Search

Introduction Electrical spinal cord neuromodulation has emerged as a leading intervention for restoring autonomic functions, such as blood pressure, lower urinary tract (LUT), bowel, and sexual functions, following spinal cord injury (SCI). While a few preliminary studies have shown the potential effect of non-invasive transcutaneous spinal cord stimulation (tSCS) on autonomic recovery following SCI, the optimal stimulation parameters, as well as real-time and long-term functional benefits of tSCS are understudied. This trial entitled “Non-invasive Neuromodulation to Treat Bladder, Bowel, and Sexual Dysfunction following Spinal Cord Injury” is a pilot trial to examine the feasibility, dosage effect and safety of tSCS on pelvic organ function for future large-scale randomized controlled trials. Methods and analysis Forty eligible participants with chronic cervical or upper thoracic motor-complete SCI will undergo stimulation mapping and assessment batteries to determine the real-time effect of tSCS on autonomic functions. Thereafter, participants will be randomly assigned to either moderate or intensive tSCS groups to test the dosage effect of long-term stimulation on autonomic parameters. Participants in each group will receive 60 minutes of tSCS per session either twice (moderate) or five (intensive) times per week, over a period of six weeks. Outcome measures include: (a) changes in bladder capacity through urodynamic studies during real-time and after long-term tSCS, and (b) resting anorectal pressure determined via anorectal manometry during real-time tSCS. We also measure assessments of sexual function, neurological impairments, and health-related quality of life using validated questionnaires and semi-structured interviews. Ethics and dissemination Ethical approval has been obtained (CREB H20-01163). All primary and secondary outcome data will be submitted to peer-reviewed journals and disseminated among the broader scientific community and stakeholders.

The design of myocontrolled devices faces particular challenges in children with dyskinetic cerebral palsy because the electromyographic signal for control contains both voluntary and involuntary components. We hypothesized that voluntary and involuntary components of movements would be uncorrelated and thus detectable as different synergistic patterns of muscle activity, and that removal of the involuntary components would improve online EMG-based control. Therefore, we performed a synergy-based decomposition of EMG-guided movements, and evaluated which components were most controllable using a Fitts' Law task. Similarly, we also tested which muscles were most controllable. We then tested whether removing the uncontrollable components or muscles improved overall function in terms of movement time, success rate, and throughput. We found that removal of less controllable components or muscles did not improve EMG control performance, and in many cases worsened performance. These results suggest that abnormal movement in dyskinetic CP is consistent with a pervasive distortion of voluntary movement rather than a superposition of separable voluntary and involuntary components of movement.

Manipulative actions involving unstable interactions with the environment require controlling mechanical impedance through muscle co-contraction. While much research has focused on how the central nervous system (CNS) selects the muscle patterns underlying a desired movement or end-point force, the coordination strategies used to achieve a desired end-point impedance have received considerably less attention. We recorded isometric forces at the hand and electromyographic (EMG) signals in subjects performing a reaching task with an external disturbance. In a virtual environment, subjects displaced a cursor by applying isometric forces and were instructed to reach targets in 20 spatial locations. The motion of the cursor was then perturbed by disturbances whose effects could be attenuated by increasing co-contraction. All subjects could voluntarily modulate co-contraction when disturbances of different magnitudes were applied. For most muscles, activation was modulated by target direction according to a cosine tuning function with an offset and an amplitude increasing with disturbance magnitude. Co-contraction was characterized by projecting the muscle activation vector onto the null space of the EMG-to-force mapping. Even in the baseline the magnitude of the null space projection was larger than the minimum magnitude required for non-negative muscle activations. Moreover, the increase in co-contraction was not obtained by scaling the baseline null space projection, scaling the difference between the null space projections in any block and the projection of the non-negative minimum-norm muscle vector, or scaling the difference between the null space projections in the perturbed blocks and the baseline null space projection. However, the null space projections in the perturbed blocks were obtained by linear combination of the baseline null space projection and the muscle activation used to increase co-contraction without generating any force. The failure of scaling rules in explaining voluntary modulation of arm co-contraction suggests that muscle pattern generation may be constrained by muscle synergies.

In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition.

While neddylation is known to activate cullin (CUL)-RING ubiquitin ligases (CRLs), its role in regulating T cell signaling is poorly understood. Using the investigational NEDD8 activating enzyme (NAE) inhibitor, MLN4924, we found that neddylation negatively regulates T cell receptor (TCR) signaling, as its inhibition increases IL-2 production, T cell proliferation and Treg development in vitro. We also discovered that loss of CUL neddylation occurs upon TCR signaling, and CRLs negatively regulate IL-2 production. Additionally, we found that tyrosine kinase signaling leads to CUL deneddylation in multiple cell types. These studies indicate that CUL neddylation is a global regulatory mechanism for tyrosine kinase signaling.

Background: In Switzerland, 20,000 people are hospitalized each year as result of drug related problems (DRPs). The sources of DRPs can be related to patients' behavior (i.e., wrong administration) or to health processes (i.e., drug-drug interaction). No community pharmacy (CP) service focus on DRPs related to patients' behavior is currently recognized or remunerated in Switzerland. A medication review with follow up (MRF) has been developed to evaluate prescription and non-prescription medication., Objective: To evaluate the impact of MRF service for the identification and management DRPs associated to patients' behavior and to describe pharmaceutical interventions carried out through MRF., Methods: A pre-post intervention study with a cluster design and one intervention group will be carried out in CPs in the canton of Vaud (Switzerland) for 15 months. Volunteer pharmacists will be trained on the identification and management of DRPs related to patients' behavior. After training, they will include randomly selected adults taking four or more chronic drugs prescribed for at least three months prior to recruitment. Then, they will conduct three pharmacist-patient face-to-face consultations at 6-month intervals. Tasks will be differentiated by pharmacy technician or pharmacist to triage expired medication or to manage DRPs in a structured manner, respectively. The primary outcome is the identification of DRPs associated to patients' behavior. Secondary outcomes are to assess patients' medication knowledge, number of expired medications, interventions carried out by pharmacists and pharmacists' satisfaction. The study will begin in April 2023 in 19 to 35 pharmacies that will recruit at least 162 patients. A sub analysis will be carried out for patients with 65 years old or over., Conclusions: The MRF intervention features a training designed for an enhanced evaluation of patient's behavior towards their medication. The study will allow the assessment and management of DRPs in Swiss CPs with the support of the local health authorities and pharmacist association., Trial Registration: Clinicaltrials.gov NCT05348538., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Amador-Fernández et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Countless social problems demand solutions, from climate change and gun control to poverty and systemic racism. But while some of these problems inspire action (e.g., "Black Lives Matter" and "Me Too" movements), most fail to gain traction or inspire new policy. Why do some problems garner more attention and response? We suggest that the relative timing of related events may play an important role. Specifically, action may be more likely when related events are concentrated in time. A multi-method investigation tests this possibility. Study 1 borrows a modeling strategy from the economics and marketing literatures to examine a particularly important domain: gun control. Analysis of over 40 years of gun control legislation finds that, even after controlling for the frequency of mass shootings, bills are more likely to be proposed (and passed) when shootings are concentrated in time. Study 2 further tests concentration's causal impact and demonstrates that concentration increases support against sexual assault. These findings illustrate how a modeling approach commonly used to study advertising goodwill can be applied to a broader set of situations, suggest why some social problems are more likely to catalyze action, and shed light on drivers of social movements and collective action., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Berger et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Self-perceived minor ailments might conceal other health conditions if patients are not appropriately assisted by health care professionals. The aim of the study was to evaluate the patient-related outcomes of a community pharmacy Minor Ailment Service (MAS) compared to usual pharmacist care (UC)., Methods: A cluster randomised controlled trial was conducted over six months in community pharmacy in the province of Valencia (Spain). Patients seeking care or requesting a product for a minor ailments considered in the study (dermatological problems, gastrointestinal disturbance, pain and upper respiratory tract related symptoms) were included. The intervention consisted of a standardised pharmacist-patient consultation guided by a web-based program using co-developed management protocols and patients' educational material. Patients were followed up by phone ten days later. Primary clinical outcomes were appropriate medical referral and modification of direct product request. Secondary outcomes were symptom resolution and reconsultation rates., Results: A total of 808 patients (323 MAS and 485 UC) were recruited in 27 pharmacies of 21 municipalities. Patients visiting MAS pharmacies had higher odds for being referred to a physician (OR = 2.343, CI95% = [1.146-4.792]) and higher reconsultation rates (OR = 1.833, CI95% = [1.151-2.919]) compared to UC. No significant differences between groups were observed for modification of direct product request and symptom resolution., Conclusions: The use of management protocols through the MAS strengthened the identification of referral criteria such as red flags in patients suffering minor ailments. These patients with symptoms of minor ailments possibly due to more severe illness were to be referred and evaluated by physicians. Results reinforce that MAS increases safety for those patients consulting in community pharmacy for minor ailments., Trial Registration: Trial registration number: ISRCTN17235323. Retrospectively registered 07/05/2021, https://www.isrctn.com/ISRCTN17235323., Competing Interests: The authors have declared that no competing interests exist.

