Processing of staphylococcus aureus and zymosan particles by human leukocytes measured by flow cytometry

Staphylococcus aureus were labeled with fluorescein-isothiocyanate (FITC), stained by ethidium bromide (EB), and measured by flow cytometry (FCM). Bacteria were identified by their FITC-fluorescence and discriminated from the leukocyte cell nuclei by the much higher EB fluorescence and lower coefficient of variation of the latter. Following phagocytosis, both the bacterial FITC- and EB-fluorescence decayed. The mean FITC-fluorescence was reduced about 20% after 15 min and 30-50% after 60 min. Zymosan particles were labeled by FITC and incubated with leukocytes for 15 min. Phagocytes were sorted by FCM and the zymosan particles were liberated by sonication. Their forward angle light scatter was reduced by about 50.6 +/- 2.1% and the FITC-fluorescence by 8.7 +/- 1.0% (mean +/- SD). The reduced FITC-fluorescence and light scatter suggests degradation of proteins, and the decay of EB-fluorescence degradation of DNA, but the specificity remains to be established. By this method phagocytes from a patient with systemic lupus erythematosus seemed to have a selective defect in DNA degradation, whereas phagocytes from a patient with acute myelogenous leukemia had a low capacity to degrade bacterial proteins. This technique offers opportunities for automatic measurements of bacteria and zymosan particle degradation by phagocytes.     

Ontogenetic induced shifts in the ecology of sunbleak Leucaspius delineatus during early development

Using the non‐native sunbleak Leucaspius delineatus as a model, the relationship between ontogeny and ecology was studied with a view to identifying specific morphological and physiological processes involved in influencing ecological niche shifts. Following a predefined saltatory model for the early ontogeny of sunbleak, field studies examined the temporal use of microhabitat, diet and morphological changes throughout early development. Following a dramatic shift in both morphology and ecology between the free embryo phase and the larval period, habitat use and diet showed little change during the larval period, with habitat use confined to marginal, vegetated areas and prey items associated with these habitats well represented in the diet. During the final larval step (L5), transition to the juvenile period resulted in the stabilization of relative growth, acquisition of the adult morphotype and was associated with a clear shift in diet and habitat use. During this period, sunbleak moved for the first time into open, deeper water, away from the banks, and utilized a similar range of food items to the adults. Specific relationships between form and function are further discussed.     

A Transdiagnostic Community-Based Mental Health Treatment for Comorbid Disorders: Development and Outcomes of a Randomized Controlled Trial among Burmese Refugees in Thailand

Background

Existing studies of mental health interventions in low-resource settings have employed highly structured interventions delivered by non-professionals that typically do not vary by client. Given high comorbidity among mental health problems and implementation challenges with scaling up multiple structured evidence-based treatments (EBTs), a transdiagnostic treatment could provide an additional option for approaching community-based treatment of mental health problems. Our objective was to test such an approach specifically designed for flexible treatments of varying and comorbid disorders among trauma survivors in a low-resource setting.

Methods and Findings

We conducted a single-blinded, wait-list randomized controlled trial of a newly developed transdiagnostic psychotherapy, Common Elements Treatment Approach (CETA), for low-resource settings, compared with wait-list control (WLC). CETA was delivered by lay workers to Burmese survivors of imprisonment, torture, and related traumas, with flexibility based on client presentation. Eligible participants reported trauma exposure and met severity criteria for depression and/or posttraumatic stress (PTS). Participants were randomly assigned to CETA (n = 182) or WLC (n = 165). Outcomes were assessed by interviewers blinded to participant allocation using locally adapted standard measures of depression and PTS (primary outcomes) and functional impairment, anxiety symptoms, aggression, and alcohol use (secondary outcomes). Primary analysis was intent-to-treat (n = 347), including 73 participants lost to follow-up. CETA participants experienced significantly greater reductions of baseline symptoms across all outcomes with the exception of alcohol use (alcohol use analysis was confined to problem drinkers). The difference in mean change from pre-intervention to post-intervention between intervention and control groups was −0.49 (95% CI: −0.59, −0.40) for depression, −0.43 (95% CI: −0.51, −0.35) for PTS, −0.42 (95% CI: −0.58, −0.27) for functional impairment, −0.48 (95% CI: −0.61, −0.34) for anxiety, −0.24 (95% CI: −0.34, −0.15) for aggression, and −0.03 (95% CI: −0.44, 0.50) for alcohol use. This corresponds to a 77% reduction in mean baseline depression score among CETA participants compared to a 40% reduction among controls, with respective values for the other outcomes of 76% and 41% for anxiety, 75% and 37% for PTS, 67% and 22% for functional impairment, and 71% and 32% for aggression. Effect sizes (Cohen''s d) were large for depression (d = 1.16) and PTS (d = 1.19); moderate for impaired function (d = 0.63), anxiety (d = 0.79), and aggression (d = 0.58); and none for alcohol use. There were no adverse events. Limitations of the study include the lack of long-term follow-up, non-blinding of service providers and participants, and no placebo or active comparison intervention.

