not have AF, the risk of heart failure was higher in those with AF (HR = 3.18, 95% CI = 2.78-3.64), and the magnitude of this association was greater than the other outcomes of interest (CHD: HR = 1.76, 95% CI = 1.54-2.03; MI: 1.40, 95% CI = 1.14-1.71; ischemic stroke: HR = 1.98, 95% CI = 1.63-2.39)., Conclusions: AF is associated with several adverse cardiovascular outcomes and heart failure is the most frequently detected event. Potentially, risk factor modification strategies for the primary prevention of heart failure will reduce the morbidity and mortality associated with AF., (© 2016 Wiley Periodicals, Inc.)

Purpose: The purpose of this study was to describe neurovascular structures-at-risk during establishment of five portals for access to distal biceps tendon (DBT) in cubital fossa, and to establish relative safety of these portal sites for such access. We hypothesized that all five portals are safe for endoscopic DBT exploration., Methods: Ten fresh frozen cadaveric elbows were dissected after placement of portals at five potential sites (four anterior, one posterior). Nine neurovascular structures (CV, cephalic vein; LCN, lateral cutaneous nerve; LV, leash of vessels; RN, radial nerve; SRN, superficial radial nerve; PIN, posterior interosseous nerve; RA, radial artery; BA, brachial artery; MN, median nerve) were dissected, and their distances from portal sites were measured. Statistical analysis was performed to determine relative portal safety, and risk of injury to neurovascular structures in relation to each portal was analyzed., Results: Structures that were significantly "at risk" were RA (p = 0.006), SRN (p = 0.002), and PIN (p = 0.004). RA was significantly "at risk" of injury from portal 4 (p = 0.009). Similarly, SRN was "at risk" from portal 3 (p = 0.036), and the PIN was "at risk" from portal 2 (p = 0.003)., Conclusions: Portal 1 (parabiceps portal) was safe for all neurovascular structures, however, portals 2-4 were significantly closer to neurovascular structures. RA, SRN, and PIN were significantly "at risk" as compared to other structures amongst the portals studied. Portal 5 was relatively safe for SRN and PIN., Clinical Relevance: Portals 1 (parabiceps portal) and 5 (distal posterior) can be safely placed for endoscopic access to the DBT. Portal 4 (open distal anterior) may be used after careful open dissection and under direct vision. Portals 2 and 3 are not recommended for elbow endoscopy.

Background and Aims: Psoriasis is a chronic inflammatory disorder associated with vascular inflammation, measured by 18-fluorodeoxyglucose positron emission tomography/computed tomography (18-FDG PET/CT), and an increased risk of myocardial infarction. Patients with psoriasis are also more likely to suffer from comorbid depression. Whether depression accelerates the development of subclinical atherosclerosis in psoriasis is unknown., Methods: Patients were selected from within a larger psoriasis cohort. Those who reported a history of depression (N = 36) on survey were matched by age and gender to patients who reported no history of psychiatric illness (N = 36). Target-to-background ratio from FDG PET/CT was used to assess aortic vascular inflammation and coronary CT angiography scans were analyzed to determine coronary plaque burden. Multivariable linear regression was performed to understand the effect of self-reported depression on vascular inflammation and coronary plaque burden after adjustment for Framingham risk (standardized β reported)., Results: In unadjusted analyses, vascular inflammation and coronary plaque burden were significantly increased in patients with self-reported depression as compared to patients with psoriasis alone. After adjustment for Framingham Risk Score, vascular inflammation (β = 0.26, p = 0.02), total plaque burden (β = 0.17, p = 0.03), and non-calcified burden (β = 0.17, p = 0.03) were associated with self-reported depression., Conclusions: Self-reported depression in psoriasis is associated with increased vascular inflammation and coronary plaque burden. Depression may play an important role in promoting subclinical atherosclerosis beyond traditional cardiovascular risk factors., Competing Interests: The authors confirm that there are no other potential conflicts of interest., (Published by Elsevier Ireland Ltd.)

Aims: To examine the relationship between peripheral arterial disease (PAD) and atrial fibrillation (AF) in a population-based cohort study of older adults., Methods and Results: We examined the relationship between PAD and AF in 5143 participants (85% white, 43% male) in the Cardiovascular Health Study (CHS), a longitudinal, observational study of adults aged 65 years and older. Peripheral arterial disease was defined by abnormal ankle-brachial index (ABI) values (<1.0 or >1.4). Incident AF events were ascertained by self-reported history, study electrocardiograms, and hospitalization discharge records. Cox regression was used to compute hazard ratios (HRs) and 95% confidence intervals (CIs) for the association between PAD and AF. Over a median follow-up of 11.7 years, a total of 1521 participants developed AF. The incidence rate (per 1000 person-years) of AF was higher in those with PAD (incidence rate = 32.9, 95% CI = 29.5, 36.7) than those without PAD (incidence rate = 23.3, 95% CI = 22.0, 24.6). In a multivariate Cox regression analysis, PAD was associated with an increased risk for AF (HR = 1.52, 95% CI = 1.34, 1.72). Each 0.1 decrease in the ABI was associated with a 6% increase in the risk for AF (HR = 1.06, 95% CI = 1.02, 1.10). The associations of high (>1.4) and low (<1.0) ABI values with AF were examined separately and were in the same direction as the main result for PAD (ABI < 1.0: HR = 1.24, 95% CI = 1.08, 1.42; ABI > 1.4: HR = 1.33, 95% CI = 0.95, 1.86)., Conclusion: The presence of PAD should alert practitioners to the increased risk of AF. Elderly patients with PAD possibly will benefit from routine electrocardiographic screening to identify AF events., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Two-pore channels (TPCs) are intracellular Ca(2+)-permeable ion channels that are expressed on acidic Ca(2+) stores. They are co-regulated by voltage and Ca(2+) in plant vacuoles and by the second messenger NAADP in animal endo-lysosomes. Two new studies of plant TPC structures reveal essential features of their architecture and provide mechanistic insight into their workings., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Left atrial enlargement (LAE) is independently associated with an increased risk of stroke and atrial fibrillation (AF). The combination of both LAE and AF possibly increases the risk of stroke beyond that observed with AF., Methods: This analysis included 4572 (43% men, 95% white) participants from the Cardiovascular Health Study. LAE was defined using transthoracic echocardiographic 2-dimensional M-mode measurements of the left atrial diameter using sex-specific cut-points (men: ≥4.1 cm, women: ≥3.9 cm). AF cases were identified during the initial study electrocardiogram or by self-reported history. We examined the association between baseline AF and incident ischemic stroke stratified by the presence of LAE. Incident cases of ischemic stroke were identified by adjudication of medical records, including hospitalization data, through December 31, 2010., Results: At baseline, a total of 253 (5.5%) participants had AF and 1947 (43%) had LAE. Participants with AF (n = 163, 64%) were more likely to have LAE than those without AF (n = 1784, 41%; P < .001). Over a median follow-up of 13 years, 739 (16%) ischemic stroke events were identified. Both AF (hazard ratio [HR] = 2.12, 95% confidence interval [CI] = 1.64-2.74) and left atrial diameter (per 1-cm increase: HR = 1.14, 95% CI = 1.01-1.28) were associated with an increased risk for ischemic stroke. The association between AF and ischemic stroke was not modified by the presence of LAE (LAE: HR = 2.13, 95% CI = 1.42-3.19; no LAE: HR = 1.91, 95% CI = 1.36-2.68; P interaction = .86)., Conclusion: Our results suggest that echocardiographic LAE does not modify the stroke risk observed with AF., (Copyright © 2016 National Stroke Association. Published by Elsevier Inc. All rights reserved.)