Objectives: Unfractionated heparin (UFH) is the commonly used anticoagulant to prevent clotting of the ECMO circuit and thrombosis of the cannulated vessels. A side effect of UFH is heparin-induced thrombocytopenia (HIT). Little is known about HIT during ECMO and the impact of changing anticoagulation in ECMO patients with newly diagnosed HIT. The aim of the study was to determine the prevalence, complications, impact of switching anticoagulation to argatroban and outcomes of patients developing heparin-induced thrombocytopenia (HIT) during either veno-venous (VV) or veno-arterial (VA) ECMO., Methods: Retrospective observational single centre study of prospectively collected data of consecutive patients receiving VV ECMO therapy for severe respiratory failure and VA ECMO for circulatory failure from January 2006 to December 2016 of the Medical intensive care unit (ICU) of the University Hospital of Regensburg. Treatment of HIT on ECMO was done with argatroban., Results: 507 patients requiring ECMO were included. Further HIT-diagnostic was conducted if HIT-4T-score was ≥4. The HIT-confirmed group had positive HIT-enzyme-linked-immunosorbent-assay (ELISA) and positive heparin-induced-platelet-activation (HIPA) test, the HIT-suspicion group a positive HIT-ELISA and missing HIPA but remained on alternative anticoagulation until discharge and the HIT-excluded group a negative or positive HIT-ELISA, however negative HIPA. These were compared to group ECMO-control without any HIT suspicion. The prevalence of HIT-confirmed was 3.2%, of HIT-suspicion 2.0% and HIT-excluded 10.8%. Confirmed HIT was trendwise more frequent in VV than in VA (3.9 vs. 1.7% p = 0.173). Compared to the ECMO control group, patients with confirmed HIT were longer on ECMO (median 13 vs. 8 days, p = 0.002). Different types of complications were higher in the HIT-confirmed than in the ECMO-control group, but in-hospital mortality was not different (31% vs. 41%, p = 0.804)., Conclusion: HIT is rare on ECMO, should be suspected, if platelets are decreasing, but seems not to increase mortality if treated promptly., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: ML received lecture honoraria from Fresenius Medical Care. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The interrelationship between wasting and stunting has been poorly investigated. We assessed the association between two indicators of linear growth, height-for-age Z-score (HAZ) change and occurrence of accelerated linear growth, and selected indicators of wasting and wasting reversal in 5,172 Cambodian children aged less than 24 months at enrolment in the 'MyHealth' study. The specific objectives were to evaluate the relationship between temporal changes in wasting and 1) change in HAZ and 2) episodes of accelerated linear growth. At enrolment, the stunting and wasting prevalence were 22.2 (21.0;23.3) % and 9.1 (8.1;10.1) %, respectively, and reached 41.4 (39.3;43.6) %, and 12.4 (11.5;13.3) % respectively, two years later. Between 14-19% of stunted children were also wasted throughout the whole study period. For each centimetre increase in Mid-Upper Arm Circumference (MUAC) from the previous assessment, the HAZ increased by 0.162 (0.150; 0.174) Z-score. We also observed a delayed positive association between the weight for height Z score (WHZ) unit increase and HAZ change of +0.10 to +0.22 units consistent with a positive relationship between linear growth and an increase in WHZ occurring with a lag of approximately three months. A similar positive correlation was observed for the occurrence of an episode of accelerated linear growth. These results show that interventions to prevent and treat wasting can contribute to stunting reduction and call for integrated wasting and stunting programming., Competing Interests: The authors have declared that no competing interests exist.

Purpose: To compare the cost of two patient management strategies with similar efficacies for chronic inflammatory demyelinating polyneuropathy (CIDP) patients in the chronic phase: hospital-based IV immunoglobulin G (IVIg) and home-based subcutaneous immunoglobulin G (SCIg) associated with an interprofessional drug therapy management programme (initial training and follow-up)., Methods: A 48-week model-based cost-minimization analysis from a societal perspective was performed. Resources included immunoglobulin (IVIg: 1 g/kg/3 weeks; SCIg: 0.4 g/kg/week initially and 0.2 g/kg/week in the maintenance phase), hospital charges, time of professionals, infusion material, transport and losses of productivity for patients. Costs were expressed in Swiss francs (CHF) (1 CHF = 0.93€ = US$1.10, www.xe.com, 2020/10/28)., Results: The total costs of IVIg were higher than those of SCIg for health insurance and other payers: 114,747 CHF versus 86,558 CHF and 8,762 CHF versus 2,401 CHF, respectively. The results were sensitive to the immunoglobulin doses, as this was the main cost driver. The SCIg daily cost in the initial phase was higher for health insurance than hospital-based IVIg was, but the additional costs were compensated during the maintenance phase (from week 28). The professional costs associated with the switch were not fully covered by the insurance and were borne by the pharmacist and the nurse., Conclusions: SCIg for CIDP patients reinforced by an interprofessional drug therapy management programme may be a cost-effective and sustainable alternative to IVIg in the Swiss system context. From an economic perspective, this therapy alternative should be more widely supported by healthcare systems and proposed to eligible patients by professionals., Competing Interests: The authors have declared that no competing interests exist.

