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Patterns of proliferation are templated by both gradients of mechanical stress as well as by gradients in membrane voltage (Vm), which is defined as the electric potential difference between the cytoplasm and the extracellular medium. Either gradient could regulate the emergence of the other, or they could arise independently and synergistically affect proliferation within a tissue. Here, we examined the relationship between endogenous patterns of mechanical stress and the generation of bioelectric gradients in mammary epithelial tissues. We observed that the mechanical stress gradients in the tissues presaged gradients in both proliferation and depolarization, consistent with previous reports correlating depolarization with proliferation. Furthermore, disrupting the Vm gradient blocked the emergence of patterned proliferation. We found that the bioelectric gradient formed downstream of mechanical stresses within the tissues and depended on connexin-43 (Cx43) hemichannels, which opened preferentially in cells located in regions of high mechanical stress. Activation of Cx43 hemichannels was necessary for nuclear localization of Yap/Taz and induction of proliferation. Together, these results suggest that mechanotransduction triggers the formation of bioelectric gradients across a tissue, which are further translated into transcriptional changes that template patterns of growth.

Melanoma tumors are highly heterogeneous, comprising of many cell populations that vary in their potential for growth and invasion. Differential transcription factor expression contributes to these phenotypic traits. BRN2, a member of the POU domain family of transcription factors is thought to play important roles in melanoma invasion and metastasis. However, the function of BRN2 during the metastatic process of melanoma remains largely unknown. We therefore investigated the effect of BRN2 expression in melanoma cells with no or low constitutive expression using a doxycycline-inducible system. Induction of BRN2 expression led to reduced proliferation and partial resistance to an inhibitor of mutated BRAF. Whole-genome profiling analysis revealed novel targets and signaling pathway changes related to prevention of cell death induced by detachment from the extracellular matrix, known as anoikis resistance. Further investigation confirmed increased survival of BRN2-expressing cell lines in non-adherent conditions. Functionally, expression of BRN2 promoted induction of c-MET levels as well as increased phosphorylation of STAT3. Treatment with crizotinib, a c-MET inhibitor, decreased cellular viability of BRN2-expressing cells under non-adherent conditions to death by anoikis. Alternative inhibitors of c-MET showed similar results. These results highlight the importance of a largely overlooked transcription factor in the progression and metastasis of melanoma, and may suggest a strategy to target BRN2-expressing cells resistant to therapy and cell death by anoikis.

Background Information on the extent of high-risk prescribing for nonsteroidal anti-inflammatory drugs (NSAIDs) across developing countries is scarce. Objectives This study examines the prescribing pattern for NSAIDs in primary care, assesses the extent of high-risk NSAIDs prescribing and identifies associated factors. Setting 129 public and 416 private primary care clinics in Malaysia. Methods Data were derived from the National Medical Care Survey 2014, a cross-sectional survey on primary care morbidity patterns and clinical activities in Malaysia. Types of NSAIDs, indications for NSAIDs use and proportion of high-risk NSAIDs prescribing were assessed. Factors associated with high-risk NSAIDs prescribing were identified with a multivariable logistic regression. Weighted results, adjusted for sampling design and non-response were presented. Main outcome measures Prescribing pattern of NSAIDs, proportion of high-risk NSAIDs prescribing and its associated factors. Results Among the 55,489 patients who received NSAIDs, diclofenac was the most frequently prescribed NSAID (40.5%, 95% CI 40.1-40.9%), followed by mefenamic acid (29.2%, 95% CI 28.8-29.6%). The commonest indications for NSAIDs use were musculoskeletal condition and respiratory tract infection, both at 17.8% (95% CI 17.4-18.1%). A total of 22.9% (95% CI 22.6-23.3%) patients received high-risk NSAID prescriptions. Of these, 47.8% (95% CI 46.9-48.7%) did not receive adequate gastroprotection despite being at risk, 24.8% (95% CI 24.0-25.5%) were prescribed NSAIDs despite having cardiovascular comorbidities and 22.4% (95% CI 21.7-23.2%) were prescribed high-dose NSAIDs. The odds of receiving high-risk NSAID prescriptions increased with the number of drugs prescribed (OR 1.23, 95% CI 1.06-1.43) and the number of diagnoses in one visit (OR 2.21, 95% CI 1.71-2.86). The odds of being prescribed high-risk NSAID prescriptions were lower in patients with secondary (OR 0.52, 95% CI 0.35-0.77) and tertiary education (OR 0.39, 95% CI 0.22-0.68) compared to patients without formal education. Patients' citizenship, indication for NSAID prescriptions and whether a medical certificate was issued were also significantly associated with the likelihood of receiving high-risk NSAID prescriptions. Conclusions A quarter of NSAIDs prescribed in Malaysian primary care setting is categorised as high-risk prescribing. Targeted strategies are necessary to improve patient safety.

The prevalence of house dust mites (HDM)-sensitization is high in allergic asthma (AA), especially in tropical/subtropical countries in Asia and South America. As HDM-sensitization can contribute to the subsequent development of HDM-driven asthma, poor asthma control and exacerbations, allergen immunotherapy (AIT) might reduce the growing global burden of allergic asthma. AIT builds immunotolerance via upregulation of T-regulatory (reg) cells, leading to increase secretion of IL-10 and TGF-β which suppress the Th2 response to allergens. IL-10 also induces the class-switch to plasma cells that produce IgG4 that competes with Ig E’s binding on mast cell. Encouragingly, 2 recent multicentre trials showed that HDM AIT could lead to improvements in clinically relevant health outcomes: reduction in asthma exacerbations and inhaled corticosteroid usage. The uptake of AIT remains low due to difficulties with long-term adherence and lack of robust cost-effectiveness studies. There is limited standardization of techniques to compare the efficacy between different AIT administration routes and formulation. In clinical practice, it is challenging to differentiate “asthma with HDM sensitization” from “HDM-driven asthma”. Additionally, Blomia tropicalis (Blo.t) play an important allergenic role especially in tropics, in addition to the Dermatophagoides species but is not found in many AIT formulations. It is uncertain if Blo.t sensitised patients will respond equally to these HDM AIT. We will summarise (a) global differences in HDM sensitization, (b) disease-modifying mechanisms behind HDM AIT (c) differences between the subcutaneous and sublingual route (d) therapeutic strategies in HDM-driven asthma to maximise cost-effectiveness.

Background The prevalence rate of reflux esophagitis (RE) in Asia, including Taiwan, has increased dramatically in recent years. However, few studies have discussed on its relationship with metabolic syndrome (MetS). This study aimed to evaluate the correlation between RE and MetS and its components. Methods We conducted a cross-sectional study during 2013 to 2014 in Taiwan. A total of 4895 subjects who completed upper gastrointestinal endoscopy at the Health Examination Center of Changhua Christian Hospital were enrolled. RE was defined according to the upper gastrointestinal endoscopic findings and MetS was defined according to the Taiwanese criteria. Univariate and multivariate logistic regression analyses were applied to calculate odds ratios and 95% confidence intervals for each variable to assess the associated features for RE. We analyzed the relationship between the number of MetS components and the severity of RE using the chi-square test for trend. Results The prevalence rates of MetS and RE were respectively 28.5 and 59.6%. According to univariate logistic regression analysis, MetS was significantly associated with RE and remained a positive association in multivariate logistic regression analysis (adjusted ORß = 1.251; 95% CI = 1.071–1.462; p = 0.005). Furthermore, among the five MetS components, elevated blood pressure (adjusted ORγ = 1.163; 95% CI = 1.023–1.323; p = 0.021), abdominal obesity (adjusted ORγ = 1.173; 95% CI = 1.020–1.349; p = 0.026) and hyperglycemia (adjusted ORγ = 1.306; 95% CI = 1.142–1.495; p