Residual trapping, a key CO2 geo-storage mechanism during the first decades of a sequestration project, immobilizes micrometre sized CO2 bubbles in the pore network of the rock. This mechanism has been proven to work in clean sandstones and carbonates; however, this mechanism has not been proven for the economically most important storage sites into which CO2 will be initially injected at industrial scale, namely oil reservoirs. The key difference is that oil reservoirs are typically oil-wet or intermediate-wet, and it is clear that associated pore-scale capillary forces are different. And this difference in capillary forces clearly reduces the capillary trapping capacity (residual trapping) as we demonstrate here. For an oil-wet rock (water contact angle θ=130°) residual CO2 saturation SCO2,r (≈8%) was approximately halved when compared to a strongly water-wet rock (θ=0°; SCO2,r≈15%). Consequently, residual trapping is less efficient in oil-wet reservoirs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Gankyrin is an ankyrin-repeat oncoprotein whose overexpression has been implicated in the development of many cancer types. Elevated gankyrin levels are linked to aberrant cellular events including enhanced degradation of tumour suppressor protein p53, and inhibition of gankyrin activity has therefore been identified as an attractive anticancer strategy. Gankyrin interacts with several partner proteins, and a number of these protein-protein interactions (PPIs) are of relevance to cancer. Thus, molecules that bind the PPI interface of gankyrin and interrupt these interactions are of considerable interest. Herein, we report the discovery of a small molecule termed cjoc42 that is capable of binding to gankyrin. Cell-based experiments demonstrate that cjoc42 can inhibit gankyrin activity in a dose-dependent manner: cjoc42 prevents the decrease in p53 protein levels normally associated with high amounts of gankyrin, and it restores p53-dependent transcription and sensitivity to DNA damage. The results represent the first evidence that gankyrin is a "druggable" target with small molecules.

Concentration of various metals, i.e., zinc (Zn), copper (Cu), lead (Pb), nickel (Ni), iron (Fe), manganese (Mn), chromium (Cr), and silver (Ag), was evaluated in five indigenous fish species (namely, silver carp, common carp, mahseer, thela fish, and rainbow trout), by using atomic absorption spectrophotometer. It is proved from this study that, overall, mahseer and rainbow trout had high amount of zinc, whereas thela fish and silver carp had high concentration of copper, chromium, silver, nickel, and lead, while common carp had highest amount of iron contents. Furthermore, a tissue-specific discrimination among various fish species was observed, where higher metal concentrations were noticed in fish liver, with decreasing concentration in other organs like skin, gills, and finally the least contents in fish muscle. Multivariate data analysis showed not only a variation in heavy metals among the tissues but also discrimination among the selected fish species.

Importance: The literature focuses on mortality among children younger than 5 years. Comparable information on nonfatal health outcomes among these children and the fatal and nonfatal burden of diseases and injuries among older children and adolescents is scarce., Objective: To determine levels and trends in the fatal and nonfatal burden of diseases and injuries among younger children (aged <5 years), older children (aged 5-9 years), and adolescents (aged 10-19 years) between 1990 and 2013 in 188 countries from the Global Burden of Disease (GBD) 2013 study., Evidence Review: Data from vital registration, verbal autopsy studies, maternal and child death surveillance, and other sources covering 14,244 site-years (ie, years of cause of death data by geography) from 1980 through 2013 were used to estimate cause-specific mortality. Data from 35,620 epidemiological sources were used to estimate the prevalence of the diseases and sequelae in the GBD 2013 study. Cause-specific mortality for most causes was estimated using the Cause of Death Ensemble Model strategy. For some infectious diseases (eg, HIV infection/AIDS, measles, hepatitis B) where the disease process is complex or the cause of death data were insufficient or unavailable, we used natural history models. For most nonfatal health outcomes, DisMod-MR 2.0, a Bayesian metaregression tool, was used to meta-analyze the epidemiological data to generate prevalence estimates., Findings: Of the 7.7 (95% uncertainty interval [UI], 7.4-8.1) million deaths among children and adolescents globally in 2013, 6.28 million occurred among younger children, 0.48 million among older children, and 0.97 million among adolescents. In 2013, the leading causes of death were lower respiratory tract infections among younger children (905.059 deaths; 95% UI, 810,304-998,125), diarrheal diseases among older children (38,325 deaths; 95% UI, 30,365-47,678), and road injuries among adolescents (115,186 deaths; 95% UI, 105,185-124,870). Iron deficiency anemia was the leading cause of years lived with disability among children and adolescents, affecting 619 (95% UI, 618-621) million in 2013. Large between-country variations exist in mortality from leading causes among children and adolescents. Countries with rapid declines in all-cause mortality between 1990 and 2013 also experienced large declines in most leading causes of death, whereas countries with the slowest declines had stagnant or increasing trends in the leading causes of death. In 2013, Nigeria had a 12% global share of deaths from lower respiratory tract infections and a 38% global share of deaths from malaria. India had 33% of the world's deaths from neonatal encephalopathy. Half of the world's diarrheal deaths among children and adolescents occurred in just 5 countries: India, Democratic Republic of the Congo, Pakistan, Nigeria, and Ethiopia., Conclusions and Relevance: Understanding the levels and trends of the leading causes of death and disability among children and adolescents is critical to guide investment and inform policies. Monitoring these trends over time is also key to understanding where interventions are having an impact. Proven interventions exist to prevent or treat the leading causes of unnecessary death and disability among children and adolescents. The findings presented here show that these are underused and give guidance to policy makers in countries where more attention is needed., Competing Interests: Conflict of Interest Disclosures: Dr. Kassebaum reports personal fees and non-financial support from Vifor Pharmaceuticals, Axon Communications LLC and Merck & Co outside the submitted work. KPG was awarded the NHMRC-Gustav Nossal Postgraduate Award sponsored by CSL; this award is peer reviewed and CSL had no part in selecting the awardee. Prof. Lotufo reports honoraria (modest) from Abbvie for one lecture. Walter Mendoza is program analyst at the UNFPA country office in Peru, which not necessarily endorses the study. Prof. Santos reports receiving a grant from São Paulo Research Foundation/FAPESP (Brazilian governmental research agency) for research purposes. In the past 3 years, Dr. Stein has received research grants and/or consultancy honoraria from AMBRF, Biocodex, Cipla, Lundbeck, National Responsible Gambling Foundation, Novartis, Servier, and Sun. No other conflicts are reported.