Background: A debate about the scientific quality of COVID-19 themed research has emerged. We explored whether the quality of evidence of COVID-19 publications is lower when compared to nonCOVID-19 publications in the three highest ranked scientific medical journals., Methods: We searched the PubMed Database from March 12 to April 12, 2020 and identified 559 publications in the New England Journal of Medicine, the Journal of the American Medical Association, and The Lancet which were divided into COVID-19 (cases, n = 204) and nonCOVID-19 (controls, n = 355) associated content. After exclusion of secondary, unauthored, response letters and non-matching article types, 155 COVID-19 publications (including 13 original articles) and 130 nonCOVID-19 publications (including 52 original articles) were included in the comparative analysis. The hierarchical level of evidence was determined for each publication included and compared between cases and controls as the main outcome. A quantitative scoring of quality was carried out for the subgroup of original articles. The numbers of authors and citation rates were also compared between groups., Results: The 130 nonCOVID-19 publications were associated with higher levels of evidence on the level of evidence pyramid, with a strong association measure (Cramer's V: 0.452, P <0.001). The 155 COVID-19 publications were 186-fold more likely to be of lower evidence (95% confidence interval [CI] for odds ratio, 7.0-47; P <0.001). The quantitative quality score (maximum possible score, 28) was significantly different in favor of nonCOVID-19 (mean difference, 11.1; 95% CI, 8.5-13.7; P <0.001). There was a significant difference in the early citation rate of the original articles that favored the COVID-19 original articles (median [interquartile range], 45 [30-244] vs. 2 [1-4] citations; P <0.001)., Conclusions: We conclude that the quality of COVID-19 publications in the three highest ranked scientific medical journals is below the quality average of these journals. These findings need to be verified at a later stage of the pandemic., Competing Interests: Marko Zdravkovic, Bogdan Zdravkovic and Joana Berger-Estilita have declared that no competing interests exist. David Berger has read the journal’s policy and the authors of this manuscript have the following competing interests: The Department of Intensive Care Medicine at Inselspital has, or has had in the past, research contracts with Abionic SA, AVA AG, CSEM SA, Cube Dx GmbH, Cyto Sorbents Europe GmbH, Edwards Lifesciences LLC, GE Healthcare, ImaCor Inc., MedImmune LLC, Orion Corporation, Phagenesis Ltd. and research & development/consulting contracts with Edwards Lifesciences LLC, Nestec SA, Wyss Zurich. The money was paid into a departmental fund; Dr Berger received no personal financial gain. The Department of Intensive Care Medicine has received unrestricted educational grants from the following organizations for organizing a quarterly postgraduate educational symposium, the Berner Forum for Intensive Care (until 2015): Abbott AG, Anandic Medical Systems, Astellas, AstraZeneca, Bard Medica SA, Baxter, B | Braun, CSL Behring, Covidien, Fresenius Kabi, GSK, Lilly, Maquet, MSD, Novartis, Nycomed, Orion Pharma, Pfizer, Pierre Fabre Pharma AG (formerly known as RobaPharm). The Department of Intensive Care Medicine has received unrestricted educational grants from the following organizations for organizing bi-annual postgraduate courses in the fields of critical care ultrasound, management of ECMO and mechanical ventilation: Abbott AG, Anandic Medical Systems, Bard Medica SA., Bracco, Dräger Schweiz AG, Edwards Lifesciences AG, Fresenius Kabi (Schweiz) AG, Getinge Group Maquet AG, Hamilton Medical AG, Pierre Fabre Pharma AG (formerly known as RobaPharm), PanGas AG Healthcare, Pfizer AG, Orion Pharma, Teleflex Medical GmbH. here are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Interprofessional Education (IPE) aims to improve students' attitudes towards collaboration, teamwork, and leads to improved patient care upon graduation. However, the best time to introduce IPE into the undergraduate curriculum is still under debate., Methods: We used a mixed-methods design based on a sequential explanatory model. Medical students from all six years at the University of Bern, Switzerland (n = 683) completed an online survey about attitudes towards interprofessional learning using a scale validated for German speakers (G-IPAS). Thirty-one medical students participated in nine semi-structured interviews focusing on their experience in interprofessional learning and on the possible impact it might have on their professional development., Results: Women showed better attitudes in the G-IPAS across all years (p = 0,007). Pre-clinical students showed more positive attitudes towards IPE [Year 1 to Year 3 (p = 0.011)]. Students correctly defined IPE and its core dimensions. They appealed for more organized IPE interventions throughout the curriculum. Students also acknowledged the relevance of IPE for their future professional performance., Conclusions: These findings support an early introduction of IPE into the medical curriculum. Although students realise that interprofessional learning is fundamental to high-quality patient care, there are still obstacles and stereotypes to overcome., Trial Registration: ISRCTN 41715934., Competing Interests: RG is the Board Director of Training and Education for the European Resuscitation Council, the Task Force Chair Education, Implementation, and Team of ILCOR, and member of the direction of the MME Programme of the University of Bern. SM is the Programme Director and Senior Lecturer at the Centre for Medical Education, University of Dundee. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The remaining authors report no competing interests. We confirm that this manuscript is not under consideration by another journal. It is our own work and was not sponsored by the industry.

Early identification of children <5 years with severe acute malnutrition (SAM) is a high priority to reduce child mortality and improved health outcomes. Current WHO guidelines for community screening for SAM recommend a Mid-Upper-Arm Circumference (MUAC) of less than 115 mm to identify children with SAM, but this cut-off does not identify a significant number of children with a weight-for-height Z-score <-3. To establish new specific MUAC cut-offs, pooled data was obtained for 25,755 children from 49 SMART recent surveys in Ethiopia (2016-2019). Sensitivity, proportion of false positive, and areas under receiver-operator characteristic curves (AUC) were calculated. MUAC below 115mm alone identified 55% of children with SAM identified with both methodologies. MUAC was worse in identifying older children (21%), those from a pastoral region (42%) and boys (41%). Using current WHO cut-offs, the sensitivity (Se) of MUAC below 115mm to identify the children severly malnourished screened through Weight-for-height below-3 was 16%. Analysing the ROC curve and Youden Index, Se and Specificity (Sp) were maximal at a MUAC < 133 mm cut-off to identify SAM (respectively Se 61.1%, Sp 81.4%). However, given the high proportion of false-positive children, according to gender, region and age groups, a cut-off around 125 mm to screen SAM could be the optimal one. In Ethiopia, implementation of a MUAC-only screening program for the identification of severe acute malnutrition with the actual cut-off of 115 mm would be unethical as it will lead to many children remaining undiagnosed and untreated. In addition, future study on implementation challenge on screening children with a higher cut-off or gender/age sensitive ones should be assessed with the collection of mortality and morbidity data to ensure that the most in need are being taking care of., Competing Interests: The authors have declared that no competing interests exist.

The world glaciers and areas of persistent summer snowpack are being lost due to warming temperatures. For cold-adapted species, habitat features may offer opportunities for cooling during summer heat yet the loss of snow and ice may compromise derived thermoregulatory benefits. Herein we offer insights about habitat selection for snow and the extent to which other behavioral adjustments reduce thermal debt among high elevation mammals. Specifically, we concentrate on respiration in mountain goats (Oreamnos americanus), a species whose native distribution is currently tied to northern mountain ranges of North America, where large patches of persistent summer snow are declining, and which became extinct during geologically warmer epochs. To examine sensitivity to possible thermal stressors and use of summer snow cover, we tracked marked and unmarked mountain goats in Glacier National Park, Montana, USA, to test hypotheses about selection for cold microclimates including shade and snow during periods of relatively high temperature. To understand functional responses of habitat choices, we measured microhabitat temperatures and a component of goat physiology-breaths per minute-as an index for metabolic expenditure. Individuals 1) selected areas closer to snow on warmer summer days, and 2) on snow had a 15% mean reduction in respiration when accounting for other factors, which suggests remnant snow plays an important role in mediating effects of air temperature. The use of shade was not as an important variable in models explaining respiration. Despite the loss of 85% of glaciers in in Glacier National Park, summer's remnant snow patches are an important reservoir by which animals reduce heat stress and potential hyperthermia. Our findings, when contextualized with behavioral strategies deployed by other high elevation mammalian taxa help frame how ambient temperatures may be modulated, and they offer a direct way by which to assess susceptibility to increasing heat in cold-adapted species., Competing Interests: The authors have declared that no competing interests exist.

Introduction: For optimal treatment of femoral fractures, it is essential to understand the anatomical antecurvation of the human femur. Recent clinical studies have highlighted the problem of distal anterior encroachment or even perforation of the nail tip. The aim of this study was to accurately describe the femoral antecurvation in a large cohort. Another objective was to identify the most important influences on femoral antecurvation, such as age, femur length, gender and ethnicity., Methods: A three dimensional modelling and analytical technology was applied for the analysis of 1,232 femurs. Individual femoral antecurvation was precisely computed to determine whether gender, femur length, age, ethnicity or body mass index influence the radius of curvature (ROC)., Results: The calculated mean ROC for all femurs was 943 mm. The lowest ROC of 826 mm was found in female Asian femurs. A regression analysis demonstrated that age and femur length could predict the variability of the curvature, with femoral length as most powerful predictor. A matched pair subgroup analysis between Asians and Caucasians could not show any significant differences of ROC values., Conclusions: The mean radius of the femoral antecurvation may be smaller than previously reported revealing a significant mismatch between the actual individual anatomy and existing implants. In opposite to existing literature, this study suggests, that antecurvation differences between various ethnicities may exclusively be attributed to differences in femoral length and age. The findings of this study may be found helpful in the development of novel designs for intra- and extramedullary implants., Competing Interests: Stryker Trauma GmbH provided support in the form of salaries for authors [AP and BH], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors confirm that there are no conflicting interests that could inappropriately influence the content of this work. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

[This corrects the article DOI: 10.1371/journal.pone.0194056.].