Conclusions

CETA provided by lay counselors was highly effective across disorders among trauma survivors compared to WLCs. These results support the further development and testing of transdiagnostic approaches as possible treatment options alongside existing EBTs.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01459068","term_id":"NCT01459068"}}NCT01459068 Please see later in the article for the Editors'' Summary     

Challenges in Developing a Validated Biomarker for Angiogenesis Inhibitors: The Motesanib Experience

Purpose

We sought to develop placental growth factor as a predictive pharmacodynamic biomarker for motesanib efficacy as first-line therapy in patients with advanced nonsquamous non–small-cell lung cancer.

Experimental Design

Placental growth factor was evaluated at baseline and study week 4 (after 3 weeks treatment) in an exploratory analysis of data from a randomized phase 2 study of motesanib 125 mg once daily plus carboplatin/paclitaxel and in a prespecified analysis of data from a randomized, double-blind phase 3 study of motesanib 125 mg once daily plus carboplatin/paclitaxel vs placebo plus carboplatin/paclitaxel (MONET1). Associations between fold-change from baseline in placental growth factor and overall survival were evaluated using Cox proportional hazards models.

Results

In the phase 2 study, serum placental growth factor increased from baseline a mean 2.8-fold at study week 4. Patients with ≥2.2-fold change from baseline in placental growth factor (n = 18) had significantly longer overall survival than those with <2.2-fold change (n = 19; 22.9 vs 7.9 months; hazard ratio, 0.30; 95% CI, 0.12–0.74; P = 0.009). Consequently, placental growth factor was investigated as a pharmacodynamic biomarker in the phase 3 MONET1 study. There was no association between log-transformed placental growth factor fold-change from baseline to week 4 (continuous variable) and overall survival (hazard ratio, 0.98; 95% CI, 0.79–1.22; P = 0.868). MONET1 did not meet its primary endpoint of overall survival. Likewise, median overall survival was similar among patients with ≥2.0-fold change in placental growth factor (n = 229) compared with <2.0-fold change (n = 127; 14.8 vs 13.8 months; hazard ratio, 0.88; 95% CI, 0.67–1.15, P = 0.340).

Conclusions

Our results illustrate the challenges of successfully translating phase 2 biomarker results into phase 3 studies.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00460317","term_id":"NCT00460317"}}NCT00460317, {"type":"clinical-trial","attrs":{"text":"NCT00369070","term_id":"NCT00369070"}}NCT00369070     

Syndecan-4 Phosphorylation Is a Control Point for Integrin Recycling

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Highlights? c-Src phosphorylates syndecan-4 in response to extracellular stimuli ? Syndecan-4 phosphorylation and engagement regulate Arf6 activity ? Syndecan-4-mediated Arf6 activity regulates differential integrin recycling ? Syndecan-4-mediated integrin recycling controls FA dynamics and cell migration     

Dicer's helicase domain discriminates dsRNA termini to promote an altered reaction mode

The role of Dicer's helicase domain is enigmatic, but in?vivo it is required for processing certain endogenous siRNA, but not miRNA. By using Caenorhabditis elegans extracts or purified Drosophila Dicer-2 we compared activities of wild-type enzymes and those containing mutations in the helicase domain. We found the helicase domain was essential for cleaving dsRNA with blunt or 5'-overhanging termini, but not those with 3' overhangs, as found on miRNA precursors. Further, blunt termini, but not 3' overhangs, led to increased siRNAs from internal regions of dsRNA; this activity required ATP and a functional helicase domain. Our data suggest that blunt or 5'-overhanging termini engage Dicer's helicase domain to facilitate accumulation of siRNAs from internal regions of a dsRNA, an activity suited for processing long siRNA precursors of low abundance, but not necessary for the single cleavage required for miRNA processing.     