Cell-based therapy has been studied as an attractive strategy for therapeutic angiogenesis. However, obtaining a stable vascular structure remains a challenge due to the poor interaction of transplanted cells with native tissue and the difficulty in selecting the optimal cell source. In this study, we developed a cell patch of cocultured human umbilical vein endothelial cells (HUVECs) and smooth muscle cells (SMCs) using thermosensitive hydrogels for regeneration of mature vasculatures. In vitro characterization of HUVECs in the cocultured group revealed the formation of a mesh-like morphology over 5 days of culture. Vascular endothelial growth factor expression was also upregulated in the cocultured group compared with HUVECs only. The cell patch seeded with HUVECs, SMCs, or both cell type was prepared on the synthetic thermosensitive and cell interactive hydrogels, and readily detached from the hydrogel within 10 min by expansion of the hydrogel when the temperature was decreased to 4°C. We then investigated the therapeutic effect of the cell patch using a hind limb ischemic model of an athymic mouse. Overall, the group that received a cell patch of cocultured HUVECs and SMCs had a significantly retarded rate of necrosis with a significant increase in the number of arterioles and capillaries for 4 weeks compared with the groups transplanted with only HUVECs or SMCs. Dual staining of smooth muscle alpha actin and human nuclear antigen showed that the implanted cell patch was partially involved in vessel formation. In summary, the simple transplantation of a cocultured cell patch using a hydrogel system could enhance therapeutic angiogenesis through the regeneration of matured vascular structures.

Objective: To understand whether directly measured psoriasis severity is associated with vascular inflammation assessed by (18)F-fluorodeoxyglucose positron emission tomography computed tomography., Approach: In-depth cardiovascular and metabolic phenotyping was performed in adult psoriasis patients (n=60) and controls (n=20). Psoriasis severity was measured using psoriasis area severity index. Vascular inflammation was measured using average aortic target-to-background ratio using (18)F-fluorodeoxyglucose positron emission tomography computed tomography., Results: Both the psoriasis patients (28 men and 32 women, mean age 47 years) and controls (13 men and 7 women, mean age 41 years) were young with low cardiovascular risk. Psoriasis area severity index scores (median 5.4; interquartile range 2.8-8.3) were consistent with mild-to-moderate skin disease severity. Increasing psoriasis area severity index score was associated with an increase in aortic target-to-background ratio (β=0.41, P=0.001), an association that changed little after adjustment for age, sex, and Framingham risk score. We observed evidence of increased neutrophil frequency (mean psoriasis, 3.7±1.2 versus 2.9±1.2; P=0.02) and activation by lower neutrophil surface CD16 and CD62L in blood. Serum levels of S100A8/A9 (745.1±53.3 versus 195.4±157.8 ng/mL; P<0.01) and neutrophil elastase-1 (43.0±2.4 versus 30.8±6.7 ng/mL; P<0.001) were elevated in psoriasis. Finally, S100A8/A9 protein was related to both psoriasis skin disease severity (β=0.53; P=0.02) and vascular inflammation (β=0.48; P=0.02)., Conclusions: Psoriasis severity is associated with vascular inflammation beyond cardiovascular risk factors. Psoriasis increased neutrophil activation and neutrophil markers, and S100A8/A9 was related to both skin disease severity and vascular inflammation., (© 2015 American Heart Association, Inc.)