Objective To determine the extent of self-management support (SMS) provided to primary care patients with type 2 diabetes (T2D) and hypertension and its associated factors. Design Cross-sectional survey conducted between April and May 2017. Setting Forty public clinics in Malaysia. Participants A total of 956 adult patients with T2D and/or hypertension were interviewed. Main Outcome Measures Patient experience on SMS was evaluated using a structured questionnaire of the short version Patient Assessment of Chronic Illness Care instrument, PACIC-M11. Linear regression analysis adjusting for complex survey design was used to determine the association of patient and clinic factors with PACIC-M11 scores. Results The overall PACIC-M11 mean was 2.3(SD,0.8) out of maximum of 5. The subscales’ mean scores were lowest for patient activation (2.1(SD,1.1)) and highest for delivery system design/decision support (2.9(SD,0.9)). Overall PACIC-M11 score was associated with age, educational level and ethnicity. Higher overall PACIC-M11 ratings was observed with increasing difference between actual and expected consultation duration [β = 0.01; 95% CI (0.001, 0.03)]. Better scores were also observed among patients who would recommend the clinic to friends and family [β = 0.19; 95% CI (0.03, 0.36)], when health providers were able to explain things in ways that were easy to understand [β = 0.34; 95% CI (0.10, 0.59)] and knew about patients’ living conditions [β = 0.31; 95% CI (0.15, 0.47)]. Conclusions Our findings indicated patients received low levels of SMS. PACIC-M11 ratings were associated with age, ethnicity, educational level, difference between actual and expected consultation length, willingness to recommend the clinic and provider communication skills.

Natural products are prolific producers of diverse chemical scaffolds, which have yielded several clinically useful drugs. However, the complex features of natural products present challenges for identifying bioactive molecules using high-throughput screens. For most assays, measured endpoints are either colorimetric or luminescence based. Thus, the presence of the major metabolites, tannins, and chlorophylls, in natural products could potentially interfere with these measurements to give either false-positive or false-negative hits. In this context, zebrafish phenotypic assays provide an alternative approach to bioprospect naturally occurring bioactive compounds. Whether tannins and/or chlorophylls interfere in zebrafish phenotypic assays, is unclear. In this study, we evaluated the interference potential of tannins and chlorophylls against efficacy of known small-molecule inhibitors that are known to cause phenotypic abnormalities in developing zebrafish embryos. First, we fractionated tannin-enriched fraction (TEF) and chlorophyll-enriched fraction (CEF) from Camellia sinensis and cotreated them with PD0325901 [mitogen-activated protein kinase-kinase (MEK) inhibitor] and sunitinib malate (SM; anti-[lymph]angiogenic drug). While TEF and CEF did not interfere with phenotypic or molecular endpoints of PD0325901, TEF at 100 μg/mL partially masked the antiangiogenic effect of SM. On the other hand, CEF (100 μg/mL) was toxic when treated up to 6 dpf. Furthermore, CEF at 100 μg/mL potentially enhanced the activity of γ-secretase inhibitors, resulting in toxicity of treated embryos. Our study provides evidence that the presence of tannin and/or chlorophyll in natural products do interfere with zebrafish phenotype assays used for identifying potential hits. However, this may be target/assay dependent and thus requiring additional optimization steps to assess interference potential of tannins and chlorophylls before performing any screening assay.

A 54-year-old autoclave technician was referred to the ambulatory care department of a UK district general hospital via his general practitioner (GP) with a 5-week history of a ‘profuse, papery’ rash ([figure 1A][1]), left groin lymphadenopathy, malaise, myalgia and fevers. He had a medical

Headache can be a primary or secondary disorder. The characteristics of headache and its associated features, especially the presence of red flag signs, are important in distinguishing secondary from primary causes. Hemicrania continua is a type of primary headache disorder characterized by a continuous unilateral headache with episodes of exacerbations and association with cranial autonomic symptoms, which include several ocular symptoms. The absolute response to indomethacin remains the hallmark of this disease. We would like to report a rare case of hemicrania continua with scintillating scotoma during exacerbations apart from the typical autonomic features of conjunctival injection, ptosis, eyelid edema, and lacrimation.

Introduction There are few sources of published data on intra-cluster correlation coefficients (ICCs) amongst patients with type 2 diabetes (T2D) and/or hypertension in primary care, particularly in low- and middle-income countries. ICC values are necessary for determining the sample sizes of cluster randomized trials. Hence, we aim to report the ICC values for a range of measures from a cluster-based interventional study conducted in Malaysia. Method Baseline data from a large study entitled Evaluation of Enhanced Primary Health Care interventions in public health clinics (EnPHC-EVA: Facility) were used in this analysis. Data from 40 public primary care clinics were collected through retrospective chart reviews and a patient exit survey. We calculated the ICCs for processes of care, clinical outcomes and patient experiences in patients with T2D and/or hypertension using the analysis of variance approach. Results Patient experience had the highest ICC values compared to processes of care and clinical outcomes. The ICC values ranged from 0.01 to 0.48 for processes of care. Generally, the ICC values for processes of care for patients with hypertension only are higher than those for T2D patients, with or without hypertension. However, both groups of patients have similar ICCs for antihypertensive medications use. In addition, similar ICC values were observed for clinical outcomes, ranging from 0.01 to 0.09. For patient experience, the ICCs were between 0.03 (proportion of patients who are willing to recommend the clinic to their friends and family) and 0.25 (for Patient Assessment of Chronic Illness Care item 9, Given a copy of my treatment plan). Conclusion The reported ICCs and their respective 95% confidence intervals for T2D and hypertension will be useful for estimating sample sizes and improving efficiency of cluster trials conducted in the primary care setting, particularly for low- and middle-income countries.

Aim: This paper describes the study protocol, which aims to evaluate the effectiveness of a multifaceted intervention package called ‘Enhanced Primary Healthcare’ (EnPHC) on the process of care and intermediate clinical outcomes among patients with Type 2 diabetes mellitus (T2DM) and hypertension. Other outcome measures include patients’ experience and healthcare providers’ job satisfaction. Background: In 2014, almost two-thirds of Malaysia’s adult population aged 18 years or older had T2DM, hypertension or hypercholesterolaemia. An analysis of health system performance from 2016 to 2018 revealed that the control and management of diabetes and hypertension in Malaysia was suboptimal with almost half of the patients not diagnosed and just one-quarter of patients with diabetes appropriately treated. EnPHC framework aims to improve diagnosis and effective management of T2DM, hypertension or hypercholesterolaemia and their risk factors by increasing prevention, optimising management and improving surveillance of diagnosed patients. Methods: This is a quasi-experimental controlled study which involves 20 intervention and 20 control clinics in two different states in Malaysia, namely Johor and Selangor. The clinics in the two states were matched and randomly allocated to ‘intervention’ and ‘control’ arms. The EnPHC framework targets different levels from community to primary healthcare clinics and integrated referral networks. Data are collected via a retrospective chart review (RCR), patient exit survey, healthcare provider survey and an intervention checklist. The data collected are entered into tablet computers which have installed in them an offline survey application. Interrupted time series and difference-in-differences (DiD) analyses will be conducted to report outcomes.

