Detection of harmful cyanobacteria and their toxins by both PCR amplification and LC-MS during a bloom event.

We briefly report here the occurrence of toxic blooms in the eutrophic reservoir Billings, S?o Paulo city, Brazil. Water samples were collected in May 2004, during a cyanobacterial bloom. The presence of toxic species was confirmed by using PCR amplifications of a fragment region of genes encoding microcystin synthetase-mcyB. The determination of toxins was performed by liquid chromatography coupled with mass spectrometry (LC-MS). LC-MS analyses of the toxins from the bloom revealed variants of microcystins (MC), such as MC-LR, MC-RR and MC-YR. HPLC-FLD was used to determine the paralytic shellfish poisoning (PSP) saxitoxin (STX), neosaxitoxin (NEO), gonyautoxins 2 (GTX2) and 3 (GTX3). GTX2, GTX3 and NEO were detected for the first time in a natural sample from Billings reservoir. These results are a contribution to the knowledge of the biogeography of toxic cyanobacteria and their toxins, specifically in S?o Paulo.     

Molecular mechanism for differential recognition of membrane phosphatidylserine by the immune regulatory receptor Tim4

Recognition of phosphatidylserine (PS) lipids exposed on the extracellular leaflet of plasma membranes is implicated in both apoptotic cell removal and immune regulation. The PS receptor T cell immunoglobulin and mucin-domain-containing molecule 4 (Tim4) regulates T-cell immunity via phagocytosis of both apoptotic (high PS exposure) and nonapoptotic (intermediate PS exposure) activated T cells. The latter population must be removed at lower efficiency to sensitively control immune tolerance and memory cell population size, but the molecular basis for how Tim4 achieves this sensitivity is unknown. Using a combination of interfacial X-ray scattering, molecular dynamics simulations, and membrane binding assays, we demonstrate how Tim4 recognizes PS in the context of a lipid bilayer. Our data reveal that in addition to the known Ca²⁺-coordinated, single-PS binding pocket, Tim4 has four weaker sites of potential ionic interactions with PS lipids. This organization makes Tim4 sensitive to PS surface concentration in a manner capable of supporting differential recognition on the basis of PS exposure level. The structurally homologous, but functionally distinct, Tim1 and Tim3 are significantly less sensitive to PS surface density, likely reflecting the differences in immunological function between the Tim proteins. These results establish the potential for lipid membrane parameters, such as PS surface density, to play a critical role in facilitating selective recognition of PS-exposing cells. Furthermore, our multidisciplinary approach overcomes the difficulties associated with characterizing dynamic protein/membrane systems to reveal the molecular mechanisms underlying Tim4’s recognition properties, and thereby provides an approach capable of providing atomic-level detail to uncover the nuances of protein/membrane interactions.Immunological recognition of the lipid phosphatidylserine (PS) has predominately been associated with the phagocytic removal of apoptotic cells (1). In a healthy cell, PS is typically confined to the intracellular leaflet of the plasma membrane by ATP-dependent, lipid-sorting processes (2). Apoptosis induces extracellular exposure of PS, whereupon immune scavenger cells bearing PS-specific receptors can recognize and remove the dying cells (1). This seemingly simple paradigm is complicated by the fact that extracellular PS exposure is not just a hallmark of cell death but is also functionally relevant during the activation of various immune cells, including T cells (3–7). Moreover, there exists a wide array of structurally diverse PS receptors, the expression of which is not strictly limited to immune cells capable of phagocytosis (8, 9). Accordingly, recognition of exposed PS is not always sufficient for phagocytosis (10) and can actually elicit a nonphagocytic response, such as immune cell proliferation (11).Thus, the question remains: How does the immune system appropriately interpret apoptotic versus nonapoptotic PS exposure? Additional factors, such as the “don’t eat me” signals CD31 (12) and CD47 (13, 14), can, in some cases, modulate the immune response to PS exposure. However, it remains unclear whether all PS receptors recognize any instance of exposed PS equally or whether the physiological context of PS exposure can influence recognition. Addressing this central question has been exceedingly difficult, given that the standard tools of structural immunology are not well suited to characterizing dynamic membrane systems. Nevertheless, it appears that the ability to discern apoptotic versus nonapoptotic instances of PS exposure may indeed be a required feature of at least one PS receptor, T cell immunoglobulin and mucin-domain-containing molecule 4 (Tim4) (15, 16).Tim4 is a member of the Tim family of proteins that has collectively been associated with a variety of immune regulatory disorders including allergy, asthma, autoimmunity, and transplant intolerance (9, 17, 18). Correspondingly, each of the Tim proteins, which includes Tim1–4 in mouse and the homologous Tim1, 3, and 4 in humans (Tim2 is not expressed in humans), are associated with regulating T-cell immunity, although with distinctly different roles (9). Tim1, for example, can provide a costimulatory signal leading to T-cell proliferation (19), whereas Tim4 mediates the recognition of PS-exposing T cells for the purposes of phagocytic removal (15, 16, 20–22). The crystal structure of Tim4 in complex with a single-PS head group has provided invaluable insight into how Tim4 specifically recognizes a single-PS molecule (23). However, the molecular details of how Tim4 recognizes PS within the context of a lipid membrane remain unknown.It is now apparent that these details may be critical to understanding the immunological function of Tim4. Tim4 has recently been shown to regulate T-cell populations during the resolution of an immune response by mediating the PS-dependent phagocytosis of both apoptotic and nonapoptotic antigen-specific T cells (15, 16). In one capacity, Tim4 initiates the highly selective and thorough clearance of apoptotic T cells by virtue of the exposure of high levels of PS on their surfaces (15, 20, 21). Equally important, however, is that Tim4 is also instrumental in pruning the population size of activated, but nonapoptotic, T cells. This activated T-cell removal is important for both immune tolerance in the lungs (16) and the maintenance of an appropriately sized pool of memory T cells after viral infection (15). Although nonapoptotic T-cell removal is believed to occur via the same PS-dependent mechanisms as apoptotic cell phagocytosis, it must necessarily be done with less vigor to ensure the appropriate level of controlled regulation (15, 16).Activated, nonapoptotic T cells have been shown to expose PS at lower whole-cell surface densities than apoptotic T cells (3, 16, 24), but the role of these differences in level of PS surface exposure is unknown. Importantly, Tim4 ignores healthy, resting T cells, presumably because of their low basal levels of PS exposure. We therefore hypothesized that Tim4 may discriminate among these three states of T cells by virtue of their different levels of cell-surface PS exposure. Given the diversity in immunological function of the Tim proteins, we further hypothesized that Tim4 might have evolved a specific sensitivity to PS surface density distinct from that of other Tim proteins.In this study, we develop a multidisciplinary approach to overcome the significant technological barriers associated with studying the molecular basis of membrane PS recognition by Tim4. Using a combination of interfacial X-ray scattering, molecular dynamics simulations, and membrane binding assays, we show that in addition to the known Ca²⁺-dependent, single-PS binding pocket (23), Tim4 also possesses four peripheral basic residues, each capable of relatively weaker ionic interactions with membrane PS lipids. Unlike other multi-PS binding proteins, such as Annexin V, which has a sharply sigmoidal, all-or-none response (25, 26), the structural characteristics of Tim4 produce a gently sigmoidal binding response. The nature of this response confers a functional sensitivity to PS surface density that is well-suited to mediating a differentiated phagocytic response to diverse populations of PS-exposing T cells. The distinctly different sensitivity of both Tim1 and Tim3 support the conclusion that Tim4 has evolved a specific sensitivity to variations in PS surface density to facilitate its unique function. These results offer a new paradigm for understanding immune recognition of PS lipid membranes by demonstrating that key characteristics of the lipid bilayer, such as PS surface density, have the potential to play an integral role in determining the appropriate immunological response.     