Aims: Cholesterol efflux capacity (CEC) was recently shown to predict future cardiovascular (CV) events. Psoriasis both increases CV risk and impairs CEC. However, whether having poor CEC is associated with coronary plaque burden is currently unknown. We aimed to assess the cross-sectional relationship between coronary plaque burden assessed by quantitative coronary computed tomography angiography (CCTA) with CEC in a well-phenotyped psoriasis cohort., Methods and Results: Total burden and non-calcified burden (NCB) plaque indices were assessed in 101 consecutive psoriasis patients using quantitative software. Cholesterol efflux capacity was quantified using a cell-based ex vivo assay measuring the ability of apoB-depleted plasma to mobilize cholesterol from lipid-loaded macrophages. Cholesterol efflux capacity was inversely correlated with NCB (unadjusted β-coefficient -0.33; P < 0.001), and this relationship persisted after adjustment for CV risk factors (β -0.24; P < 0.001), HDL-C levels (β -0.22; P < 0.001), and apoA1 levels (β -0.19; P < 0.001). Finally, we observed a significant gender interaction (P < 0.001) whereby women with low CEC had higher NCB compared to men with low CEC., Conclusions: We show that CEC is inversely associated with prevalent coronary plaque burden measured by quantitative CCTA. Low CEC may therefore be an important biomarker for subclinical coronary atherosclerosis in psoriasis., Clinicaltrialsgov: NCT01778569., (Published by Oxford University Press on behalf of the European Society of Cardiology 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Background: Spontaneous intracerebral hemorrhage (SICH) has a high morbidity and mortality and places a huge significant economic burden on health care and social services. The role of surgery is still controversial as evidenced by wide variation internationally in management of SICH. Traditional surgery for SICH involved open craniotomy with hematoma evacuation. Using available evidence, this article assesses the efficacy of stereotactic-guided evacuation compared with medical treatment., Methods: A systematic review was performed comparing stereotactic-guided evacuation of SICH with conservative medical management. Eligible studies were identified using a text word search of an electronic journal database for randomized controlled trials. Extracted data outcomes were subjected to meta-analysis with a forest plot. Quality was assessed using Cochrane risk of bias analysis tools., Results: There were 5 studies with 740 patients. There was a nonsignificant reduction in odds ratio (OR) for death at the end of the follow-up period (OR = 0.74, 95% confidence interval = 0.45-1.21) with no significant heterogeneity. Nonsignificant benefits were observed for dependent survival (OR = 2.14, 95% confidence interval = 0.31-0.58). In the subgroup analysis, stereotactic evacuation showed improved outcomes in patients with hematoma volume <50 mL. In this review, the effectiveness of stereotactic evacuation plus subsequent thrombolysis was insignificant (OR = 1.34, 95% confidence interval = 0.57-3.12)., Conclusions: The outcome of patients who had stereotactic-guided evacuation of SICH was not better compared with patients who received medical treatment; however, there was a trend toward better quality of survival and chance of survival in the stereotactic-guided evacuation group. This study identified areas for further research., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Engineering bone tissue is particularly challenging because of the distinctive structural features of bone within a complex biochemical environment. In the present study, we fabricated poly(L-lactic acid) (PLLA) electrospun nanofibers with random and aligned morphology immobilized with bone morphogenic protein-2 (BMP-2) and investigated how these signals modulate (1) in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs) and (2) in vivo bone growth rate, mechanical properties, and collagen assembly of newly formed bone. The orientation of adherent cells followed the underlying nanofiber morphology; however, nanofiber alignment did not show any difference in alkaline phosphate activity or in calcium mineralization of hMSCs after 14 days of in vitro culture in osteogenic differentiation media. In vivo bone regeneration was significantly higher in the nanofiber implanted groups (approximately 65-79%) as compared to the defect-only group (11.8 ± 0.2%), while no significant difference in bone regeneration was observed between random and aligned groups. However, nanoindentation studies of regenerated bone revealed Young's modulus and contact hardness with anisotropic feature for aligned group as compared to random group. More importantly, structural analysis of collagen at de novo bone showed the ability of nanofiber morphology to guide collagen deposition. SEM and TEM images revealed regular, highly ordered collagen assemblies on aligned nanofibers as compared to random fibers, which showed irregular, randomly organized collagen deposition. Taken together, we conclude that nanofibers in the presence of osteoinductive signals are a potent tool for bone regeneration, and nanofiber alignment can be used for engineering bone tissues with structurally assembled collagen fibers with defined direction.

Drug efflux pumps confer resistance upon bacteria to a wide range of antibiotics from various classes. The expression of efflux pumps are also implicated in virulence and biofilm formation. Moreover, organisms can only acquire resistance in the presence of active drug efflux pumps. Therefore, efflux pump inhibitors (EPIs) are attractive compounds to reverse multidrug resistance and to prevent the development of resistance in clinically relevant bacterial pathogens. We investigated the potential of pure compounds isolated from plants to act as EPIs. In silico screening was used to predict the bioactivity of plant compounds and to compare that with the known EPI, phe-arg-β-naphthylamide (PAβN). Subsequently, promising products have been tested for their ability to inhibit efflux. Plumbagin nordihydroguaretic acid (NDGA) and to a lesser degree shikonin, acted as sensitizers of drug-resistant bacteria to currently used antibiotics and were able to inhibit the efflux pump-mediated removal of substrate from cells. We demonstrated the feasibility of in silico screening to identify compounds that potentiate the action of antibiotics against drug-resistant strains and which might be potentially useful lead compounds for an EPI discovery program., (© 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Four-domain voltage-gated Ca(2+) and Na(+) channels (CaV, NaV) underpin nervous system function and likely emerged upon intragenic duplication of a primordial two-domain precursor. To investigate if two-pore channels (TPCs) may represent an intermediate in this evolutionary transition, we performed molecular docking simulations with a homology model of TPC1, which suggested that the pore region could bind antagonists of CaV or NaV. CaV or NaV antagonists blocked NAADP (nicotinic acid adenine dinucleotide phosphate)-evoked Ca(2+) signals in sea urchin egg preparations and in intact cells that overexpressed TPC1. By sequence analysis and inspection of the model, we predicted a noncanonical selectivity filter in animal TPCs in which the carbonyl groups of conserved asparagine residues are positioned to coordinate cations. In contrast, a distinct clade of TPCs [TPCR (for TPC-related)] in several unicellular species had ion selectivity filters with acidic residues more akin to CaV. TPCRs were predicted to interact strongly with CaV antagonists. Our data suggest that acquisition of a "blueprint" pharmacological profile and changes in ion selectivity within four-domain voltage-gated ion channels may have predated intragenic duplication of an ancient two-domain ancestor., (Copyright © 2014, American Association for the Advancement of Science.)

Background: This article reviews the epidemiology of polio, acute flaccid paralysis (AFP) surveillance, and the implementation of supplemental immunization activities (SIAs) in Afghanistan from 1997 thru 2013., Methods: Published reports and unpublished national data on polio cases, AFP surveillance, and SIAs were analyzed. Recommendations from independent advisory groups and Afghan government informed the conclusions., Results: From 1997 thru 2013, the annual number of confirmed polio cases fluctuated from a low of 4 in 2004 to a high of 80 in 2011. Wild poliovirus types 2 and 3 were last reported in 1997 and 2010, respectively. Circulating vaccine-derived poliovirus type 2 emerged in 2009. AFP surveillance quality in children aged <15 years improved over time, achieving rates>8 per 100,000 population. Since 2001, at least 6 SIAs have been conducted annually., Conclusions: Afghanistan has made progress moving closer to eliminating polio. The program struggles to reach all children because of management and accountability problems in the field, inaccessible populations, and inadequate social mobilization. Consequently, too many children are missed during SIAs. Afghanistan adopted a national emergency action plan in 2012 to address these issues, but national elimination will require consistent and complete implementation of proven strategies., (Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Mangrove plants are specialized woody plants growing in the swamps of tidal-coastal areas and river deltas of tropical and subtropical parts of the world. They have been utilized for medicinal and other purposes by the coastal people over the years. Heritiera fomes Buch. Ham. (family: Sterculiaceae) commonly known as Sundari (Bengali) is a preeminent mangrove plant occurring in the Sundarbans forest located in the southern part of Bangladesh and adjoining West Bengal province of India. The plant has applications in traditional folk medicine as evidenced by its extensive use for treating diabetes, hepatic disorders, gastrointestinal disorders, goiter, and skin diseases by the local people and traditional health practitioners. A number of investigations indicated that the plant possesses significant antioxidant, antinociceptive, antihyperglycemic, antimicrobial, and anticancer activities. Phytochemical analyses have revealed the presence of important chemical constituents like saponins, alkaloids, glycosides, tannins, steroids, flavonoids, gums, phytosterols, and reducing sugars. The present study is aimed at compiling information on phytochemical, biological, pharmacological, and ethnobotanical properties of this important medicinal plant, with a view to critically assess the legitimacy of the use of this plant in the aforementioned disorders as well as providing directions for further research.