A syndecan-4 hair trigger initiates wound healing through caveolin- and RhoG-regulated integrin endocytosis

Cell migration during wound healing requires adhesion receptor turnover to enable the formation and disassembly of cell-extracellular matrix contacts. Although recent advances have improved our understanding of integrin trafficking pathways, it is not known how extracellular ligand engagement controls receptor dynamics. Using atomic force microscopy, we have measured cell avidity for fibronectin and defined a mechanism for the outside-in regulation of α(5)β(1)-integrin. Surprisingly, adhesive strength was attenuated by the syndecan-4-binding domain of fibronectin due to a rapid triggering of α(5)β(1)-integrin endocytosis. Association of syndecan-4 with PKCα was found to trigger RhoG activation and subsequent dynamin- and caveolin-dependent integrin uptake. Like disruption of syndecan-4 or caveolin, gene disruption of RhoG in mice was found to retard closure of dermal wounds due to a migration defect of the fibroblasts and keratinocytes of RhoG null mice. Thus, this syndecan-4-regulated integrin endocytic pathway appears to play a key role in tissue repair.     

Phylogenetic Estimation of Community Composition and Novel Eukaryotic Lineages in Base Mine Lake: An Oil Sands Tailings Reclamation Site in Northern Alberta

Reclamation of anthropogenically impacted environments is a critical issue worldwide. In the oil sands extraction industry of Alberta, reclamation of mining‐impacted areas, especially areas affected by tailings waste, is an important aspect of the mining life cycle. A reclamation technique currently under study is water‐capping, where tailings are capped by water to create an end‐pit lake (EPL). Base Mine Lake (BML) is the first full‐scale end‐pit lake in the Alberta oil sands region. In this study, we sequenced eukaryotic 18S rRNA genes recovered from 92 samples of Base Mine Lake water in a comprehensive sampling programme covering the ice‐free period of 2015. The 565 operational taxonomic units (OTUs) generated revealed a dynamic and diverse community including abundant Microsporidia, Ciliata and Cercozoa, though 41% of OTUs were not classifiable below the phylum level by comparison to 18S rRNA databases. Phylogenetic analysis of five heterotrophic phyla (Cercozoa, Fungi, Ciliata, Amoebozoa and Excavata) revealed substantial novel diversity, with many clusters of OTUs that were more similar to each other than to any reference sequence. All of these groups are entirely or mostly heterotrophic, as a relatively small number of definitively photosynthetic clades were amplified from the BML samples.     

Differential freshwater flagellate community response to bacterial food quality with a focus on Limnohabitans bacteria

Different bacterial strains can have different value as food for heterotrophic nanoflagellates (HNF), thus modulating HNF growth and community composition. We examined the influence of prey food quality using four Limnohabitans strains, one Polynucleobacter strain and one freshwater actinobacterial strain on growth (growth rate, length of lag phase and growth efficiency) and community composition of a natural HNF community from a freshwater reservoir. Pyrosequencing of eukaryotic small subunit rRNA amplicons was used to assess time-course changes in HNF community composition. All four Limnohabitans strains and the Polynucleobacter strain yielded significant HNF community growth while the actinobacterial strain did not although it was detected in HNF food vacuoles. Notably, even within the Limnohabitans strains we found significant prey-related differences in HNF growth parameters, which could not be related only to size of the bacterial prey. Sequence data characterizing the HNF communities showed also that different bacterial prey items induced highly significant differences in community composition of flagellates. Generally, Stramenopiles dominated the communities and phylotypes closely related to Pedospumella (Chrysophyceae) were most abundant bacterivorous flagellates rapidly reacting to addition of the bacterial prey of high food quality.