Knowledge of current genetic diversity and mating systems of crop wild relatives (CWR) in the Fertile Crescent is important in crop genetic improvement, because western agriculture began in the area after the cold-dry period known as Younger Dryas about 12,000 years ago and these species are also wild genepools of the world's most important food crops. Wild pea (Pisum sativum subsp. elatius) is an important source of genetic diversity for further pea crop improvement harbouring traits useful in climate change context. The genetic structure was assessed on 187 individuals of Pisum sativum subsp. elatius from fourteen populations collected in the northern part of the Fertile Crescent using 18,397 genome wide single nucleotide polymorphism DARTseq markers. AMOVA showed that 63% of the allelic variation was distributed between populations and 19% between individuals within populations. Four populations were found to contain admixed individuals. The observed heterozygosity ranged between 0.99 to 6.26% with estimated self-pollination rate between 47 to 90%. Genetic distances of wild pea populations were correlated with geographic but not environmental (climatic) distances and support a mixed mating system with predominant self-pollination. Niche modelling with future climatic projections showed a local decline in habitats suitable for wild pea, making a strong case for further collection and ex situ conservation.

Background: Reduction of neonatal and under-five mortality rates remains a primary target in the achievement of universal health goals, as evident in renewed investments of Sustainable Development Goals. Various studies attribute declines in mortality to the combined effects of improvements in health care practices and changes in socio-economic factors. Since the early nineties, Cambodia has managed to evolve from a country devastated by war to a nation soon to enter the group of middle income countries. Cambodia's development efforts are reflected in some remarkable health outcomes such as a significant decline in child mortality rates and the early achievement of related Millennium Development Goals. An achievement acknowledged through the inclusion of Cambodia as one of the ten fast-track countries in the Partnership for Maternal, Newborn and Child Health. This study aims to highlight findings from the field so to provide evidence for future programming and policy efforts. It will be argued that to foster further advances in health, Cambodia will need to keep neonatal survival and health high on the agenda and tackle exacerbating inequities that arise from a pluralistic health system with considerable regional differences and socio-economic disparities., Methods/findings: Data was drawn from Demographic Health Surveys (2000, 2005, 2010, 2014). Information on a series of demographic and socio-economic household characteristics and on child anthropometry, feeding practices and child health were collected from nationally representative samples. To reach the required sample size, live-births that occurred over the past 10 years before the date of the interview were included. Demographic variables included: gender of the child, living area (urban or rural; four ecological regions (constructed by merging provinces and the capital), mother's age at birth (<20, 20-35, 35+), birth interval (long, short) and birth order (1st, 2-3, 4-6, 7+). Socio-economic variables included: mother education level (none, primary, secondary+) and household wealth (asset-based index). Data on antenatal care, tetanus injection and skilled assistance at birth were used for the mother's last child. Between 2000 and 2014, Cambodia achieved a considerable reduction in neonatal mortality (46% reduction rate). By 2014, gender inequities became almost non-existent (for all measures of equality); inequity related to mother's education decreased for all time periods; improvements were observed for differences in neonatal mortality by preceding birth interval; and a reduction in neonatal mortality rates could be noted among all the regional subgroups. Inequities increased between mothers who had limited antenatal care and those who received more than four antenatal care visits. In most scale indicators, the Slope Index of Inequality and Relative Index of Inequality estimates for all four rounds of the survey suggest inequity exacerbated in deprived communities. Also, wealth and residence (urban/rural divide) continued to be major determinants in neonatal mortality rates and related inequity trends., Conclusion: Analysis highlighted some of the complex patterns and determinants of neonatal mortality, in Cambodia. There has been a considerable decline in neonatal mortality which echoes global trends. Our analysis reveals that despite these advances, additional socio-economic and demographic characteristics considerably affected neonatal mortality rates and its inequities. There continue to be pockets of vulnerable groups that are lagging behind. This analysis highlights the determinants along the urban-rural and rich-poor divides in neonatal mortality inequities and how these affect access to and utilization of quality basic health services. This calls for future policy and programming efforts to be deliberate in their equity approach. Quality improvements in health services and targeted interventions for specific socio-economic groups will be required to further accelerate progress in reducing neonatal mortality and address Cambodia's pressing unfinished agenda in health.

Costly signaling theory provides an explanation for why humans are willing to a pay a premium for conspicuous products such as luxury brand-labeled clothing or conspicuous environmentally friendly cars. According to the theory, the extra cost of such products is a signal of social status and wealth and leads to advantages in social interactions for the signaler. A previous study found positive evidence for the case of luxury brand labels. However, an issue of this study was that some of the experiments were not conducted in a perfectly double-blind manner. I resolved this by replicating variations of the original design in a double-blind procedure. Additionally, besides the luxury label condition, I introduced a "green" label condition. Thus, the hypothesis that signaling theory is able to explain pro-environmental behavior was tested for the first time in a natural field setting. Further, I conducted experiments in both average and below-average socioeconomic neighborhoods, where, according to signaling theory, the effects of luxury signals should be even stronger. In contrast to the original study, I did not find positive effects of the luxury brand label in any of the five experiments. Nor did I find evidence for a green-signaling effect. Moreover, in poor neighborhoods a negative tendency of the luxury label actually became evident. This suggests that a signaling theory explanation of costly labels must take into account the characteristics of the observers, e.g. their social status., Competing Interests: The author has declared that no competing interests exist.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is considered to be the main variant surface antigen (VSA) of Plasmodium falciparum and is mainly localized on electron-dense knobs in the membrane of the infected erythrocyte. Switches in PfEMP1 expression provide the basis for antigenic variation and are thought to be critical for parasite persistence during chronic infections. Recently, strain transcending anti-PfEMP1 immunity has been shown to develop early in life, challenging the role of PfEMP1 in antigenic variation during chronic infections. In this work we investigate how P. falciparum achieves persistence during a chronic asymptomatic infection. The infected individual (MOA) was parasitemic for 42 days and multilocus var gene genotyping showed persistence of the same parasite population throughout the infection. Parasites from the beginning of the infection were adapted to tissue culture and cloned by limiting dilution. Flow cytometry using convalescent serum detected a variable surface recognition signal on isogenic clonal parasites. Quantitative real-time PCR with a field isolate specific var gene primer set showed that the surface recognition signal was not correlated with transcription of individual var genes. Strain transcending anti-PfEMP1 immunity of the convalescent serum was demonstrated with CD36 selected and PfEMP1 knock-down NF54 clones. In contrast, knock-down of PfEMP1 did not have an effect on the antibody recognition signal in MOA clones. Trypsinisation of the membrane surface proteins abolished the surface recognition signal and immune electron microscopy revealed that antibodies from the convalescent serum bound to membrane areas without knobs and with knobs. Together the data indicate that PfEMP1 is not the main variable surface antigen during a chronic infection and suggest a role for trypsin sensitive non-PfEMP1 VSAs for parasite persistence in chronic infections., Competing Interests: The authors have declared that no competing interests exist.

A surveillance study in 1996 identified the USA100 clone (ST5/SCCmecII)-also known as the "New York/Japan" clone-as the most prevalent MRSA causing infections in 12 New York City hospitals. Here we update the epidemiology of MRSA in seven of the same hospitals eighteen years later in 2013/14. Most of the current MRSA isolates (78 of 121) belonged to the MRSA clone USA300 (CC8/SCCmecIV) but the USA100 clone-dominant in the 1996 survey-still remained the second most frequent MRSA (25 of the 121 isolates) causing 32% of blood stream infections. The USA300 clone was most common in skin and soft tissue infections (SSTIs) and was associated with 84.5% of SSTIs compared to 5% caused by the USA100 clone. Our data indicate that by 2013/14, the USA300 clone replaced the New York/Japan clone as the most frequent cause of MRSA infections in hospitals in Metropolitan New York. In parallel with this shift in the clonal type of MRSA, there was also a striking change in the types of MRSA infections from 1996 to 2014.

