Sphingosine 1-phosphate rapidly increases endothelial barrier function independently of VE-cadherin but requires cell spreading and Rho kinase

Sphingosine 1-phosphate (S1P) rapidly increases endothelial barrier function and induces the assembly of the adherens junction proteins vascular endothelial (VE)-cadherin and catenins. Since VE-cadherin contributes to the stabilization of the endothelial barrier, we determined whether the rapid, barrier-enhancing activity of S1P requires VE-cadherin. Ca(2+)-dependent, homophilic VE-cadherin binding of endothelial cells, derived from human umbilical veins and grown as monolayers, was disrupted with EGTA, an antibody to the extracellular domain of VE-cadherin, or gene silencing of VE-cadherin with small interfering RNA. All three protocols caused a reduction in the immunofluorescent localization of VE-cadherin at intercellular junctions, the separation of adjacent cells, and a decrease in basal endothelial electrical resistance. In all three conditions, S1P rapidly increased endothelial electrical resistance. These findings demonstrate that S1P enhances the endothelial barrier independently of homophilic VE-cadherin binding. Junctional localization of VE-cadherin, however, was associated with the sustained activity of S1P. Imaging with phase-contrast and differential interference contrast optics revealed that S1P induced cell spreading and closure of intercellular gaps. Pretreatment with latrunculin B, an inhibitor of actin polymerization, or Y-27632, a Rho kinase inhibitor, attenuated cell spreading and the rapid increase in electrical resistance induced by S1P. We conclude that S1P rapidly closes intercellular gaps, resulting in an increased electrical resistance across endothelial cell monolayers, via cell spreading and Rho kinase and independently of VE-cadherin.     

Decolonizing Primate Conservation Practice: A Case Study from North Morocco

Understanding the historical context of an area enables an incoming conservationist to reflect on their role in communities and to better position themselves both politically and socially within them. Here, we explore how outside agencies and institutions, including a former colonial power, have affected and influenced local communities who share their landscape with Barbary macaques (Macaca sylvanus) in Bouhachem forest, north Morocco. In the context of initiating Barbary macaque conservation activities, we interviewed representatives from local governmental and nongovernmental organizations, city dwellers, and villagers about the historical, political, and social context of the study site. We found that villages around Bouhachem were politically and socially marginalized and discriminated against by the state and urban society. The existence of these divisions and the outside agencies’ simplistic view of villages as homogeneous communities negatively influenced conservation interventions, because people resisted initiatives imposed on them without prior consultation. We found that Bouhachem villagers have been, and still are, excluded from meaningful participation in the conservation of the forest and this finding encouraged us to decolonize our own practice. We engaged meaningfully with members of the surrounding communities and responded to news of erroneous stories about our activities by developing a project working in three villages that included all households. Based on our experiences, we recommend that all conservationists conduct historical and qualitative research to gain a deeper, more nuanced understanding of the communities they work in. This understanding should encourage conservationists to recognize their own social and cultural biases and to decolonize their practice. Attending to our own position may help us to avoid underestimating and alienating people who view conservation actions through a very different but equally valid lens.

التخلص من التداعيات الاستعمارية في المحافظة على الرئيسيات :دراسة حالة من شمال المغربخلاصة : إن فهم السياق التاريخي لمنطقة معينة، يمكّن المحافظ البيئي الوافد من التفكير في دوره في المجتمعات، ولانسجامٍ سياسيٍ واجتماعي أفضل داخلها. هنا نكتشف كيف أثرت الوكالات والمؤسسات الخارجية ، بما في ذلك القوة الاستعمارية السابقة، على المجتمعات المحلية التي تتعايش مع قرود المكاك البربري (Macaca sylvanus) في غابة بوهاشم شمال المغرب. في سياق بدء أنشطة الحفاظ على المكاك البربري، أجرينا مقابلات مع ممثلين من المنظمات الحكومية وغير الحكومية المحلية وسكان المدن والقرويين حول السياق التاريخي والسياسي والاجتماعي لموقع الدراسة. وجدنا أن القرى المحيطة بغابة بوهاشم كانت مهمشة و معرضة للتمييز ضدها سياسياً واجتماعياً من قبل الدولة والمجتمع الحضري. و أثر بشكل سلبي وجود هذه الانقسامات والنظرة السطحية للوكالات الخارجية للقرى على أنها مجتمعات متجانسة، على تدخلات الحفظ البيئي، لأن الناس رفضوا أي مبادرة مفروضة عليهم دون استشارة مسبقة. و وجدنا أن القرويين في بوهاشم كانوا ولا يزالون مستبعدين من المشاركة الهادفة في الحفاظ على الغابة، وهذا الاكتشاف شجعنا على التخلص من التداعيات الاستعمارية في ممارستنا. لقد تواصلنا بشكل هادف بأعضاء من المجتمعات المحيطة، واستجبنا لأخبار القصص الخاطئة حول أنشطتنا من خلال تطوير مشروع يعمل في ثلاث قرى شمل جميع الأسر. بناءً على تجاربنا، نوصي جميع دعاة الحفاظ على البيئة بإجراء بحث تاريخي ونوعي لاكتساب فهم أعمق وأكثر دقة للمجتمعات التي يعملون فيها. يجب أن يشجع هذا الفهم دعاة الحفاظ على البيئة على التعرف على التحيزات الاجتماعية والثقافية الخاصة بهم، والتخلص من التداعيات الاستعمارية في ممارساتهم.قد يساعدنا الاهتمام بموقفنا الخاص على تجنب الإبعاد و التقليل من شأن الأشخاص، الذين ينظرون إلى إجراءات الحفاظ على البيئة من منظور مختلف تمامًا لكن عادل .