Patients with severe asthma that remain uncontrolled incur significant medical burden and healthcare costs. Severe asthma is a heterogeneous airway disorder with complex pathophysiological mechanisms which can be broadly divided into type 2 (T2)-high and T2-low inflammatory pathways. Recent advances in asthma therapeutics with the advent of biologics have heralded an era of promising targeted therapy in this group of patients. The current available biologics, including anti-IgE mAb, anti-IL-5/IL-5R mAb and anti-IL-4Rα mAb, mainly target patients with an asthma endotype characterised by T2-high inflammation. While they have delivered positive outcomes in terms of reduction in exacerbations, improving lung function and quality of life, as well as reducing the dependence on oral corticosteroids, they have not functioned as the "panacea" as a significant proportion of patients do not respond completely to these targeted therapies. In addition, there is a lack of markers that can predict treatment response and clinicians are guided only by subjective asthma symptom scores. Suboptimal treatment response is common for individual patients. There has also been a dearth of effective targeted therapy for patients with T2-low asthma and treatment options remain limited for these patients. There is a pipeline of newer biologics targeting cytokines that operate at the interface between innate and adaptive immunity (e.g. IL-17A, thymic stromal lymphopoietin (TSLP), IL-25, IL-33, IL-32 and IL-36γ) with potential of modifying and reducing the severity of asthma. This commentary provides an overview of treatment with the current biologics and highlights the limitations, challenges and unmet needs in clinical management. We also summarise up-and-coming potential targets and therapeutic biologics for severe asthma.

Purpose The purpose of this paper is to assess National Medical Care Survey data quality. Design/methodology/approach Data completeness and representativeness were computed for all observations while other data quality measures were assessed using a 10 per cent sample from the National Medical Care Survey database; i.e., 12,569 primary care records from 189 public and private practices were included in the analysis. Findings Data field completion ranged from 69 to 100 per cent. Error rates for data transfer from paper to web-based application varied between 0.5 and 6.1 per cent. Error rates arising from diagnosis and clinical process coding were higher than medication coding. Data fields that involved free text entry were more prone to errors than those involving selection from menus. The authors found that completeness, accuracy, coding reliability and representativeness were generally good, while data timeliness needs to be improved. Research limitations/implications Only data entered into a web-based application were examined. Data omissions and errors in the original questionnaires were not covered. Practical implications Results from this study provided informative and practicable approaches to improve primary health care data completeness and accuracy especially in developing nations where resources are limited. Originality/value Primary care data quality studies in developing nations are limited. Understanding errors and missing data enables researchers and health service administrators to prevent quality-related problems in primary care data.

Globally, group A streptococcal infections are responsible for over 500,000 deaths per year. A safe vaccine that does not induce autoimmune pathology and that affords coverage for most GAS serotypes is highly desired. We have previously demonstrated that a vaccine based on the conserved M-protein epitope, J8 was safe and immunogenic in a pilot Phase I study. We subsequently improved vaccine efficacy by incorporation of a B-cell epitope from the IL-8 protease, SpyCEP, which protected IL-8 and enhanced neutrophil ingress to the site of infection. We have now substituted the carrier protein, diphtheria toxoid with its superior analogue, CRM197 which provides better immunogenicity and is widely used in licenced human vaccines. The new vaccine was compared with the DT conjugate vaccine to confirm that these modifications have not altered the physicochemical properties of the vaccine. This vaccine, when tested in an animal model of GAS infection, demonstrated significant reduction in systemic and local GAS burden, with comparable efficacy to the DT conjugate vaccine. The vaccine was shown to be equally effective in the presence of human plasma and in the presence of pre-existing DT-specific antibodies, thus minimising concerns regarding its potential efficacy in humans.

Babesia spp. are tick-transmitted intra-erythrocytic protozoan parasites that infect humans and animals, causing a flu-like illness and hemolytic anemia. There is currently no human vaccine available. People most at risk of severe disease are the elderly, immunosuppressed, and asplenic individuals. B. microti and B. divergens are the predominant species affecting humans. Here, we present a whole-parasite Babesia vaccine. To establish proof-of-principle, we employed chemically attenuated B. microti parasitized red blood cells from infected mice. To aid clinical translation, we produced liposomes containing killed parasite material. Vaccination significantly reduces peak parasitemia following challenge. B cells and anti-parasite antibodies do not significantly contribute to vaccine efficacy. Protection is abrogated by the removal of CD4+ T cells or macrophages prior to challenge. Importantly, splenectomized mice are protected by vaccination. To further facilitate translation, we prepared a culture-based liposomal vaccine and demonstrate that this performs as a universal vaccine inducing immunity against different human Babesia species.

Reciprocal epithelial-mesenchymal signaling is essential for morphogenesis, including branching of the lung. In the mouse, mesenchymal cells differentiate into airway smooth muscle that wraps around epithelial branches, but this contractile tissue is absent from the early avian lung. Here, we found that branching morphogenesis in the embryonic chicken lung requires extracellular matrix (ECM) remodeling driven by reciprocal interactions between the epithelium and mesenchyme. Before branching, the basement membrane wraps the airway epithelium as a spatially uniform sheath. After branch initiation, however, the basement membrane thins at branch tips; this remodeling requires mesenchymal expression of matrix metalloproteinase-2, which is necessary for branch extension but not branch initiation. As branches extend, tenascin-C (TNC) accumulates in the mesenchyme several cell diameters away from the epithelium. Despite its pattern of accumulation, TNC is expressed exclusively by epithelial cells. Branch extension coincides with deformation of adjacent mesenchymal cells, which correlates with an increase in mesenchymal fluidity at branch tips that may transport TNC away from the epithelium. These data reveal novel epithelial-mesenchymal interactions that direct ECM remodeling during airway branching morphogenesis.

The development of a blood-stage malaria vaccine has largely focused on the subunit approach. However, the limited success of this strategy, mainly due to antigenic polymorphism and the failure to maintain potent parasite-specific immune responses, indicates that other approaches must be considered. Whole parasite (WP) vaccines offer many advantages over sub-units; they represent every antigen on the organism, thus limiting the effects of antigenic polymorphism, and similarly they compensate for individual Immune-Response (Ir) gene-regulated non-responsiveness to any particular antigen. From a development perspective, they negate the need to identify and compare the relative efficacies of individual candidate antigens. WP vaccines induce protective immunity that is largely cell-mediated. However, WP blood-stage vaccines present a number of challenges for the development pathway. Key issues are cryopreservation and storage and the possible induction of antibodies against red blood cell surface antigens, even if the parasites are grown in blood group O, Rh negative blood. Here, we used a novel adaptation of an immunomagnetic method from STEMCELL™ Technologies to remove the red cell membranes from human red blood cells parasitized with P. falciparum. We then used these antigens to construct liposomes which were modified to present mannose on their membrane to target the liposome to antigen presenting cells. We then compared the immunogenicity of freshly prepared and lyophilized liposome vaccines. Following vaccination of mice, liposomes induced significantly lower antibody responses to human red cells but potent strain- and species-transcending cell-mediated immune responses to parasite antigens. These data support transitioning the P. falciparum liposomal vaccine into clinical studies.

Background A major outbreak of the Zika virus (ZIKV) has been reported in Brazil in 2015. Since then, it spread further to other countries in the Americas and resulted in declaration of the Public Health Emergency of International Concern (PHEIC) by World Health Organization. In 2016, Singapore reported its first minor ZIKV epidemic. Malaysia shares similar ecological environment as Brazil and Singapore which may also favor ZIKV transmission. However, no ZIKV outbreak has been reported in Malaysia to date. This study aimed to discuss all confirmed ZIKV cases captured under Malaysia ZIKV surveillance system after declaration of the PHEIC; and explore why Malaysia did not suffer a similar ZIKV outbreak as the other two countries. Methods This was an observational study reviewing all confirmed ZIKV cases detected in Malaysia through the ZIKV clinical surveillance and Flavivirus laboratory surveillance between June 2015 and December 2017. All basic demographic characteristics, co-morbidities, clinical, laboratory and outcome data of the confirmed ZIKV cases were collected from the source documents. Results Only eight out of 4043 cases tested positive for ZIKV infection during that period. The median age of infected patients was 48.6 years and majority was Chinese. Two of the subjects were pregnant. The median interval between the onset of disease and the first detection of ZIKV Ribonucleic Acid (RNA) in body fluid was 3 days. Six cases had ZIKV RNA detected in both serum and urine samples. Phylogenetic analysis suggests that isolates from the 7 cases of ZIKV infection came from two clusters, both of which were local circulating strains. Conclusion Despite similar ecological background characteristics, Malaysia was not as affected by the recent ZIKV outbreak compared to Brazil and Singapore. This could be related to pre-existing immunity against ZIKV in this population, which developed after the first introduction of the ZIKV in Malaysia decades ago. A serosurvey to determine the seroprevalence of ZIKV in Malaysia was carried out in 2017. The differences in circulating ZIKV strains could be another reason as to why Malaysia seemed to be protected from an outbreak.