Background: Early identification of children <5 yrs with acute malnutrition is a priority. Acute malnutrition is defined by the World Health Organization as a mid-upper-arm circumference (MUAC) <12.5 cm or a weight-for-height Z-score (WHZ) <-2. MUAC is a simple and low-cost indicator to screen for acute malnutrition in communities, but MUAC cut-offs currently recommended by WHO do not identify the majority of children with weight-for-height Z-score (<-2 (moderate malnourished) or r<-3 (severe malnourished). Also, no cut-offs for MUAC are established for children >5 yrs. Therefore, this study aimed at defining gender and age-specific cut-offs to improve sensitivity of MUAC as an indicator of acute malnutrition., Methods: To establish new age and gender-specific MUAC cut-offs, pooled data was obtained for 14,173 children from 5 surveys in Cambodia (2011-2013). Sensitivity, false positive rates, and areas under receiver-operator characteristic curves (AUC) were calculated using wasting for children <5yrs and thinness for children ≥5yrs as gold standards. Among the highest values of AUC, the cut-off with the highest sensitivity and a false positive rate ≤33% was selected as the optimal cut-off., Results: Optimal cut-off values increased with age. Boys had higher cut-offs than girls, except in the 8-10.9 yrs age range. In children <2yrs, the cut-off was lower for stunted children compared to non stunted children. Sensitivity of MUAC to identify WHZ<-2 and <-3 z-scores increased from 24.3% and 8.1% to >80% with the new cut-offs in comparison with the current WHO cut-offs., Conclusion: Gender and age specific MUAC cut-offs drastically increased sensitivity to identify children with WHZ-score <-2 z-scores. International reference of MUAC cut-offs by age group and gender should be established to screen for acute malnutrition at the community level.

Background: Fortification of staple foods is considered an effective and safe strategy to combat micronutrient deficiencies, thereby improving health. While improving micronutrient status might be expected to have positive effects on immunity, some studies have reported increases in infections or inflammation after iron supplementation., Objective: To study effects of micronutrient-fortified rice on hookworm infection in Cambodian schoolchildren., Methods: A double-blinded, cluster-randomized trial was conducted in 16 Cambodian primary schools partaking in the World Food Program school meal program. Three types of multi-micronutrient fortified rice were tested against placebo rice within the school meal program: UltraRice&#95;original, UltraRice&#95;improved and NutriRice. Four schools were randomly assigned to each study group (placebo n = 492, UltraRice&#95;original n = 479, UltraRice&#95;improved n = 500, NutriRice n = 506). Intestinal parasite infection was measured in fecal samples by Kato-Katz method at baseline and after three and seven months. In a subgroup (N = 330), fecal calprotectin was measured by ELISA as a marker for intestinal inflammation., Results: Baseline prevalence of hookworm infection was 18.6%, but differed considerably among schools (range 0%- 48.1%).Micronutrient-fortified rice significantly increased risk of new hookworm infection. This effect was modified by baseline hookworm prevalence at the school; hookworm infection risk was increased by all three types of fortified rice in schools where baseline prevalence was high (>15%), and only by UltraRice&#95;original in schools with low baseline prevalence. Neither hookworm infection nor fortified rice was related to fecal calprotectin., Conclusions: Consumption of rice fortified with micronutrients can increase hookworm prevalence, especially in environments with high infection pressure. When considering fortification of staple foods, a careful risk-benefit analysis is warranted, taking into account severity of micronutrient deficiencies and local prevalence of parasitic infections., Trial Registration: ClinicalTrials.gov NCT01706419.

Background: Nutrition is one of many factors affecting the cognitive development of children. In Cambodia, 55% of children <5 y were anemic and 40% stunted in 2010. Currently, no data exists on the nutritional status of Cambodian school-aged children, or on how malnutrition potentially affects their cognitive development., Objective: To assess the anthropometric and micronutrient status (iron, vitamin A, zinc, iodine) of Cambodian schoolchildren and their associations with cognitive performance., Methods: School children aged 6-16 y (n = 2443) from 20 primary schools in Cambodia were recruited. Anthropometry, hemoglobin, serum ferritin, transferrin receptors, retinol-binding protein and zinc concentrations, inflammation status, urinary iodine concentration and parasite infection were measured. Socio-economic data were collected in a sub-group of children (n = 616). Cognitive performance was assessed using Raven's Colored Progressive Matrices (RCPM) and block design and picture completion, two standardized tests from the Wechsler Intelligence Scale for Children (WISC-III)., Results: The prevalence of anemia, iron, zinc, iodine and vitamin A deficiency were 15.7%; 51.2%, 92.8%, 17.3% and 0.7% respectively. The prevalence of stunting was 40.0%, including 10.9% of severe stunting. Stunted children scored significantly lower than non-stunted children on all tests. In RCPM test, boys with iron-deficiency anemia had lower scores than boys with normal iron status (-1.46, p<0.05). In picture completion test, children with normal iron status tended to score higher than iron-deficient children with anemia (-0.81; p = 0.067) or without anemia (-0.49; p = 0.064). Parasite infection was associated with an increase in risk of scoring below the median value in block design test (OR = 1.62; p<0.05), and with lower scores in other tests, for girls only (both p<0.05)., Conclusion: Poor cognitive performance of Cambodian school-children was multifactorial and significantly associated with long-term (stunting) and current nutritional status indicators (iron status), as well as parasite infection. A life-cycle approach with programs to improve nutrition in early life and at school-age could contribute to optimal cognitive performance.

Background: Vietnamese Living Standard Surveys showed that the rate of overweight and obese in Vietnamese adults doubled between 1992 and 2002, from 2% to 5.5%, respectively with no significant difference in the proportions of overweight/obesity between men and women., Objectives: Considering the increasing public health concern over the double burden of malnutrition in Vietnam, we investigated micronutrient deficiencies among women of reproductive age according to their Body Mass Index., Methods: A transversal study was conducted in 2010 among 1530 women of reproductive age from 19 provinces. Participating women were asked to give a non-fasting blood sample for plasma iron, vitamin A, folate, vitamin B12 and zinc assessment., Results: Although % body fat was associated with haemoglobin, ferritin, retinol and zinc concentrations, BMI category was only associated with marginal vitamin A status (19% among underweight vs 7% among overweight/obese; p<0.0001) and not with iron deficiency anemia, zinc deficiency, vitamin B12 deficiency or folate status. The prevalence of iron, and vitamin B12 deficiencies was respectively 11.4% and 15% among the 20% overweight/obese women; prevalence of zinc deficiency and marginal/deficient folate status was much higher, affecting respectively 61.1% and 25.8%. Intra-individual double burden of malnutrition (overweight/obesity (OW) and micronutrient deficiency) was observed among 2.0% for OW-anemia, 2.3% OW-iron deficient, 3.0% for OW-Vitamin B12 deficiency, 12.2% for OW-Zinc deficiency and 5.2% for OW-marginal/deficient folate status., Conclusions: This large, cross-sectional survey demonstrated that micronutrient deficiencies are an issue across the weight spectrum among women in Vietnam, with only vitamin A status being better among overweight than underweight women. It is therefore essential for Vietnam to actively prevent women of reproductive age from overweight/obesity and at same time to control micronutrient deficiencies in this population to limit their economic and health consequences.