Acid sensing ion channels (ASICs) are proton-gated cation channels that are expressed throughout the nervous system and have been implicated in mediating sensory perception of noxious stimuli. Amongst the six ASIC isoforms, ASIC1a, 1b, 2a and 3 form proton-gated homomers, which differ in their activation and inactivation kinetics, expression profiles and pharmacological modulation; protons do not gate ASIC2b and ASIC4. As with many other ion channels, structure-function studies of ASICs have been greatly aided by the discovery of some toxins that act in isoform-specific ways. ASIC3 is predominantly expressed by sensory neurons of the peripheral nervous system where it acts to detect acid as a noxious stimulus and thus plays an important role in nociception. ASIC3 is the only ASIC subunit that is inhibited by the sea anemone (Anthopleura elegantissima)-derived toxin APETx2. However, the molecular mechanism by which APETx2 interacts with ASIC3 remains largely unknown. In this study, we made a homology model of ASIC3 and used extensive protein-protein docking to predict for the first time, the probable sites of APETx2 interaction on ASIC3. Additionally, using computational alanine scanning, we also suggest the 'hot-spots' that are likely to be critical for ASIC3-APETx2 interaction., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Premature ectopic beats are frequently detected on routine 12-lead screening electrocardiograms (ECGs). However, their prognostic importance in subjects without known cardiovascular disease (CVD) is not well established. We evaluated prognostic value of atrial premature complexes (APCs) and ventricular premature complexes (VPCs) detected by a single 12-lead electrocardiography. A prospective cohort of 7,504 participants selected from nationally representative community-dwelling subjects living in the United States, enrolled in the Third National Health and Nutrition Examination Survey III from 1988 to 1994 with follow-up through December 2006 without known CVD. The main outcomes were all-cause mortality, CVD-related mortality, and ischemic heart disease (IHD)-related mortality. Of 7,504 participants (mean age 60 ± 14 years, 47% women, 49% whites), 89 (1.2%) had APCs and 110 (1.5%) had VPCs on 12-lead ECGs. During a follow-up of up to 18 years, 2,386 deaths occurred, of which 963 were due to CVD and 511 were due to IHD. In a multivariate analysis adjusted for demographics, clinical variables, and electrocardiographic measures, APCs were significantly associated with all-cause mortality (hazard ratio [HR] 1.41, 95% confidence interval [CI] 1.08 to 1.80), CVD death (HR 1.78, 95% CI 1.26 to 2.44), and IHD death (HR 2.40, 95% CI 1.59 to 3.47). For VPCs, however, there were no significant associations with all-cause mortality (HR 1.05, 95% CI 0.80 to 1.36), CVD death (HR 0.96, 95% CI 0.62 to 1.43), and IHD death (HR 0.89, 95% CI 0.47 to 1.52). In conclusion, APCs, but not VPCs, on routine screening ECGs are predictive of adverse events in community-dwelling subjects without known CVD., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In the 30 years since IP3 (inositol 1,4,5-trisphosphate) was first shown to release Ca2+ from intracellular stores, the importance of spatially organized interactions within IP3-regulated signalling pathways has been universally recognized. Recent evidence that addresses three different levels of the structural determinants of IP3-evoked Ca2+ signalling is described in the present review. High-resolution structures of the N-terminal region of the IP3R (IP3 receptor) have established that the two essential phosphate groups of IP3 bind to opposite sides of the IP3-binding site, pulling its two domains together. This conformational change is proposed to disrupt an interaction between adjacent subunits within the tetrameric IP3R that normally holds the channel in a closed state. Similar structural changes are thought to allow gating of ryanodine receptors. cAMP increases the sensitivity of IP3Rs and thereby potentiates the Ca2+ signals evoked by receptors that stimulate IP3 formation. We speculate that both IP3 and cAMP are delivered to IP3Rs within signalling junctions, wherein the associated IP3Rs are exposed to a saturating concentration of either messenger. The concentration-dependent effects of extracellular stimuli come from recruitment of junctions rather than from a graded increase in the activity of individual junctions. IP3Rs within 'IP3 junctions' respond directly to receptors that stimulate phospholipase C, whereas extra-junctional IP3Rs are exposed to suboptimal concentrations of IP3 and open only when they are sensitized by cAMP. These results highlight the importance of selective delivery of diffusible messengers to IP3Rs. The spatial organization of IP3Rs also allows them to direct Ca2+ to specific intracellular targets that include other IP3Rs, mitochondria and Ca2+-regulated channels and enzymes. IP3Rs also interact functionally with lysosomes because Ca2+ released by IP3Rs, but not that entering cells via store-operated Ca2+ entry pathways, is selectively accumulated by lysosomes. This Ca2+ uptake shapes the Ca2+ signals evoked by IP3 and it may regulate lysosomal behaviour.

In Afghanistan, malnutrition in children less than 60 months of age remains high despite nutritional services being offered in health facilities since 2003. Afghanistan's Ministry of Public Health solicited extensive community consultation to develop pictorial community-based growth monitoring and promotion (cGMP) tools to help illiterate community health workers (CHWs) provide nutritional assessment and counselling. The planned evaluation in the five districts where cGMP was implemented demonstrated that a mean weight-for-age (WFA) Z-score of 414 participant children was 0.3 Z-scores higher than that of matched non-participants who lived outside of cGMP programme catchment areas. The mean change in WFA Z-scores at evaluation was 0.3 (95% CI 0.3, 0.4) Z-scores higher than at entry into the programme. The most influential factor on WFA Z-score changes in participants was initial WFA Z-score. Those with an initial WFA Z-score of less than -2 experienced a mean increase of 0.33 (95% CI 0.29, 0.38) WFA Z-scores per session attended, while those with a baseline WFA Z-score of greater than zero showed a decrease of 0.19 (95% CI 0.22, 0.15) WFA Z-scores per session attended. These results are encouraging since they demonstrate that the cGMP programme in Afghanistan for illiterate women has some potential to contribute to improving nutrition, specifically in underweight children of either sex who enter the programme at less than nine months of age and attend 50% or more sessions.