Naturally acquired immunity to malaria is robust and protective against all strains of the same species of Plasmodium. This develops as a result of repeated natural infection, taking several years to develop., Naturally acquired immunity to malaria is robust and protective against all strains of the same species of Plasmodium. This develops as a result of repeated natural infection, taking several years to develop. Evidence suggests that apoptosis of immune lymphocytes due to uncontrolled parasite growth contributes to the slow acquisition of immunity. To hasten and augment the development of natural immunity, we studied controlled infection immunization (CII) using low-dose exposure to different parasite species (Plasmodium chabaudi, P. yoelii, or P. falciparum) in two rodent systems (BALB/c and C57BL/6 mice) and in human volunteers, with drug therapy commencing at the time of initiation of infection. CIIs with infected erythrocytes and in conjunction with doxycycline or azithromycin, which are delayed death drugs targeting the parasite’s apicoplast, allowed extended exposure to parasites at low levels. In turn, this induced strong protection against homologous challenge in all immunized mice. We show that P. chabaudi/P. yoelii infection initiated at the commencement of doxycycline therapy leads to cellular or antibody-mediated protective immune responses in mice, with a broad Th1 cytokine response providing the best correlate of protection against homologous and heterologous species of Plasmodium. P. falciparum CII with doxycycline was additionally tested in a pilot clinical study (n = 4) and was found to be well tolerated and immunogenic, with immunological studies primarily detecting increased cell-associated immune responses. Furthermore, we report that a single dose of the longer-acting drug, azithromycin, given to mice (n = 5) as a single subcutaneous treatment at the initiation of infection controlled P. yoelii infection and protected all mice against subsequent challenge.

Background The continuing morbidity and mortality associated with infection with malaria parasites highlights the urgent need for a vaccine. The efficacy of sub-unit vaccines tested in clinical trials in malaria-endemic areas has thus far been disappointing, sparking renewed interest in the whole parasite vaccine approach. We previously showed that a chemically attenuated whole parasite asexual blood-stage vaccine induced CD4+ T cell-dependent protection against challenge with homologous and heterologous parasites in rodent models of malaria. Methods In this current study, we evaluated the immunogenicity and safety of chemically attenuated asexual blood-stage Plasmodium falciparum (Pf) parasites in eight malaria-naïve human volunteers. Study participants received a single dose of 3 × 107 Pf pRBC that had been treated in vitro with the cyclopropylpyrolloindole analogue, tafuramycin-A. Results We demonstrate that Pf asexual blood-stage parasites that are completely attenuated are immunogenic, safe and well tolerated in malaria-naïve volunteers. Following vaccination with a single dose, species and strain transcending Plasmodium-specific T cell responses were induced in recipients. This included induction of Plasmodium-specific lymphoproliferative responses, T cells secreting the parasiticidal cytokines, IFN-γ and TNF, and CD3+CD45RO+ memory T cells. Pf-specific IgG was not detected. Conclusions This is the first clinical study evaluating a whole parasite blood-stage malaria vaccine. Following administration of a single dose of completely attenuated Pf asexual blood-stage parasites, Plasmodium-specific T cell responses were induced while Pf-specific antibodies were not detected. These results support further evaluation of this chemically attenuated vaccine in humans. Trial registration Trial registration: ACTRN12614000228684. Registered 4 March 2014.

The aim of this study was to investigate factors influencing nursing students' intention to use a simulated web-application 'Integrated Nursing Education System' for practicing electronic health documentation. The cross-sectional study was conducted at a Malaysian University that provides undergraduate nursing degree program. A total of 133 undergraduate nursing students in their year 2 to year 4 of the program were recruited. They had accessed the simulated web-application as part of the teaching-learning activities during the academic year 2016/2017. Technology Acceptance Model was used to guide the study. Validated questionnaires were used to measure the students' perceived ease of use, perceived usefulness, attitudes and intention to use the application. Data collection was done at the end of the semester. Data analysis was done using SPSS (19.0) and AMOS (23.0). Perceived usefulness was the most influential factor of the students' intention to use the simulated web-application. Perceived ease of use influenced their perceived usefulness significantly. Nurse educators should explain the usefulness of the simulated web-application before assigning students to access it to practice electronic health documentation. The availability of a stable internet access and IT support is important to influence students' perceived ease of use and intention to use the application.

Breast tumors are stiffer and more hypoxic than nonmalignant breast tissue. Here we report that stiff and hypoxic microenvironments promote the development of breast cancer stem-like cells (CSC) through modulation of the integrin-linked kinase ILK. Depleting ILK blocked stiffness and hypoxia-dependent acquisition of CSC marker expression and behavior, whereas ectopic expression of ILK stimulated CSC development under softer or normoxic conditions. Stiff microenvironments also promoted tumor formation and metastasis in ovo, where depleting ILK significantly abrogated the tumorigenic and metastatic potential of invasive breast cancer cells. We further found that the ILK-mediated phenotypes induced by stiff and hypoxic microenvironments are regulated by PI3K/Akt. Analysis of human breast cancer specimens revealed an association between substratum stiffness, ILK, and CSC markers, insofar as ILK and CD44 were expressed in cancer cells located in tumor regions predicted to be stiff. Our results define ILK as a key mechanotransducer in modulating breast CSC development in response to tissue mechanics and oxygen tension. Cancer Res; 76(18); 5277–87. ©2016 AACR.

Central serous chorioretinopathy (CSCR) is a retinal condition characterized by fluid accumulation in the subretinal space, resulting in neurosensory detachment or pigment epithelial detachment. The risk factors associated with this condition include male gender, middle age, smoking, stress and use of corticosteroids. We report a case of CSCR in a 37-year-old policeman with hypertension and bronchial asthma. He presented with sudden onset of bilateral blurring of central vision for 1 day, worse over the left eye. There were no other significant eye complaints. He is an active smoker and has been on long-term corticosteroids for asthma. On examination, the visual acuity was 6/9 bilaterally. The anterior segment of both eyes was normal. Examination of the right fundus revealed a dome-shaped swelling inferior to the fovea, while the left fundus showed circular detachment of the neurosensory retina at the macula. He was diagnosed to have bilateral central serous chorioretinopathy. He was also counseled to stop smoking. The corticosteroids were continued due to the risk of precipitating an asthma attack if they were withheld. Upon his next review two months later, his condition remained stable. CSCR is usually a self-limited condition, with good visual outcome. A thorough medical, social and drug history should be obtained, and patients advised to modify their lifestyle to eliminate or reduce risk factors such as smoking, stress and corticosteroid use.

We report a case series of dengue maculopathy with different ocular manifestations, managements, visual outcomes and sequelae of patients. All three cases were diagnosed to have dengue fever. Patients presented with symptoms of central scotoma (case 1 and case 3) and blurring of vision (case 2) on Day 9 of dengue fever. Fundus examination showed intra retinal haemorrhages (case 1 and case 2) and macula thickening (case 3). Optical coherence tomography (OCT) revealed macular thickening with intra retinal fluid (case 1) and diffuse retinal thickening (case 3), while in case 2, it had irregular ellipsoid line in OCT. Fundus fluorescence angiography (FFA) revealed parafoveal vasculitis. After treatment with systemic steroids, one patient had good visual outcome (case 2), while two others had a persistent central scotoma due to macula atrophy (case 3). Although it is a self limiting disease, but the mode of treatment is variable. Treatment with corticosteroids may hasten recovery of vision and prevent permanent visual impairment.