The inherited peroxisomal disorder X-linked adrenoleukodystrophy (X-ALD), associated with neurodegeneration and inflammatory cerebral demyelination, is caused by mutations in the ABCD1 gene encoding the peroxisomal ATP-binding cassette (ABC) transporter ABCD1 (ALDP). ABCD1 transports CoA-esters of very long-chain fatty acids (VLCFA) into peroxisomes for degradation by β-oxidation; thus, ABCD1 deficiency results in VLCFA accumulation. The closest homologue, ABCD2 (ALDRP), when overexpressed, compensates for ABCD1 deficiency in X-ALD fibroblasts and in Abcd1-deficient mice. Microglia/macrophages have emerged as important players in the progression of neuroinflammation. Human monocytes, lacking significant expression of ABCD2, display severely impaired VLCFA metabolism in X-ALD. Here, we used thioglycollate-elicited primary mouse peritoneal macrophages (MPMΦ) from Abcd1 and Abcd2 single- and double-deficient mice to establish how these mutations affect VLCFA metabolism. By quantitative RT-PCR, Abcd2 mRNA was about half as abundant as Abcd1 mRNA in wild-type and similarly abundant in Abcd1-deficient MPMΦ. VLCFA (C26∶0) accumulated about twofold in Abcd1-deficient MPMΦ compared with wild-type controls, as measured by gas chromatography-mass spectrometry. In Abcd2-deficient macrophages VLCFA levels were normal. However, upon Abcd1/Abcd2 double-deficiency, VLCFA accumulation was markedly increased (sixfold) compared with Abcd1-deficient MPMΦ. Elovl1 mRNA, encoding the rate-limiting enzyme for elongation of VLCFA, was equally abundant across all genotypes. Peroxisomal β-oxidation of C26∶0 amounted to 62% of wild-type activity in Abcd1-deficient MPMΦ and was significantly more impaired (29% residual activity) upon Abcd1/Abcd2 double-deficiency. Single Abcd2 deficiency did not significantly compromise β-oxidation of C26∶0. Thus, the striking accumulation of VLCFA in double-deficient MPMΦ compared with single Abcd1 deficiency was due to the loss of ABCD2-mediated, compensatory transport of VLCFA into peroxisomes. We propose that moderate endogenous expression of Abcd2 in Abcd1-deficient murine macrophages prevents the severe metabolic phenotype observed in human X-ALD monocytes, which lack appreciable expression of ABCD2. This supports upregulation of ABCD2 as a therapeutic concept in X-ALD.

X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disorder, is a clinically heterogeneous disease that can manifest as devastating inflammatory cerebral demyelination (CALD) leading to death of affected males. Currently, the only curative treatment is allogeneic hematopoietic stem cell transplantation (HSCT). However, HSCT is only effective when performed at an early stage because the inflammation may progress for eighteen months after HSCT. Thus, alternative treatment options able to immediately halt the progression are urgently needed. X-ALD is caused by mutations in the ABCD1 gene, encoding the peroxisomal membrane protein ABCD1, resulting in impaired very long-chain fatty acid metabolism. The related ABCD2 protein is able to functionally compensate for ABCD1-deficiency both in vitro and in vivo. Recently, we demonstrated that of the cell types derived from CD34+ stem cells, predominantly monocytes but not lymphocytes are metabolically impaired in X-ALD. As ABCD2 is virtually not expressed in these cells, we hypothesize that a pharmacological up-regulation of ABCD2 should compensate metabolically and halt the inflammation in CALD. Retinoids are anti-inflammatory compounds known to act on ABCD2. Here, we investigated the capacity of selected retinoids for ABCD2 induction in human monocytes/macrophages. In THP-1 cells, 13-cis-retinoic acid reached the highest, fivefold, increase in ABCD2 expression. To test the efficacy of retinoids in vivo, we analyzed ABCD2 mRNA levels in blood cells isolated from acne patients receiving 13-cis-retinoic acid therapy. In treated acne patients, ABCD2 mRNA levels were comparable to pre-treatment levels in monocytes and lymphocytes. Nevertheless, when primary monocytes were in vitro differentiated into macrophages and treated with 13-cis-retinoic acid, we observed a fourfold induction of ABCD2. However, the level of ABCD2 induction obtained by retinoids alone is probably not of therapeutic relevance for X-ALD. In conclusion, our results suggest a change in promoter accessibility during macrophage differentiation allowing induction of ABCD2 by retinoids.

Background: Urban areas in West Africa are not immune to undernutrition with recent urbanization and high food prices being important factors. School children often have a poor nutritional status, potentially affecting their health and schooling performance. Yet, generally school children do not benefit from nutrition programs. The objective of the study was to assess the anthropometric and micronutrient status of children from state schools in the Dakar area., Methods: School children (n = 604) aged from 5 to 17 y (52.5% girls, 47.5% ≥ 10 y) were selected through a two-stage random cluster sample of children attending urban primary state schools in the Dakar area (30 schools × 20 children). The prevalence of stunting (height-for-age<-2 z-scores) and thinness (BMI-for-age<-2 z-scores, WHO 2006, and three grades of thinness corresponding to BMI of 18.5, 17.0 and 16.0 kg/m2 in adults) were calculated from weight and height. Hemoglobin, plasma concentrations of ferritin (FER), transferrin receptors (TfR), retinol binding protein (RBP), and zinc, and urinary iodine concentrations were measured. Correction factors were used for FER and RBP in subjects with inflammation determined with C-reactive protein and α1-acid-glycoprotein., Results: 4.9% of children were stunted, 18.4% were thin, 5.6% had severe thinness (BMI-for-age<-3 z-scores). Only one child had a BMI-for-age>2 z-scores. Prevalence of anemia, iron deficiency and iron deficiency anemia was 14.4%, 39.1% and 10.6% respectively. 3.0% had vitamin A deficiency, 35.9% a marginal vitamin A status, and 25.9% zinc deficiency. Urinary iodine was <50 µg/L in 7.3% of children and ≥ 200 µg/L in 22.3%. The prevalence of marginal vitamin A, zinc deficiency, high TfR was significantly higher in boys than in girls (P<0.05). Height-for-age and retinol were significantly lower in participants ≥ 10 y and <10 y respectively., Conclusion: Undernutrition, especially thinness, iron and zinc deficiencies in school children in the Dakar area requires special targeted nutrition interventions.

Background: Marfan syndrome is associated with ventricular arrhythmia but risk factors including FBN1 mutation characteristics require elucidation., Methods and Results: We performed an observational cohort study of 80 consecutive adults (30 men, 50 women aged 42±15 years) with Marfan syndrome caused by FBN1 mutations. We assessed ventricular arrhythmia on baseline ambulatory electrocardiography as >10 premature ventricular complexes per hour (>10 PVC/h), as ventricular couplets (Couplet), or as non-sustained ventricular tachycardia (nsVT), and during 31±18 months of follow-up as ventricular tachycardia (VT) events (VTE) such as sudden cardiac death (SCD), and sustained ventricular tachycardia (sVT). We identified >10 PVC/h in 28 (35%), Couplet/nsVT in 32 (40%), and VTE in 6 patients (8%), including 3 with SCD (4%). PVC>10/h, Couplet/nsVT, and VTE exhibited increased N-terminal pro-brain natriuretic peptide serum levels(P<.001). All arrhythmias related to increased NT-proBNP (P<.001), where PVC>10/h and Couplet/nsVT also related to increased indexed end-systolic LV diameters (P = .024 and P = .020), to moderate mitral valve regurgitation (P = .018 and P = .003), and to prolonged QTc intervals (P = .001 and P = .006), respectively. Moreover, VTE related to mutations in exons 24-32 (P = .021). Kaplan-Meier analysis corroborated an association of VTE with increased NT-proBNP (P<.001) and with mutations in exons 24-32 (P<.001)., Conclusions: Marfan syndrome with causative FBN1 mutations is associated with an increased risk for arrhythmia, and affected persons may require life-long monitoring. Ventricular arrhythmia on electrocardiography, signs of myocardial dysfunction and mutations in exons 24-32 may be risk factors of VTE.