Background: O(6)-methylguanine-DNA methyltransferase is one of the few proteins to directly remove alkylating agents in the human DNA direct reversal repair pathway. A large number of case-control studies have been conducted to explore the association between MGMT Leu84Phe polymorphism and cancer risk. However, the results were not consistent., Methods: We carried out a meta-analysis of 44 case-control studies to clarify the association between the Leu84Phe polymorphism and cancer risk., Results: Overall, significant association of the T allele with cancer susceptibility was verified with meta-analysis under a recessive genetic model (P<0.001, OR=1.30, 95%CI 1.24-1.50) and TT versus CC comparison (P=0.001, OR=1.29, 95% CI 1.12-1.50). In subgroup analysis, a significant increased risk was found for lung cancer (TT versus CC, P=0.027, OR=1.67, 95% CI 1.06-2.63; recessive genetic model, P=0.32, OR=1.64, 95% CI 1.04-2.58), whereas risk of colorectal cancer was significantly low under a dominant genetic model (P=0.019, OR=0.84, 95% CI 0.72-0.97). Additionally, a significant association between TT genetic model and total cancer risk was found in the Caucasian population (TT versus CC, P=0.014, OR=1.29, 95% CI 1.05-1.59; recessive genetic model, P=0.009, OR=1.31, 95% CI 1.07-1.61), but not in the Asian population. An increased risk for lung cancer was also verified in the Caucasian population (TT versus CC, P=0.035, OR=1.62, 95% CI 1.04-2.53; recessive genetic model, P=0.048, OR=1.57, 95% CI 1.01-2.45)., Conclusions: These results suggest that MGMT Leu84Phe polymorphism might contribute to the susceptibility of certain cancers.

TPCs (two-pore channels) are NAADP (nicotinic acid-adenine dinucleotide phosphate)-sensitive Ca2+-permeable ion channels expressed on acidic organelles. In the present study we show that deletion of the N-terminal region redirects TPC1 to the ER (endoplasmic reticulum). The introduction of fluorophores at the N-terminus of TPC1 does not affect its subcellular location, but does reversibly abolish NAADP sensitivity. Our results reveal a dual role for the N-terminus in localization and function of TPC1.

Interleukin-10 (IL-10) plays an important role in a host's defense against human papillomavirus (HPV) infection. IL-10 promoter variants may affect its expression level or functional efficiency and, subsequently, susceptibility to and survival of HPV16-associated squamous cell carcinoma of oropharynx (SCCOP). We determined tumor HPV16 DNA and genotyped three IL-10 promoter polymorphisms in 309 incident patients with SCCOP. Compared with the patients with corresponding common homozygous genotypes, patients carrying variant genotypes of IL-10 rs1800871 and rs1800872 were ~2.5 times more likely to have HPV16(+) tumors among patients with SCCOP. Among HPV16(+) patients with SCCOP only, compared to those with the corresponding variant genotypes, the patients with IL-10 rs1800871 and rs1800872 CC genotypes had significantly better survival and ~70-80% reduced risk of death/recurrence after multivariable adjustment. Additionally, functional relevance of these variants was characterized to explore the genotype-phenotype correlation. Our findings indicate that IL-10 genetic variants may be associated with tumor HPV16(+) SCCOP and predict survival of HPV16(+) patients with SCCOP. Larger studies are needed to validate our findings.

Calcium-binding protein 1 (CaBP1) is a neuron-specific member of the calmodulin superfamily that regulates several Ca(2+) channels, including inositol 1,4,5-trisphosphate receptors (InsP3Rs). CaBP1 alone does not affect InsP3R activity, but it inhibits InsP3-evoked Ca(2+) release by slowing the rate of InsP3R opening. The inhibition is enhanced by Ca(2+) binding to both the InsP3R and CaBP1. CaBP1 binds via its C lobe to the cytosolic N-terminal region (NT; residues 1-604) of InsP3R1. NMR paramagnetic relaxation enhancement analysis demonstrates that a cluster of hydrophobic residues (V101, L104, and V162) within the C lobe of CaBP1 that are exposed after Ca(2+) binding interact with a complementary cluster of hydrophobic residues (L302, I364, and L393) in the β-domain of the InsP3-binding core. These residues are essential for CaBP1 binding to the NT and for inhibition of InsP3R activity by CaBP1. Docking analyses and paramagnetic relaxation enhancement structural restraints suggest that CaBP1 forms an extended tetrameric turret attached by the tetrameric NT to the cytosolic vestibule of the InsP3R pore. InsP3 activates InsP3Rs by initiating conformational changes that lead to disruption of an intersubunit interaction between a "hot-spot" loop in the suppressor domain (residues 1-223) and the InsP3-binding core β-domain. Targeted cross-linking of residues that contribute to this interface show that InsP3 attenuates cross-linking, whereas CaBP1 promotes it. We conclude that CaBP1 inhibits InsP3R activity by restricting the intersubunit movements that initiate gating.

Binding of IP3 (inositol 1,4,5-trisphosphate) to the IP3-binding core (residues 224-604) of IP3Rs (IP3 receptors) initiates opening of these ubiquitous intracellular Ca2+ channels. The mechanisms are unresolved, but require conformational changes to pass through the suppressor domain (residues 1-223). A calmodulin-binding peptide derived from myosin light chain kinase uncouples these events. We identified a similar conserved 1-8-14 calmodulin-binding motif within the suppressor domain of IP3R1 and, using peptides and mutagenesis, we demonstrate that it is essential for IP3R activation, whether assessed by IP3-evoked Ca2+ release or patch-clamp recoding of nuclear IP3R. Mimetic peptides specifically inhibit activation of IP3R by uncoupling the IP3-binding core from the suppressor domain. Mutations of key hydrophobic residues within the endogenous 1-8-14 motif mimic the peptides. Our results show that an endogenous 1-8-14 motif mediates conformational changes that are essential for IP3R activation. The inhibitory effects of calmodulin and related proteins may result from disruption of this essential interaction.