     

The imprecision of heterozygosity-fitness correlations hinders the detection of inbreeding and inbreeding depression in a threatened species

In nonpedigreed wild populations, inbreeding depression is often quantified through the use of heterozygosity-fitness correlations (HFCs), based on molecular estimates of relatedness. Although such correlations are typically interpreted as evidence of inbreeding depression, by assuming that the marker heterozygosity is a proxy for genome-wide heterozygosity, theory predicts that these relationships should be difficult to detect. Until now, the vast majority of empirical research in this area has been performed on generally outbred, nonbottlenecked populations, but differences in population genetic processes may limit extrapolation of results to threatened populations. Here, we present an analysis of HFCs, and their implications for the interpretation of inbreeding, in a free-ranging pedigreed population of a bottlenecked species: the endangered takahe (Porphyrio hochstetteri). Pedigree-based inbreeding depression has already been detected in this species. Using 23 microsatellite loci, we observed only weak evidence of the expected relationship between multilocus heterozygosity and fitness at individual life-history stages (such as survival to hatching and fledging), and parameter estimates were imprecise (had high error). Furthermore, our molecular data set could not accurately predict the inbreeding status of individuals (as 'inbred' or 'outbred', determined from pedigrees), nor could we show that the observed HFCs were the result of genome-wide identity disequilibrium. These results may be attributed to high variance in heterozygosity within inbreeding classes. This study is an empirical example from a free-ranging endangered species, suggesting that even relatively large numbers (>20) of microsatellites may give poor precision for estimating individual genome-wide heterozygosity. We argue that pedigree methods remain the most effective method of quantifying inbreeding in wild populations, particularly those that have gone through severe bottlenecks.     

Expression of functional recombinant human growth hormone in transgenic soybean seeds

We produced human growth hormone (hGH), a protein that stimulates growth and cell reproduction, in genetically engineered soybean [Glycine max (L.) Merrill] seeds. Utilising the alpha prime (α') subunit of β-conglycinin tissue-specific promoter from soybean and the α-Coixin signal peptide from Coix lacryma-jobi, we obtained transgenic soybean lines that expressed the mature form of hGH in their seeds. Expression levels of bioactive hGH up to 2.9% of the total soluble seed protein content (corresponding to approximately 9?g?kg(-1)) were measured in mature dry soybean seeds. The results of ultrastructural immunocytochemistry assays indicated that the recombinant hGH in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Specific bioassays demonstrated that the hGH expressed in the soybean seeds was fully active. The recombinant hGH protein sequence was confirmed by mass spectrometry characterisation. These results demonstrate that the utilisation of tissue-specific regulatory sequences is an attractive and viable option for achieving high-yield production of recombinant proteins in stable transgenic soybean seeds.     

Antibiotic dosing in the 'at risk' critically ill patient: Linking pathophysiology with pharmacokinetics/pharmacodynamics in sepsis and trauma patients

Background

Critical illness, mediated by trauma or sepsis, can lead to physiological changes that alter the pharmacokinetics of antibiotics and may result in sub-therapeutic concentrations at the sites of infection. The first aim of this project is to identify the clinical characteristics of critically ill patients with significant trauma that have been recently admitted to ICU that may predict the dosing requirements for the antibiotic, cefazolin. The second aim of this is to identify the clinical characteristics of critically ill patients with sepsis that may predict the dosing requirements for the combination antibiotic, piperacillin-tazobactam.