Background Burkholderia pseudomallei is a gram negative bacteria that causes a spectrum of human diseases in the tropics. Although melioidosis is endemic in Southeast Asia, large clinical case series were rarely reported from metropolitan Singapore. Methods This is a retrospective study of 219 consecutive patients with culture proven infections due to Burkholderia pseudomallei between the years 2001 to 2016 managed in Singapore General Hospital (SGH). We aimed to review local patients’ characteristics and identify clinical factors associated with mortality and recurrent melioidosis. Results Culture proven melioidosis occurred in 219 patients, 83.1% were male with a mean age of 55.7 ± 14.3 years and 63.0% had diabetes mellitus. Most patients (71.7%) present within 4 weeks of symptom onset and the most common symptom was fever. The majority of patients had bacteremia (67.6%) and had infection involving the respiratory system (71.2%), presenting most frequently with multi-lobar pneumonia. Thirty-four (15.5%) deaths occurred during the initial hospitalisation with a median time from presentation to death of 6.0 days (interquartile range: 2.8–16.3). Twelve patients demised before the diagnosis of melioidosis was made. Univariate analysis identified patients with symptom duration of longer than 4 weeks, bacteremia, and disease requiring mechanical ventilation, inotropic support or temporary dialysis as factors that were significantly associated with mortality. Having bacteremia and disease requiring mechanical ventilation remained statistically significant factors in the multivariable analysis. Twenty-one (11.4%) patients developed at least 1 episode of culture proven recurrent infection, with 15 recurring within the first 12 months of their initial infection. Eight patients developed more than 1 episode of culture proven recurrent infection. Patients with multifocal infection were more likely to develop recurrent infection. Conclusion In metropolitan Singapore, melioidosis was associated with mortality in excess of 15%, where more than a third occurred before diagnosis. This study reminds local physicians that melioidosis is still a serious infection affecting local male diabetic patients and an important differential diagnosis in a patient presenting with severe multi-lobar pneumonia and septic shock. Recurrent infections occurred in 11.4% and the weight-based dosing of oral eradication antibiotics may improve the management of this disease locally.

Introduction Understanding the potential determinants of community healthcare seeking behaviour helps in improving healthcare utilisation and health outcomes within different populations. This in turn will aid the development of healthcare policies and planning for prevention, early diagnosis and management of health conditions. Objective To evaluate patients’ perception of community healthcare seeking behaviour towards both acute and preventive physical and psychosocial health concerns by sex, age and type of primary care setting (as a proxy for affordability of healthcare). Methods A total of 3979 patients from 221 public and 239 private clinics in Malaysia were interviewed between June 2015 and February 2016 using a patient experience survey questionnaire from the Quality and Cost of Primary Care cross-sectional study. Multivariable logistic regression analysis adjusted for the complex survey design was used. Results After adjusting for covariates, more women than men perceived that most people would see their general practitioners for commonly consulted acute and preventive physical and some psychosocial health concerns such as stomach pain (adjusted odds ratio (AOR), 1.64; 95% confidence interval (CI), 1.22–2.21), sprained ankle (AOR, 1.29; 95% CI, 1.06–1.56), anxiety (AOR, 1.32; 95% CI, 1.12–1.55), domestic violence (AOR, 1.35; 95% CI, 1.13–1.62) and relationship problems (AOR, 1.24; 95% CI, 1.02–1.51). There were no significant differences in perceived healthcare seeking behaviour by age groups except for the removal of a wart (AOR, 1.41; 95% CI, 1.12–1.76). Patients who visited the public clinics had generally higher perception of community healthcare seeking behaviour for both acute and preventive physical and psychosocial health concerns compared to those who went to private clinics. Conclusions Our findings showed that sex and healthcare affordability differences were present in perceived community healthcare seeking behaviour towards primary care services. Also perceived healthcare seeking behaviour were consistently lower for psychosocial health concerns compared to physical health concerns.

Background Polypharmacy is particularly important in older persons as they are more likely to experience adverse events compared to the rest of the population. Despite the relevance, there is a lack of studies on the possible association of patient, prescriber and practice characteristics with polypharmacy. Thus, the aim of this study was to determine the rate of polypharmacy among older persons attending public and private primary care clinics, and its association with patient, prescriber and practice characteristics. Methods We used data from The National Medical Care Survey (NMCS), a national cross-sectional survey of patients’ visits to primary care clinics in Malaysia. A weighted total of 22,832 encounters of patients aged ≥65 years were analysed. Polypharmacy was defined as concomitant use of five medications and above. Multilevel logistic regression was performed to examine the association of polypharmacy with patient, prescriber and practice characteristics. Results A total of 20.3% of the older primary care attenders experienced polypharmacy (26.7%% in public and 11.0% in private practice). The adjusted odds ratio (OR) of polypharmacy were 6.37 times greater in public practices. Polypharmacy was associated with patients of female gender (OR 1.49), primary education level (OR 1.61) and multimorbidity (OR 14.21). The variation in rate of polypharmacy was mainly found at prescriber level. Conclusion Polypharmacy is common among older persons visiting primary care practices. Given the possible adverse outcomes, interventions to reduce the burden of polypharmacy are best to be directed at individual prescribers.

Cells communicate constantly with their surrounding extracellular matrix (ECM) to maintain homeostasis, using both mechanical and chemical signals. In cancer, abnormal signaling leads to stiffening of the ECM. A stiff microenvironment affects many aspects of the cell, including internal molecular signaling as well as behaviors such as motility and proliferation. Thus, cells and ECM interact in a feedback loop to drive matrix deposition and cross-linking, which alter the mechanical properties of the tissue. Stiffer tissue enhances the invasive potential of a tumor and decreases therapeutic efficacy. This chapter describes how specific molecular effects caused by an abnormally stiff tissue drive macroscopic changes that help determine disease outcome. A complete understanding may foster the generation of new cancer therapies.

Yoga has recently been touted as a means to improve physical and mental well-being. However, no form of exercise is without its risks. A 32-year-old Chinese female with moderate myopia complained of right eye sudden onset of floaters and mild blurring of vision after the head-down posture. The visual acuity was 6/12 in the right eye and 6/9 in the left eye. A right eye fundus examination showed posterior vitreous detachment, with a small blood clot located at the inferior margin of the optic disc. The patient was diagnosed with right eye vitreous hemorrhage secondary to acute posterior vitreous detachment and was managed conservatively. Acute changes in posture, especially between an upright and a head-down position, may cause acute posterior vitreous detachment. As yoga practitioners may be required to assume this head-down position, myopic patients should be warned of the possible ocular complications of this exercise.

This study investigates the long-term effects of deoxynivalenol (DON) consumption on avian growth performance, on the proliferation, apoptosis, and DNA damage of spleen cells, and on intestinal integrity. Two hundred and eight 5-day-old black-feathered Taiwan country chickens were fed diets containing 0, 2, 5, and 10 mg/kg of DON for 16 weeks. Body weight gain of male birds in the 2 mg/kg group was significantly lower than that in the 5 mg/kg group. At the end of trial, feeding DON-contaminated diets of 5 mg/kg resulted in heavier spleens. Moreover, the increase in DON induced cellular proliferation, apoptosis, and DNA damage signals in the spleen, the exception being female birds fed 10 mg/kg of DON showing reduced proliferation. Expression of claudin-5 was increased in jejunum of female birds fed 2 and 5 mg/kg of DON, whereas decreased expression levels were found in male birds. In conclusion, our results verified that DON may cause a disturbance to the immune system and alter the intestinal barrier in Taiwan country chickens, and may also lead to discrepancies in growth performances in a dose- and sex-dependent manner.