PRE-OPERATIVE CORE BIOPSY OF SOFT-TISSUE TUMOURS FACILITATES THEIR SURGICAL MANAGEMENT

Background: Soft-tissue sarcomas are rare, and clinical differentiation of benign tumours from sarcomas is sometimes impossible. Further, the diagnosis of soft-tissue sarcomas may be unsuspected pre-operatively, and the presenting mass enucleated. While enucleation (excisional biopsy) is acceptable for benign lesions, it is inappropriate for sarcomas, because the opportunity for the most effective management resulting in both adequate local control and functional limb salvage surgery is compromised. A high rate of wound complications following open incisional biopsy may also compromise local treatment. Inappropriate siting of the incision for both incisional and excisional biopsies may adversely affect subsequent surgery and radiotherapy. Methods: We therefore assessed the accuracy of core biopsy in the diagnosis of soft-tissue tumours, and planning of definitive surgery. All patients with primary soft-tissue tumours managed by two surgeons with a special interest in soft-tissue sarcomas since 1991 were reviewed. More than half (53%) were referred from other specialists. Results: Of 45 cases, 37 (82%) were referred with the tumour intact, and of these 31 (84%) underwent core biopsy. The overall accuracy of core biopsy was 84%. The sensitivity was 94%, with 100% specificity. In most patients this allowed planning of definitive one-stage surgery (P < 0.005). Of the remaining five non-diagnostic cores, four were benign and one was a non-specific malignancy. Conclusions: Core biopsy has a high degree of accuracy in the diagnosis of soft-tissue tumours, particularly malignant lesions, and is not misleading. Core biopsy avoids the complications of open biopsy, and enables planning of one-stage surgery when used in combination with appropriate imaging.     