In the past decade, DNA barcoding became increasingly common as a method for species identification in biodiversity inventories and related studies. However, mainly due to technical obstacles, squamate reptiles have been the target of few barcoding studies. In this article, we present the results of a DNA barcoding study of squamates of the Comoros archipelago, a poorly studied group of oceanic islands close to and mostly colonized from Madagascar. The barcoding dataset presented here includes 27 of the 29 currently recognized squamate species of the Comoros, including 17 of the 18 endemic species. Some species considered endemic to the Comoros according to current taxonomy were found to cluster with non-Comoran lineages, probably due to poorly resolved taxonomy. All other species for which more than one barcode was obtained corresponded to distinct clusters useful for species identification by barcoding. In most species, even island populations could be distinguished using barcoding. Two cryptic species were identified using the DNA barcoding approach. The obtained barcoding topology, a Bayesian tree based on COI sequences of 5 genera, was compared with available multigene topologies, and in 3 cases, major incongruences between the two topologies became evident. Three of the multigene studies were initiated after initial screening of a preliminary version of the barcoding dataset presented here. We conclude that in the case of the squamates of the Comoros Islands, DNA barcoding has proven a very useful and efficient way of detecting isolated populations and promising starting points for subsequent research.

Lung cancer is the leading cause of cancer deaths worldwide. In the United States, only one in six lung cancer patients survives five years after diagnosis. These statistics may improve if new therapeutic targets are identified. We previously reported that an enzyme of fatty acid metabolism, very long-chain acyl-CoA synthetase 3 (ACSVL3), is overexpressed in malignant glioma, and that depleting glioblastoma cells of ACSVL3 diminishes their malignant properties. To determine whether ACSVL3 expression was also increased in lung cancer, we studied tumor histologic sections and lung cancer cell lines. Immunohistochemical analysis of normal human lung showed moderate ACSVL3 expression only in bronchial epithelial cells. In contrast, all of 69 different lung tumors tested, including adeno-, squamous cell, large cell, and small cell carcinomas, had robustly elevated ACSVL3 levels. Western blot analysis of lung cancer cell lines derived from these tumor types also had significantly increased ACSVL3 protein compared to normal bronchial epithelial cells. Decreasing the growth rate of lung cancer cell lines did not change ACSVL3 expression. However, knocking down ACSVL3 expression by RNA interference reduced cell growth rates in culture by 65-76%, and the ability of tumor cells to form colonies in soft agar suspension by 65-80%. We also conducted studies to gain a better understanding of the biochemical properties of human ACSVL3. ACSVL3 mRNA was detected in many human tissues, but the expression pattern differed somewhat from that of the mouse. The enzyme activated long- and very long-chain saturated fatty acid substrates, as well as long-chain mono- and polyunsaturated fatty acids to their respective coenzyme A derivatives. Endogenous human ACSVL3 protein was found in a punctate subcellular compartment that partially colocalized with mitochondria as determined by immunofluorescence microscopy and subcellular fractionation. From these studies, we conclude that ACSVL3 is a promising new therapeutic target in lung cancer.

Polyamines are important regulators of basal cellular functions but also subserve highly specific tasks in the mammalian brain. With this respect, polyamines and the synthesizing and degrading enzymes are clearly differentially distributed in neurons versus glial cells and also in different brain areas. The synthesis of the diamine putrescine may be driven via two different pathways. In the "classical" pathway urea and carbon dioxide are removed from arginine by arginase and ornithine decarboxylase. The alternative pathway, first removing carbon dioxide by arginine decarboxlyase and then urea by agmatinase, may serve the same purpose. Furthermore, the intermediate product of the alternative pathway, agmatine, is an endogenous ligand for imidazoline receptors and may serve as a neurotransmitter. In order to evaluate and compare the expression patterns of the two gate keeper enzymes arginase and arginine decarboxylase, we generated polyclonal, monospecific antibodies against arginase-1 and arginine decarboxylase. Using these tools, we immunocytochemically screened the rat brain and compared the expression patterns of both enzymes in several brain areas on the regional, cellular and subcellular level. In contrast to other enzymes of the polyamine pathway, arginine decarboxylase and arginase are both constitutively and widely expressed in rat brain neurons. In cerebral cortex and hippocampus, principal neurons and putative interneurons were clearly labeled for both enzymes. Labeling, however, was strikingly different in these neurons with respect to the subcellular localization of the enzymes. While with antibodies against arginine decarboxylase the immunosignal was distributed throughout the cytoplasm, arginase-like immunoreactivity was preferentially localized to Golgi stacks. Given the apparent congruence of arginase and arginine decarboxylase distribution with respect to certain cell populations, it seems likely that the synthesis of agmatine rather than putrescine may be the main purpose of the alternative pathway of polyamine synthesis, while the classical pathway supplies putrescine and spermidine/spermine in these neurons.

Spermine oxidase is a FAD-containing enzyme involved in polyamines catabolism, selectively oxidizing spermine to produce H2O2, spermidine, and 3-aminopropanal. Spermine oxidase is highly expressed in the mouse brain and plays a key role in regulating the levels of spermine, which is involved in protein synthesis, cell division and cell growth. Spermine is normally released by neurons at synaptic sites where it exerts a neuromodulatory function, by specifically interacting with different types of ion channels, and with ionotropic glutamate receptors. In order to get an insight into the neurobiological roles of spermine oxidase and spermine, we have deregulated spermine oxidase gene expression producing and characterizing the transgenic mouse model JoSMOrec, conditionally overexpressing the enzyme in the neocortex. We have investigated the effects of spermine oxidase overexpression in the mouse neocortex by transcript accumulation, immunohistochemical analysis, enzymatic assays and polyamine content in young and aged animals. Transgenic JoSMOrec mice showed in the neocortex a higher H2O2 production in respect to Wild-Type controls, indicating an increase of oxidative stress due to SMO overexpression. Moreover, the response of transgenic mice to excitotoxic brain injury, induced by kainic acid injection, was evaluated by analysing the behavioural phenotype, the immunodistribution of neural cell populations, and the ultrastructural features of neocortical neurons. Spermine oxidase overexpression and the consequently altered polyamine levels in the neocortex affects the cytoarchitecture in the adult and aging brain, as well as after neurotoxic insult. It resulted that the transgenic JoSMOrec mouse line is more sensitive to KA than Wild-Type mice, indicating an important role of spermine oxidase during excitotoxicity. These results provide novel evidences of the complex and critical functions carried out by spermine oxidase and spermine in the mammalian brain.

Introduction: In many developing countries including Vietnam, data are lacking on vitamin D and calcium deficiencies whereas those deficiencies can play an important role in the development of bone health and possibly non-communicable diseases. The purpose of this study was to determine the overall prevalence of vitamin D and calcium deficiencies in women and young children and their nutritional related risk factors., Methods: A cross-sectional study conducted among 595 women of reproductive age and 532 children <5 years from 19 provinces of Vietnam. For each individual, data concerning daily diet, socioeconomic group, anthropometric status were obtained, and plasma concentrations of calcium and vitamin D were measured., Results: The prevalence of hypovitaminosis D status was very high, with the prevalence of vitamin D deficiency (25(OH)D<30 nmol/L) and insufficiency (25(OH)D between 30-49.9 nmol/L) being 17% and 40% in women and 21% and 37% in children, respectively. Using more liberal cut-off of 75 nmol/L, approximately 90% of the women and children were classified as having hypovitaminosis D. Overweight/obese women had a 2 times lower risk (OR = 0.46, [0.24-0.90]) for vitamin D deficiency than non-overweight and non-obese women. No participant had severe calcium deficiency but moderate and mild hypocalcaemia (plasma calcium concentrations between 1.15-0.9 mmol/L for mild deficiency and between 0.9-0.8 mmol/L for moderate deficiency) affected respectively 14% and 83% of the women with 97% of the children having mild hypocalcaemia. Women and children consumed about 1% of the Institute of Medicine (IOM) recommended nutrient intake (RNI) for vitamin D and less than 43% of the RNI for calcium., Conclusion: Our study suggests that calcium and vitamin D deficiencies represent a major public health concern in Vietnam. Thus, actions to improve the vitamin D and calcium status of the Vietnamese population should be considered.