SummaryThe airway epithelium develops into a tree-like structure via branching morphogenesis. Here, we show a critical role for localized differentiation of airway smooth muscle during epithelial bifurcation in the embryonic mouse lung. We found that during terminal bifurcation, changes in the geometry of nascent buds coincided with patterned smooth muscle differentiation. Evaluating spatiotemporal dynamics of α-smooth muscle actin (αSMA) in reporter mice revealed that αSMA-expressing cells appear at the basal surface of the future epithelial cleft prior to bifurcation and then increase in density as they wrap around the bifurcating bud. Disrupting this stereotyped pattern of smooth muscle differentiation prevents terminal bifurcation. Our results reveal stereotyped differentiation of airway smooth muscle adjacent to nascent epithelial buds and suggest that localized smooth muscle wrapping at the cleft site is required for terminal bifurcation during airway branching morphogenesis.

The Wnt/β-catenin pathway controls a variety of cellular behaviors, aberrant activation of which are associated with tumor progression in several types of cancer. The same cellular behaviors are also affected by the mechanical properties of the extracellular matrix (ECM) substratum, which induces signaling through integrins and integrin-linked kinase (ILK). Here, we examined the role of substratum stiffness in the regulation of cell proliferation downstream of Wnt3a. We found that treatment with Wnt3a increased proliferation of cells cultured on stiff substrata, with compliances characteristic of breast tumors, but not of cells on soft substrata, with compliances comparable to that of normal mammary tissue. Depleting ILK rendered cells unresponsive to Wnt3a on both substrata. Ectopic expression of ILK permitted Wnt3a to induce proliferation of cells on both microenvironments, although proliferation on soft substrata remained lower than that on stiff substrata. We further showed that ILK regulates expression of the Wnt receptor frizzled-1 (Fzd1), suggesting the presence of a positive feedback loop between Wnt3a, ILK and Fzd1. These findings suggest that tissue mechanics regulates the cellular response to Wnt under physiological and pathological microenvironmental conditions. This article has an associated First Person interview with the first author of the paper.

Cryptococcus gattii is an endemic fungus predominantly isolated in the tropical and subtropical regions, causing predominantly pulmonary disease with a predilection for the central nervous system. Herein, we report a case of rapidly progressing C. gattii pneumonia in an immune-deficient but virologically suppressed host with underlying human immunodeficiency viral (HIV) infection, exhibiting various fungal morphologies from bronchoalveolar lavage (BAL) cytological specimens. A 51-year-old Chinese male with known HIV disease was admitted to the Singapore General Hospital for evaluation of functional decline, febrile episodes, and a left hilar mass on chest radiograph. Computed tomography (CT) showed consolidation in the apical segment of the left lower lobe. He underwent bronchoscopy and BAL. Positron emission tomography-computed tomography done 10 days after the initial CT showed approximate doubling of the pulmonary lesion. Cytological examination of the fluid revealed yeasts of varying sizes. Subsequent fungal culture from BAL fluid grew C. gattii 10 days later.

Bidirectional co-morbidity between migraine and depression has been observed. Mood disorders are associated with an increased risk of both migraine and irritable bowel syndrome (IBS). The aim of this study was to evaluate the risk of developing IBS in patients with migraine and to compare the risks between those with and without anxiety or depression. This research used the data contained in the National Health Insurance Research Database (NHIRD). A total of 2859 subjects with migraine and 5718 age-, sex-, hypertension-, diabetes-, mood disorder-matched controls were identified. Both cohorts excluded subjects with pre-existing catastrophic illness and IBS diagnosed before the index visit or within 30days after the index visit. All individuals of both cohorts were tracked until either having the diagnosis of IBS, loss of follow-up, or IBS free up to 7years. During the 7-year follow-up period, 8.4% of patients with migraine and 5.4% of control cohort developed IBS. Migraine is associated with an increased risk of developing IBS (HR=1.58, 95% CI: 1.33-1.87). When separating the cohort into those with mood disorder and without it, migraine is a significant risk factor of IBS in patients without mood disorders, but not in patients with co-existed mood disorders. The findings of this study suggest that migraine is a risk factor of future IBS development for those without comorbid anxiety or depression. However, migraine does not contribute significantly additional risk to IBS development in patients with comorbid anxiety or depression.

The upper respiratory tract (URT) is the major entry site for human pathogens and strategies to activate this network could lead to new vaccines capable of preventing infection with many pathogens. Group A streptococcus (GAS) infections, causing rheumatic fever, rheumatic heart disease, and invasive disease, are responsible for substantial morbidity and mortality. We describe an innovative vaccine strategy to induce mucosal antibodies of significant magnitude against peptide antigens of GAS using a novel biocompatible liposomal platform technology. The approach is to encapsulate free diphtheria toxoid (DT), a standard vaccine antigen, within liposomes as a source of helper T-cell stimulation while lipidated peptide targets for B-cells are separately displayed on the liposome surface. As DT is not physically conjugated to the peptide, it is possible to develop modular epitopic constructs that simultaneously activate IgA-producing B-cells of different and complementary specificity and function that together neutralize distinct virulence factors. An inflammatory cellular immune response is also induced. The immune response provides profound protection against streptococcal infection in the URT. The study describes a new vaccine platform for humoral and cellular immunity applicable to the development of vaccines against multiple mucosal pathogens.

Zebrafish represents a powerful in vivo model for phenotype-based drug discovery to identify clinically relevant small molecules. By utilizing this model, we evaluated natural product derived compounds that could potentially modulate Notch signaling that is important in both zebrafish embryogenesis and pathogenic in human cancers. A total of 234 compounds were screened using zebrafish embryos and 3 were identified to be conferring phenotypic alterations similar to embryos treated with known Notch inhibitors. Subsequent secondary screens using HEK293T cells overexpressing truncated Notch1 (HEK293TΔE) identified 2 compounds, EDD3 and 3H4MB, to be potential Notch antagonists. Both compounds reduced protein expression of NOTCH1, Notch intracellular domain (NICD) and hairy and enhancer of split-1 (HES1) in HEK293TΔE and downregulated Notch target genes. Importantly, EDD3 treatment of human oral cancer cell lines demonstrated reduction of Notch target proteins and genes. EDD3 also inhibited proliferation and induced G0/G1 cell cycle arrest of ORL-150 cells through inducing p27KIP1. Our data demonstrates the utility of the zebrafish phenotypic screen and identifying EDD3 as a promising Notch antagonist for further development as a novel therapeutic agent.

Cells are surrounded by mechanical stimuli in their microenvironment. It is important to determine how cells respond to the mechanical information that surrounds them in order to understand both development and disease progression, as well as to be able to predict cell behavior in response to physical stimuli. Here we describe a protocol to determine the effects of interstitial fluid flow on the migratory behavior of an aggregate of epithelial cells in a three-dimensional (3D) culture model. This protocol includes detailed methods for the fabrication of a 3D cell culture chamber with hydrostatic pressure control, the culture of epithelial cells as an aggregate in a collagen gel, and the analysis of collective cell behavior in response to pressure-driven flow.