Most animal cells express mixtures of the three subtypes of inositol 1,4,5-trisphosphate receptor (IP(3)R) encoded by vertebrate genomes. Activation of each subtype by different agonists has not hitherto been examined in cells expressing defined homogenous populations of IP(3)R. Here we measure Ca(2+) release evoked by synthetic analogues of IP(3) using a Ca(2+) indicator within the lumen of the endoplasmic reticulum of permeabilized DT40 cells stably expressing single subtypes of mammalian IP(3)R. Phosphorylation of (1,4,5)IP(3) to (1,3,4,5)IP(4) reduced potency by ~100-fold. Relative to (1,4,5)IP(3), the potencies of IP(3) analogues modified at the 1-position (malachite green (1,4,5)IP(3)), 2-position (2-deoxy(1,4,5)IP(3)) or 3-position (3-deoxy(1,4,5)IP(3), (1,3,4,5)IP(4)) were similar for each IP(3)R subtype. The potency of an analogue, (1,4,6)IP(3), in which the orientations of the 2- and 3-hydroxyl groups were inverted, was also reduced similarly for all three IP(3)R subtypes. Most analogues of IP(3) interact similarly with the three IP(3)R subtypes, but the decrease in potency accompanying removal of the 1-phosphate from (1,4,5)IP(3) was least for IP(3)R3. Addition of a large chromophore (malachite green) to the 1-phosphate of (1,4,5)IP(3) only modestly reduced potency suggesting that similar analogues could be used to measure (1,4,5)IP(3) binding optically. These data provide the first structure-activity analyses of key IP(3) analogues using homogenous populations of each mammalian IP(3)R subtype. They demonstrate broadly similar structure-activity relationships for all mammalian IP(3)R subtypes and establish the potential utility of (1,4,5)IP(3) analogues with chromophores attached to the 1-position.

Background: Inositol 1,4,5-trisphosphate receptors (IP3R) are expressed in almost all animal cells. Three mammalian genes encode closely related IP3R subunits, which assemble into homo- or hetero-tetramers to form intracellular Ca2+ channels., Scope of the Review: In this brief review, we first consider a variety of complementary methods that allow the links between IP3 binding and channel gating to be defined. How does IP3 binding to the IP3-binding core in each IP3R subunit cause opening of a cation-selective pore formed by residues towards the C-terminal? We then describe methods that allow IP3, Ca2+ signals and IP3R mobility to be examined in intact cells. A final section briefly considers genetic analyses of IP3R signalling., Major Conclusions: All IP3R are regulated by both IP3 and Ca2+. This allows them to initiate and regeneratively propagate intracellular Ca2+ signals. The elementary Ca2+ release events evoked by IP3 in intact cells are mediated by very small numbers of active IP3R and the Ca2+-mediated interactions between them. The spatial organization of these Ca2+ signals and their stochastic dependence on so few IP3Rs highlight the need for methods that allow the spatial organization of IP3R signalling to be addressed with single-molecule resolution., General Significance: A variety of complementary methods provide insight into the structural basis of IP3R activation and the contributions of IP3-evoked Ca2+ signals to cellular physiology. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The versatility of Ca2+ as an intracellular messenger stems largely from the impressive, but complex, spatiotemporal organization of the Ca2+ signals. For example, the latter when initiated by IP3 (inositol 1,4,5-trisphosphate) in many cells manifest hierarchical recruitment of elementary Ca2+ release events ('blips' and then 'puffs') en route to global regenerative Ca2+ waves as the cellular IP3 concentration rises. The spacing of IP3Rs (IP3 receptors) and their regulation by Ca2+ are key determinants of these spatially organized Ca2+ signals, but neither is adequately understood. IP3Rs have been proposed to be pre-assembled into clusters, but their composition, geometry and whether clustering affects IP3R behaviour are unknown. Using patch-clamp recording from the outer nuclear envelope of DT40 cells expressing rat IP3R1 or IP3R3, we have recently shown that low concentrations of IP3 cause IP3Rs to aggregate rapidly and reversibly into small clusters of approximately four IP3Rs. At resting cytosolic Ca2+ concentrations, clustered IP3Rs open independently, but with lower open probability, shorter open duration and lesser IP3-sensitivity than lone IP3Rs. This inhibitory influence of clustering on IP3R is reversed when the [Ca2+]i (cytosolic free Ca2+ concentration) increases. The gating of clustered IP3Rs exposed to increased [Ca2+]i is coupled: they are more likely to open and close together, and their simultaneous openings are prolonged. Dynamic clustering of IP3Rs by IP3 thus exposes them to local Ca2+ rises and increases their propensity for a CICR (Ca2+-induced Ca2+ rise), thereby facilitating hierarchical recruitment of the elementary events that underlie all IP3-evoked Ca2+ signals.

The combination of white light interferometry with hyperspectral imaging ("hyperspectral interferometry") is a recently proposed technique for single-shot measurement of 3D surface profiles. We consider for the first time its application to speckled wavefronts from optically rough surfaces. The intensity versus wavenumber signal at each pixel provides unambiguous range information despite the speckle-induced random phase shifts. Experimental results with samples undergoing controlled rigid body translation demonstrate a measurement repeatability of 460 nm for a bandwidth of approximately 30 nm. Potential applications include roughness measurement and coordinate measurement machine probes where rapid data acquisition in noncooperative environments is essential.

Objective: Screening for colorectal cancer using guaiac-based fecal occult blood tests (gFOBT) is well established in Western populations, but is hampered by poor patient compliance due to the imposed dietary restrictions. Fecal immunochemical tests (FIT) do not require dietary restriction, but are more expensive than gFOBT and therefore restrict its use in developing countries in Asia. However, Asian diets being low in meat content may not require diet restriction for gFOBT to achieve equivalent results. The objective of this study was to evaluate and compare the validity and suitability of gFOBT and FIT or a combination of the two in screening for colorectal neoplasias without prior dietary restriction in an Asian population., Methods: Patients referred to the Endoscopic Unit for colonoscopy were recruited for the study. Stool samples were collected prior to bowel preparation, and tested for occult blood with both gFOBT and FIT. Dietary restriction was not imposed. To assess the validity of either tests or in combination to detect a neoplasm or cancer in the colon, their false positive rates, their sensitivity (true positive rate) and the specificity (true negative rate) were analyzed and compared., Results: One hundred and three patients were analysed. The sensitivity for picking up any neoplasia was 53% for FIT, 40% for gFOBT and 23.3% for the combination. The sensitivities for picking up only carcinoma were 77.8% , 66.7% and 55.5%, respectively. The specificity for excluding any neoplasia was 91.7% for FIT, 74% for gFOBT and 94.5% for a combination, whereas for excluding only carcinomas they were 84%, 73.4% and 93.6%. Of the 69 with normal colonoscopic findings, FOBT was positive in 4.3%, 23.2 %and 2.9% for FIT, gFOBT, or combination of tests respectively., Conclusion: FIT is the recommended method if we are to dispense with dietary restriction in our patients because of its relatively low-false positivity and better sensitivity and specificity rates.