Background: A micronutrient survey carried out in 2010 among randomly selected Vietnamese women in reproductive age indicated that anemia and micronutrient deficiencies are still prevalent. The objective of this study was thus to analyze the dietary micronutrient intakes of these women, to select the food vehicles to be fortified and to calculate their contributions to meet the recommended nutrient intake (RNI) for iron, zinc, vitamin A and folic acid., Main Findings: Consumption data showed that the median intake was 38.4% of the RNI for iron, 61.1% for vitamin A and 91.8% for zinc. However, more than 50% of the women had daily zinc consumption below the RNI. Rice and vegetable oil were consumed daily in significant amounts (median: 320.4 g/capita/day and 8.6 g/capita/day respectively) by over 90% of the women, making them suitable vehicles for fortification. Based on consumption data, fortified vegetable oil could contribute to an additional vitamin A intake of 27.1% of the RNI and fortified rice could increase the intake of iron by 41.4% of the RNI, zinc by 15.5% and folate by 34.1%. Other food vehicles, such as fish and soy sauces and flavoring powders, consumed respectively by 63% and 90% of the population could contribute to increase micronutrient intakes if they are properly fortified and promoted. Wheat flower was consumed by 39% of the women and by less than 20% women from the lowest socioeconomic strata., Conclusion: The fortification of edible vegetable oils with vitamin A and of rice with iron, zinc and folic acid are the most promising fortification strategies to increase micronutrient intakes of women in reproductive age in Vietnam. While rice fortification will be implemented, fortification of fish and soy sauces with iron, that has been proven to be effective, has to be supported and fortification of flavouring powders with micronutrients investigated.

X-linked adrenoleukodystrophy (X-ALD) is characterized by marked phenotypic variation ranging from adrenomyeloneuropathy (AMN) to childhood cerebral ALD (CCALD). X-ALD is caused by mutations in the ABCD1 gene, but no genotype-phenotype correlation has been established so far and modifier gene variants are suspected to modulate phenotypes. Specific classes of lipids, enriched in very long-chain fatty acids that accumulate in plasma and tissues from X-ALD patients are suspected to be involved in the neuroinflammatory process of CCALD. CD1 proteins are lipid- antigen presenting molecules encoded by five CD1 genes in human (CD1A-E). Association studies with 23 tag SNPs covering the CD1 locus was performed in 52 patients with AMN and 87 patients with CCALD. The minor allele of rs973742 located 4-kb downstream from CD1D was significantly more frequent in AMN patients (χ² = 7.6; P = 0.006). However, this association was no longer significant after Bonferroni correction for multiple testing. The other polymorphisms of the CD1 locus did not reveal significant association. Further analysis of other CD1D polymorphisms did not detect stronger association with X-ALD phenotypes. Although the association with rs973742 warrants further investigations, these results indicate that the genetic variants of CD1 genes do not contribute markedly to the phenotypic variance of X-ALD.

Background: The 2000 Vietnamese National Nutrition Survey showed that the population's dietary intake had improved since 1987. However, inequalities were found in food consumption between socioeconomic groups. As no national data exist on the prevalence of micronutrient deficiencies, a survey was conducted in 2010 to assess the micronutrient status of randomly selected 1526 women of reproductive age and 586 children aged 6-75 mo., Principal Findings: In women, according to international thresholds, prevalence of zinc deficiency (ZnD, 67.2 ± 2.6%) and vitamin B12 deficiency (11.7 ± 1.7%) represented public health problems, whereas prevalence of anemia (11.6 ± 1.0%) and iron deficiency (ID, 13.7 ± 1.1%) were considered low, and folate (<3%) and vitamin A (VAD, <2%) deficiencies were considered negligible. However, many women had marginal folate (25.1%) and vitamin A status (13.6%). Moreover, overweight (BMI ≥ 23 kg/m(2) for Asian population) or underweight occurred in 20% of women respectively highlighting the double burden of malnutrition. In children, a similar pattern was observed for ZnD (51.9 ± 3.5%), anemia (9.1 ± 1.4%) and ID (12.9 ± 1.5%) whereas prevalence of marginal vitamin A status was also high (47.3 ± 2.2%). There was a significant effect of age on anemia and ID prevalence, with the youngest age group (6-17 mo) having the highest risk for anemia, ID, ZnD and marginal vitamin A status as compared to other groups. Moreover, the poorest groups of population had a higher risk for zinc, anemia and ID., Conclusion: The prevalence of anemia and ID in Vietnam has been markedly reduced over the last decade, but a large part of the population is still at risk for other deficiencies such as zinc, vitamin A, folate and vitamin B(12) especially the youngest children aged 6-17 mo. Consequently specific interventions to improve food diversity and quality should be implemented, among them food fortification of staple foods and condiments and improvement of complementary feeding.

Acute bacterial meningitis is a life-threatening disease in humans. Discussed as entry sites for pathogens into the brain are the blood-brain and the blood-cerebrospinal fluid barrier (BCSFB). Although human brain microvascular endothelial cells (HBMEC) constitute a well established human in vitro model for the blood-brain barrier, until now no reliable human system presenting the BCSFB has been developed. Here, we describe for the first time a functional human BCSFB model based on human choroid plexus papilloma cells (HIBCPP), which display typical hallmarks of a BCSFB as the expression of junctional proteins and formation of tight junctions, a high electrical resistance and minimal levels of macromolecular flux when grown on transwell filters. Importantly, when challenged with the zoonotic pathogen Streptococcus suis or the human pathogenic bacterium Neisseria meningitidis the HIBCPP show polar bacterial invasion only from the physiologically relevant basolateral side. Meningococcal invasion is attenuated by the presence of a capsule and translocated N. meningitidis form microcolonies on the apical side of HIBCPP opposite of sites of entry. As a functionally relevant human model of the BCSFB the HIBCPP offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens.

Invasin and intimin are major virulence factors of enteropathogenic Yersiniae and Escherichia coli, mediating invasion into and intimate adherence to host cells, respectively. Several studies have hinted that extracellular portion of these homologous proteins might be exported via an autotransport mechanism, but rigorous experimental proof has been lacking. Here, we present a topology model for invasin and intimin, consistent with the hypothesis that the N-terminal β-barrel domain acts as a translocation pore to secrete the C-terminal passenger domain. We confirmed this topology model by inserting epitope tags into the loops of the β-barrel. We further show that obstructing the pore of β-barrel hinders the export of the passenger domain. As for classical autotransport, the biogenesis of invasin and intimin is dependent on the Bam complex and the periplasmic chaperone SurA, whereas the chaperone/protease DegP is involved in quality control. However, compared to classical autotransporters (Type Va secretion), the domain structure of intimin and invasin is inverted. We conclude that proteins of the intimin and invasin family constitute a novel group of autotransported proteins, and propose that this class of autotransporters be termed Type Ve secretion.

Background: Biofilm formation has been studied in much detail for a variety of bacterial species, as it plays a major role in the pathogenicity of bacteria. However, only limited information is available for the development of archaeal communities that are frequently found in many natural environments., Methodology: We have analyzed biofilm formation in three closely related hyperthermophilic crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii. We established a microtitre plate assay adapted to high temperatures to determine how pH and temperature influence biofilm formation in these organisms. Biofilm analysis by confocal laser scanning microscopy demonstrated that the three strains form very different communities ranging from simple carpet-like structures in S. solfataricus to high density tower-like structures in S. acidocaldarius in static systems. Lectin staining indicated that all three strains produced extracellular polysaccharides containing glucose, galactose, mannose and N-acetylglucosamine once biofilm formation was initiated. While flagella mutants had no phenotype in two days old static biofilms of S. solfataricus, a UV-induced pili deletion mutant showed decreased attachment of cells., Conclusion: The study gives first insights into formation and development of crenarchaeal biofilms in extreme environments.