A 74-year-old man presented with cough of 3 weeks' duration. Chest X-ray showed a large left-sided pleural effusion (figure 1) that was a lymphocytic exudate with normal adenosine deaminase level, and negative bacterial and mycobacterial cultures. Flexi-rigid pleuroscopy (LTF 160, Olympus, Japan) revealed candle-wax fibrotic parietal pleura (figure 2), which was sampled using the flexible forceps (FB-55CD-1, Olympus, Japan) with cusps measuring 2.4 mm when fully open, and a cryoprobe of 2.4 mm in diameter (20416-032, Erbokryo CA, Erbe, Germany). The cryoprobe was directed to the target area and 4-second freeze time was applied. The pleuroscope and cryoprobe with the pleural specimen attached …

Cancer metastasis which predominantly occurs through blood and lymphatic vessels, is the leading cause of death in cancer patients. Consequently, several anti-angiogenic agents have been approved as therapeutic agents for human cancers such as metastatic renal cell carcinoma. Also, anti-lymphangiogenic drugs such as monoclonal antibodies VGX-100 and IMC-3C5 have undergone phase I clinical trials for advanced and metastatic solid tumors. Although anti-tumor-associated angiogenesis has proven to be a promising therapeutic strategy for human cancers, this approach is fraught with toxicities and development of drug resistance. This emphasizes the need for alternative anti-(lymph)angiogenic drugs. The use of zebrafish has become accepted as an established model for high-throughput screening, vascular biology, and cancer research. Importantly, various zebrafish transgenic lines have now been generated that can readily discriminate different vascular compartments. This now enables detailed in vivo studies that are relevant to both human physiological and tumor (lymph)angiogenesis to be conducted in zebrafish. This review highlights recent advancements in the zebrafish anti-vascular screening platform and showcases promising new anti-(lymph)angiogenic compounds that have been derived from this model. In addition, this review discusses the promises and challenges of the zebrafish model in the context of anti-(lymph)angiogenic compound discovery for cancer treatment.

Non-arteritic anterior ischaemic optic neuropathy (NAION) may develop due to severe anaemia and hypotension which is seen in acute blood loss. The devastating visual loss is often irreversible. We report a case of NAION in a 20-year-old healthy woman, who presented on the third day of a heavy menstrual cycle with hypovolaemic shock. On day 2 of admission, she had sudden right eye blurring of vision at the superior field on awakening from sleep. Funduscopy revealed a pale and swollen right optic disc. There was a dense right superior altitudinal visual field defect. Her haemoglobin level was low (3.6 g/dL), but she refused blood transfusion due to her religious belief (Jehovah's Witness) and opted for conservative management. She later developed right optic atrophy with persistent visual field defect despite an improved haemoglobin level of 10.5 g/dL.

Melioidosis is a notifiable infectious disease registered with the Ministry of Health (MOH) and Agri-Food & Veterinary Authority (AVA), Singapore. From a clinical perspective, increased awareness of the disease has led to early detection and treatment initiation, thus resulting in decreasing mortality rates in recent years. However, the disease still poses a threat to local pet, zoo and farm animals, where early diagnosis is a challenge. The lack of routine environmental surveillance studies also makes prevention of the disease in animals difficult. To date, there have been no reports that provide a complete picture of how the disease impacts the local human and animal populations in Singapore. Information on the distribution of Burkholderia pseudomallei in the environment is also lacking. The aim of this review is to provide a comprehensive overview of both published and unpublished clinical, veterinary and environmental studies on melioidosis in Singapore to achieve better awareness and management of the disease.

Background Maternal consumption of alcohol during pregnancy is associated with a range of physical, cognitive and behavioural outcomes in the offspring which are collectively called foetal alcohol spectrum disorders. We and others have proposed that epigenetic modifications, such as DNA methylation and post-translational histone modifications, mediate the effects of prenatal alcohol exposure on gene expression and, ultimately, phenotype. Here we use an inbred C57BL/6J mouse model of early gestational ethanol exposure equivalent, developmentally, to the first 3–4 weeks of pregnancy in humans to examine the long-term effects on gene expression and epigenetic state in the hippocampus. Results Gene expression analysis in the hippocampus revealed sex- and age-specific up-regulation of solute carrier family 17 member 6 (Slc17a6), which encodes vesicular glutamate transporter 2 (VGLUT2). Transcriptional up-regulation correlated with decreased DNA methylation and enrichment of histone H3 lysine 4 trimethylation, an active chromatin mark, at the Slc17a6 promoter. In contrast to Slc17a6 mRNA levels, hippocampal VGLUT2 protein levels were significantly decreased in adult ethanol-exposed offspring, suggesting an additional level of post-transcriptional control. MicroRNA expression profiling in the hippocampus identified four ethanol-sensitive microRNAs, of which miR-467b-5p was predicted to target Slc17a6. In vitro reporter assays showed that miR-467b-5p specifically interacted with the 3′UTR of Slc17a6, suggesting that it contributes to the reduction of hippocampal VGLUT2 in vivo. A significant correlation between microRNA expression in the hippocampus and serum of ethanol-exposed offspring was also observed. Conclusions Prenatal ethanol exposure has complex transcriptional and post-transcriptional effects on Slc17a6 (VGLUT2) expression in the mouse hippocampus. These effects are observed following a relatively moderate exposure that is restricted to early pregnancy, modelling human consumption of alcohol before pregnancy is confirmed, and are only apparent in male offspring in adulthood. Our findings are consistent with the idea that altered epigenetic and/or microRNA-mediated regulation of glutamate neurotransmission in the hippocampus contributes to the cognitive and behavioural phenotypes observed in foetal alcohol spectrum disorders. Although further work is needed in both mice and humans, the results also suggest that circulating microRNAs could be used as biomarkers of early gestational ethanol exposure and hippocampal dysfunction. Electronic supplementary material The online version of this article (doi:10.1186/s13072-015-0032-6) contains supplementary material, which is available to authorized users.

Poor prognosis of most cancer patients is in part, due to limited therapeutic options. Furthermore, as chemotherapy remains the standard-of-care for several cancers, partial or lack of response remains a concern and compounding this are the adverse side effects of the treatment that severely impacts the quality of life and survival. In pursuit of improving treatment options, we have opted to investigate the unique chemical skeleton of natural compounds as anticancer therapies. In this study, from an initial screen of 31 crude methanol extracts from ~15 plant species using HL60 cells, the root extract of Brucea javanica (L.) Merr indicated the presence of bioactive compounds. Subsequent bioassay-guided purification on the root extract yielded two alkaloids canthin-6-one (1) and bruceolline J (2), which were further investigated for their bioactivity in representative human cancer lines and normal phenotypic counterparts. MTT assay demonstrated ED50values from 34.7–72.9 μM for 1 and 16.0–54.0 μM for 2 for the cancer cell lines panel. NP69 cells also demonstrated sensitivity to both compounds (9.3 μM and 4.5 μM). As amount of 2 isolated were limiting, we focused on 1 to further identify novel anticancer properties in PC3 and HeLa cancer lines. We observed at 30 μM, 1 induced a G2/M phase arrest coinciding with decreased cell proliferation. Furthermore, 1 was able to synergize the cytotoxic effect of cisplatin when used in combination, suggesting the potential of combination therapy for those less responsive lesions to standard chemotherapy.

The immunobiology underlying the slow acquisition of skin immunity to group A streptococci (GAS), is not understood, but attributed to specific virulence factors impeding innate immunity and significant antigenic diversity of the type-specific M-protein, hindering acquired immunity. We used a number of epidemiologically distinct GAS strains to model the development of acquired immunity. We show that infection leads to antibody responses to the serotype-specific determinants on the M-protein and profound protective immunity; however, memory B cells do not develop and immunity is rapidly lost. Furthermore, antibodies do not develop to a conserved M-protein epitope that is able to induce immunity following vaccination. However, if re-infected with the same strain within three weeks, enduring immunity and memory B-cells (MBCs) to type-specific epitopes do develop. Such MBCs can adoptively transfer protection to naïve recipients. Thus, highly protective M-protein-specific MBCs may never develop following a single episode of pyoderma, contributing to the slow acquisition of immunity and to streptococcal endemicity in at-risk populations., Author Summary GAS skin infections pose a significant health problem in the tropics. They are highly prevalent in developing countries as well as amongst the Indigenous populations of developed countries. In at-risk impoverished communities the epidemiology of GAS infections is very dynamic, leading to very high rates of streptococcal-associated serious pathology including rheumatic heart disease, glomerulonephritis and invasive GAS disease. Immunity to GAS takes over 20 years to develop and this has been attributed to sequence diversity of the type-specific surface M-protein. There are more than 250 different strains of GAS and it known that antibodies to the amino-terminal segment of the M-protein can kill organisms in a strain-specific manner in vitro. In the present study, using four different strains of GAS isolated from the skin lesions of Aboriginal patients in the Northern Territory of Australia, we make the discovery that skin infection does not induce long-lived type-specific immunity. However, following reinfection with the same strain memory B cells are generated and long-term strain-protective immunity then develops. The dependence on reinfection for the development of strain-specific immunity compounds with antigenic diversity of the M-protein and provides a rational explanation for the very slow acquisition of streptococcal immunity.