Viral antibodies in maternal and cord sera

Viral antibodies were determined in paired maternal and cord blood sera of 258 consecutive deliveries. Antibodies to Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex type 1 (HSV-1), adenovirus (ADV), and varicella zoster (VZV) were tested by indirect immunofluorescence and to rubella (RUB) by hemagglutination inhibition. Analysis of the overall pattern of differences between maternal and cord blood showed a highly significant difference for all six viruses. Analysis regarding individual viruses showed significantly higher titers to CMV and RUB in cord blood, a higher titer to VZV in maternal blood, and similar levels of antibodies to EBV, HSV-1, and ADV in maternal and cord blood. Infants with one or more risk indicators (weight < 3 kg, Apgar score 7, clinical jaundice, meconium-stained amniotic fluid, respiratory distress syndrome) were defined as at risk. Infants free of such indicators were defined as normal. Significantly lower antibody levels to all six viruses were found in both maternal and cord blood of the at risk as compared to the normal group, while the ratios between the maternal and cord blood levels remained similar. Birth defects were found to have no effect on antibody titers. These results indicate an efficient and selective transfer through the placenta of certain viral antibodies and the possible association of lower antibody production with the presence of risk indicators in the infants.     

Leishmaniasis impact and treatment access

According to disease burden estimates, leishmaniasis ranks third in disease burden in disability-adjusted life years caused by neglected tropical diseases and is the second cause of parasite-related deaths after malaria; but for a variety of reasons, it is not receiving the attention that would be justified seeing its importance. This is especially apparent in the unnecessarily and unacceptably poor access to timely and appropriate treatment for patients. To our knowledge, this is the first publication that addresses the major issues associated with poor access to drugs for leishmaniasis and that outlines a number of feasible and practical solutions.     

Astrocyte-derived VEGF-A drives blood-brain barrier disruption in CNS inflammatory disease

In inflammatory CNS conditions such as multiple sclerosis (MS), current options to treat clinical relapse are limited, and more selective agents are needed. Disruption of the blood-brain barrier (BBB) is an early feature of lesion formation that correlates with clinical exacerbation, leading to edema, excitotoxicity, and entry of serum proteins and inflammatory cells. Here, we identify astrocytic expression of VEGF-A as a key driver of BBB permeability in mice. Inactivation of astrocytic Vegfa expression reduced BBB breakdown, decreased lymphocyte infiltration and neuropathology in inflammatory and demyelinating lesions, and reduced paralysis in a mouse model of MS. Knockdown studies in CNS endothelium indicated activation of the downstream effector eNOS as the principal mechanism underlying the effects of VEGF-A on the BBB. Systemic administration of the selective eNOS inhibitor cavtratin in mice abrogated VEGF-A-induced BBB disruption and pathology and protected against neurologic deficit in the MS model system. Collectively, these data identify blockade of VEGF-A signaling as a protective strategy to treat inflammatory CNS disease.     

Changes in active travel of school children from 2004 to 2010 in New South Wales, Australia

Purpose

To describe changes in mode of commuting to school among Australia students between 2004 and 2010 and in relation to body mass index (BMI) and cardio respiratory fitness.

Methods

Representative cross-sectional survey of school children in grades 6, 8 and 10 in 2004 (n = 2750) and 2010 (n = 4273). Information on how many days students use active and passive travel modes to and from school and measured BMI and cardio-respiratory fitness test were collected as part of the New South Wales (NSW) Schools Physical Activity and Nutrition Surveys (SPANS).

Results

Active travel to school remained stable between 2004 and 2010, although there was a small increase in minutes spent on active travel. There was no association between active travel and body mass index. In 2010 there was a significant association between frequent car use and low cardio-respiratory fitness (adjusted OR = 1.7, CI 1.3-2.1).

Conclusion

It is a positive finding that the generational decline in active travel may have levelled out. Student inactivity associated with regular car use is plausibly related to lower cardio-respiratory fitness, but active commuting may not be of sufficient energy expenditure to impact upon BMI.     

Cost and Affordability of Healthy,Equitable and Sustainable Diets in Low Socioeconomic Groups in Australia

Few Australians consume a healthy, equitable and more sustainable diet consistent with the Australian Dietary Guidelines (ADGs). Low socioeconomic groups (SEGs) suffer particularly poor diet-related health problems. However, granular information on dietary intakes and affordability of recommended diets was lacking for low SEGs. The Healthy Diets Australian Standardised Affordability and Pricing protocol was modified for low SEGs to align with relevant dietary intakes reported in the National Nutrition Survey 2011–2012(which included less healthy and more discretionary options than the broader population), household structures, food purchasing habits, and incomes. Cost and affordability of habitual and recommended diets of low SEGs were calculated using prices of ‘standard brands’ and ‘cheapest options’. With ‘standard brands’, recommended diets cost less than habitual diets, but were unaffordable for low SEGs. With ‘cheapest options’, both diets were more affordable, but recommended diets cost more than habitual diets for some low SEGs, potentially contributing to perceptions that healthy food is unaffordable. The study confirms the need for an equity lens to better target dietary guidelines for low SEGs. It also highlights urgent policy action is needed to help improve affordability of recommended diets.