Methods/Design

This is an observational pharmacokinetic study of patients with trauma (cefazolin) or with sepsis (piperacillin-tazobactam). Participants will have samples from blood and urine, collected at different intervals. Patients will also have a microdialysis catheter inserted into subcutaneous tissue to measure interstitial fluid penetration of the antibiotic. Participants will be administered sinistrin, indocyanine green and sodium bromide as well as have cardiac output monitoring performed and tetrapolar bioimpedance to determine physiological changes resulting from pathology. Analysis of samples will be performed using validated liquid chromatography tandem mass-spectrometry. Pharmacokinetic analysis will be performed using non-linear mixed effects modeling to determine individual and population pharmacokinetic parameters of antibiotics.

Discussion

The study will describe cefazolin and piperacillin-tazobactam concentrations in plasma and the interstitial fluid of tissues in trauma and sepsis patients respectively. The results of this study will guide clinicians to effectively dose these antibiotics in order to maximize the concentration of antibiotics in the interstitial fluid of tissues.     

The relation between cartilage biomarkers (C2C,C1,2C,CS846, and CPII) and the long-term outcome of rheumatoid arthritis patients within the CAMERA trial

Introduction  

The aim of this study was to investigate whether serum biomarker levels of C2C, C1,2C, CS846, and CPII can predict the long-term course of disease activity and radiographic progression early in the disease course of rheumatoid arthritis (RA).     

Activation of a PAK-MEK signalling pathway in malaria parasite-infected erythrocytes

Merozoites of malaria parasites invade red blood cells (RBCs), where they multiply by schizogony, undergoing development through ring, trophozoite and schizont stages that are responsible for malaria pathogenesis. Here, we report that a protein kinase-mediated signalling pathway involving host RBC PAK1 and MEK1, which do not have orthologues in the Plasmodium kinome, is selectively stimulated in Plasmodium falciparum-infected (versus uninfected) RBCs, as determined by the use of phospho-specific antibodies directed against the activated forms of these enzymes. Pharmacological interference with host MEK and PAK function using highly specific allosteric inhibitors in their known cellular IC50 ranges results in parasite death. Furthermore, MEK inhibitors have parasiticidal effects in vitro on hepatocyte and erythrocyte stages of the rodent malaria parasite Plasmodium berghei, indicating conservation of this subversive strategy in malaria parasites. These findings have profound implications for the development of novel strategies for antimalarial chemotherapy.     

Discovery of polymorphisms in starch-related genes in rice germplasm by amplification of pooled DNA and deeply parallel sequencing

High-throughput sequencing of pooled DNA was applied to polymorphism discovery in candidate genes involved in starch synthesis. This approach employed semi- to long-range PCR (LR-PCR) followed by next-generation sequencing technology. A total of 17 rice starch synthesis genes encoding seven classes of enzymes, including ADP-glucose pyrophosphorylase (AGPase), granule starch synthase (GBSS), soluble starch synthase (SS), starch branching enzyme (BE), starch debranching enzyme (DBE) and starch phosphorylase (SPHOL) and phosphate translocator (GPT1) from 233 genotypes were PCR amplified using semi- to long-range PCR. The amplification products were equimolarly pooled and sequenced using massively parallel sequencing technology (MPS). By detecting single nucleotide polymorphism (SNP)/Indels in both coding and noncoding areas of the genes, we identified genetic differences and characterized the SNP/Indel variation and distribution patterns among individual starch candidate genes. Approximately, 60.9 million reads were generated, of which 54.8 million (90%) mapped to the reference sequences. The average coverage rate ranged from 12,708 to 38,300 times for SSIIa and SSIIIb, respectively. SNPs and single/multiple-base Indels were analysed in a total assembled length of 116,403 bp. In total, 501 SNPs and 113 Indels were detected across the 17 starch-related loci. The ratio of synonymous to nonsynonymous SNPs (Ka/Ks) test indicated GBSSI and isoamylase 1 (ISA1) as the least diversified (most purified) and conservative genes as the studied populations have been through cycles of selection. This report demonstrates a useful strategy for screening germplasm by MPS to discover variants in a specific target group of genes.