The coxsackie- and adenovirus receptor (CAR) is a cell adhesion molecule predominantly associated with epithelial tight junctions in adult tissues. CAR is also expressed in cardiomyocytes and essential for heart development up to embryonic day 11.5, but not thereafter. CAR is not expressed in vascular endothelial cells but was recently detected in neonatal lymphatic vessels, suggesting that CAR could play a role in the development of the lymphatic system. To address this, we generated mice carrying a conditional deletion of the CAR gene (Cxadr) and knocked out CAR in the mouse embryo at different time points during post-cardiac development. Deletion of Cxadr from E12.5, but not from E13.5, resulted in subcutaneous edema, hemorrhage and embryonic death. Subcutaneous lymphatic vessels were dilated and structurally abnormal with gaps and holes present at lymphatic endothelial cell-cell junctions. Furthermore, lymphatic vessels were filled with erythrocytes showing a defect in the separation between the blood and lymphatic systems. Regionally, erythrocytes leaked out into the interstitium from leaky lymphatic vessels explaining the hemorrhage detected in CAR-deficient mouse embryos. The results show that CAR plays an essential role in development of the lymphatic vasculature in the mouse embryo by promoting appropriate formation of lymphatic endothelial cell-cell junctions.

Background: Malaria rapid diagnostic tests (RDTs) offer significant potential to improve the diagnosis of malaria, and are playing an increasing role in malaria case management, control and elimination. Peru, along with other South American countries, is moving to introduce malaria RDTs as components of malaria control programmes supported by the Global Fund for AIDS, TB and malaria. The selection of the most suitable malaria RDTs is critical to the success of the programmes. Methods: Eight of nine microscopy positive P. falciparum samples collected in Iquitos, Peru tested negative or weak positive using HRP2-detecting RDTs. These samples were tested for the presence of pfhrp2 and pfhrp3 and their flanking genes by PCR, as well as the presence of HRP proteins by ELISA. To investigate for geographic extent of HRP-deleted parasites and their temporal occurrence a retrospective study was undertaken on 148 microscopy positive P. falciparum samples collected in different areas of the Amazon region of Peru. Findings: Eight of the nine isolates lacked the pfhrp2 and/or pfhrp3 genes and one or both flanking genes, and the absence of HRP was confirmed by ELISA. The retrospective study showed that 61 (41%) and 103 (70%) of the 148 samples lacked the pfhrp2 or pfhrp3 genes respectively, with 32 (21.6%) samples lacking both hrp genes. Conclusions: This is the first documentation of P. falciparum field isolates lacking pfhrp2 and/or pfhrp3. The high frequency and wide distribution of different parasites lacking pfhrp2 and/or pfhrp3 in widely dispersed areas in the Peruvian Amazon implies that malaria RDTs targeting HRP2 will fail to detect a high proportion of P. falciparum in malaria-endemic areas of Peru and should not be used. RDTs detecting parasite LDH or aldolase and quality microscopy should be use for malaria diagnosis in this region. There is an urgent need for investigation of the abundance and geographic distribution of these parasites in Peru and neighbouring countries.

Background Scabies is a parasitic skin infestation caused by the burrowing mite Sarcoptes scabiei. It is common worldwide and spreads rapidly under crowded conditions, such as those found in socially disadvantaged communities of Indigenous populations and in developing countries. Pruritic scabies lesions facilitate opportunistic bacterial infections, particularly Group A streptococci. Streptococcal infections cause significant sequelae and the increased community streptococcal burden has led to extreme levels of acute rheumatic fever and rheumatic heart disease in Australia's Indigenous communities. In addition, emerging resistance to currently available therapeutics emphasizes the need to identify potential targets for novel chemotherapeutic and/or immunological intervention. Scabies research has been severely limited by the availability of parasites, and scabies remains a truly neglected infectious disease. We report development of a tractable model for scabies in the pig, Sus domestica. Methodology/Principal Findings Over five years and involving ten independent cohorts, we have developed a protocol for continuous passage of Sarcoptes scabiei var. suis. To increase intensity and duration of infestation without generating animal welfare issues we have optimised an immunosuppression regimen utilising daily oral treatment with 0.2mg/kg dexamethasone. Only mild, controlled side effects are observed, and mange infection can be maintained indefinitely providing large mite numbers (>6000 mites/g skin) for molecular-based research on scabies. In pilot experiments we explore whether any adaptation of the mite population is reflected in genetic changes. Phylogenetic analysis was performed comparing sets of genetic data obtained from pig mites collected from naturally infected pigs with data from pig mites collected from the most recent cohort. Conclusions/Significance A reliable pig/scabies animal model will facilitate in vivo studies on host immune responses to scabies including the relations to the associated bacterial pathogenesis and more detailed studies of molecular evolution and host adaption. It is a most needed tool for the further investigation of this important and widespread parasitic disease., Author Summary Scabies, a neglected parasitic disease caused by the microscopic mite Sarcoptes scabiei, is a major driving force behind bacterial skin infections in tropical settings. Aboriginal and Torres Strait Islander peoples are nearly twenty times more likely to die from acute rheumatic fever and rheumatic heart disease than individuals from the wider Australian community. These conditions are caused by bacterial pathogens such as Group A streptococci, which have been linked to underlying scabies infestations. Community based initiatives to reduce scabies and associated disease have expanded, but have been threatened in recent years by emerging drug resistance. Critical biological questions surrounding scabies remain unanswered due to a lack of biomedical research. This has been due in part to a lack of either a suitable animal model or an in vitro culture system for scabies mites. The pig/mite model reported here will be a much needed resource for parasite material and will facilitate in vivo studies on host immune responses to scabies, including relations to associated bacterial pathogenesis, and more detailed studies of molecular evolution and host adaptation. It represents the missing tool to extrapolate emerging molecular data into an in vivo setting and may well allow the development of clinical interventions.

Background Accurate diagnosis is essential for prompt and appropriate treatment of malaria. While rapid diagnostic tests (RDTs) offer great potential to improve malaria diagnosis, the sensitivity of RDTs has been reported to be highly variable. One possible factor contributing to variable test performance is the diversity of parasite antigens. This is of particular concern for Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-detecting RDTs since PfHRP2 has been reported to be highly variable in isolates of the Asia-Pacific region. Methods The pfhrp2 exon 2 fragment from 458 isolates of P. falciparum collected from 38 countries was amplified and sequenced. For a subset of 80 isolates, the exon 2 fragment of histidine-rich protein 3 (pfhrp3) was also amplified and sequenced. DNA sequence and statistical analysis of the variation observed in these genes was conducted. The potential impact of the pfhrp2 variation on RDT detection rates was examined by analysing the relationship between sequence characteristics of this gene and the results of the WHO product testing of malaria RDTs: Round 1 (2008), for 34 PfHRP2-detecting RDTs. Results Sequence analysis revealed extensive variations in the number and arrangement of various repeats encoded by the genes in parasite populations world-wide. However, no statistically robust correlation between gene structure and RDT detection rate for P. falciparum parasites at 200 parasites per microlitre was identified. Conclusions The results suggest that despite extreme sequence variation, diversity of PfHRP2 does not appear to be a major cause of RDT sensitivity variation.