Accumulating evidence suggests that the endo-lysosomal system provides a substantial store of Ca(2+) that is tapped by the Ca(2+)-mobilizing messenger, NAADP. In this article, we review evidence that NAADP-mediated Ca(2+) release from this acidic Ca(2+) store proceeds through activation of the newly described two-pore channels (TPCs). We discuss recent advances in defining the sub-cellular targeting, topology and biophysics of TPCs. We also discuss physiological roles and the evolution of this ubiquitous ion channel family., (2011 Elsevier Ltd. All rights reserved.)

The cytotoxic activities of five new benzopyranone derivatives containing basic amino side chain are described. Their cytotoxicities against ER(+) MCF-7 and ER(-) MDA-MB-231 human breast cancer cell lines, and Ishikawa human endometrial cell line were determined after 72 h drug exposure employing CellTiter-Glo assay at concentrations ranging from 0.01-1.0 × 10(5) nM. The antiproliferative activities of these compounds were compared to tamoxifen (TAM), 4-hydroxytamoxifen (4-OHT, active metabolite of tamoxifen), and raloxifene (RAL). In-vitro results indicated that compounds 9, 10, 12, and 13 were more potent than TAM against the human breast cancer cell lines with IC(50)  < 20 µM. The in-silico structure-activity relationships of these compounds and their binding mode within the estrogen receptor (ER) binding site using AutoDock vina are discussed., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Folk medicinal practitioners form the first tier of primary health-care providers to most of the rural population of Bangladesh. They are known locally as Kavirajes and rely almost solely on oral or topical administration of whole plants or plant parts for treatment of various ailments. Also about 2% of the total population of Bangladesh are scattered among more than twenty tribes residing within the country's borders. The various tribes have their own tribal practitioners, who use medicinal plants for treatment of diseases. The objective of the present survey was to conduct an ethnomedicinal survey among the Kavirajes and tribal practitioners to determine which species of plants belonging to the Verbenaceae family are used by the practitioners. The Verbenaceae family plants are well known for constituents having important bio-active properties. The present survey indicated that 13 species belonging to 8 genera are used by the folk and tribal medicinal practitioners of Bangladesh. A comparison of their folk medicinal uses along with published reports in the scientific literature suggests that the Verbenaceae family plants used in Bangladesh can potentially be important sources of lead compounds or novel drugs for treatment of difficult to cure debilitating diseases like malaria and rheumatoid arthritis.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous messenger proposed to stimulate Ca(2+) release from acidic organelles via two-pore channels (TPCs). It has been difficult to resolve this trigger event from its amplification via endoplasmic reticulum Ca(2+) stores, fuelling speculation that archetypal intracellular Ca(2+) channels are the primary targets of NAADP. Here, we redirect TPC2 from lysosomes to the plasma membrane and show that NAADP evokes Ca(2+) influx independent of ryanodine receptors and that it activates a Ca(2+)-permeable channel whose conductance is reduced by mutation of a residue within a putative pore. We therefore uncouple TPC2 from amplification pathways and prove that it is a pore-forming subunit of an NAADP-gated Ca(2+) channel.

Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R) are intracellular Ca(2+) channels. Their opening is initiated by binding of IP(3) to the IP(3)-binding core (IBC; residues 224-604 of IP(3)R1) and transmitted to the pore via the suppressor domain (SD; residues 1-223). The major conformational changes leading to IP(3)R activation occur within the N terminus (NT; residues 1-604). We therefore developed a high-throughput fluorescence polarization (FP) assay using a newly synthesized analog of IP(3), fluorescein isothiocyanate (FITC)-IP(3), to examine the thermodynamics of IP(3) and adenophostin A binding to the NT and IBC. Using both single-channel recording and the FP assay, we demonstrate that FITC-IP(3) is a high-affinity partial agonist of the IP(3)R. Conventional [(3)H]IP(3) and FP assays provide similar estimates of the K(D) for both IP(3) and adenophostin A in cytosol-like medium at 4 degrees C. They further establish that the isolated IBC retains the ability of full-length IP(3)R to bind adenophostin A with approximately 10-fold greater affinity than IP(3). By examining the reversible effects of temperature on ligand binding, we established that favorable entropy changes (T Delta S) account for the greater affinities of both ligands for the IBC relative to the NT and for the greater affinity of adenophostin A relative to IP(3). The two agonists differ more substantially in the relative contribution of Delta H and T Delta S to binding to the IBC relative to the NT. This suggests that different initial binding events drive the IP(3)R on convergent pathways toward a similar open state.

In HEK cells stably expressing type 1 receptors for parathyroid hormone (PTH), PTH causes a sensitization of inositol 1,4,5-trisphosphate receptors (IP(3)R) to IP(3) that is entirely mediated by cAMP and requires cAMP to pass directly from type 6 adenylyl cyclase (AC6) to IP(3)R2. Using DT40 cells expressing single subtypes of mammalian IP(3)R, we demonstrate that high concentrations of cAMP similarly sensitize all IP(3)R isoforms to IP(3) by a mechanism that does not require cAMP-dependent protein kinase (PKA). IP(3) binding to IP(3)R2 is unaffected by cAMP, and sensitization is not mediated by the site through which ATP potentiates responses to IP(3). In single channel recordings from excised nuclear patches of cells expressing IP(3)R2, cAMP alone had no effect, but it increased the open probability of IP(3)R2 activated by a submaximal concentration of IP(3) alone or in combination with a maximally effective concentration of ATP. These results establish that cAMP itself increases the sensitivity of all IP(3)R subtypes to IP(3). For IP(3)R2, this sensitization results from cAMP binding to a novel site that increases the efficacy of IP(3). Using stably expressed short hairpin RNA to reduce expression of the G-protein, G alpha(s), we demonstrate that attenuation of AC activity by loss of G alpha(s) more substantially reduces sensitization of IP(3)R by PTH than does comparable direct inhibition of AC. This suggests that G alpha(s) may also specifically associate with each AC x IP(3)R complex. We conclude that all three subtypes of IP(3)R are regulated by cAMP independent of PKA. In HEK cells, where IP(3)R2 selectively associates with AC6, G alpha(s) also associates with the AC x IP(3)R signaling junction.