Autophagy protein Rubicon mediates phagocytic NADPH oxidase activation in response to microbial infection or TLR stimulation

Phagocytosis and autophagy are two important and related arms of the host's first-line defense against microbial invasion. Rubicon is a RUN domain containing cysteine-rich protein that functions as part of a Beclin-1-Vps34-containing autophagy complex. We report that Rubicon is also an essential, positive regulator of the NADPH oxidase complex. Upon microbial infection or Toll-like-receptor 2 (TLR2) activation, Rubicon interacts with the p22phox subunit of the NADPH oxidase complex, facilitating its phagosomal trafficking to induce a burst of reactive oxygen species (ROS) and inflammatory cytokines. Consequently, ectopic expression or depletion of Rubicon profoundly affected ROS, inflammatory cytokine production, and subsequent antimicrobial activity. Rubicon's actions in autophagy and in the NADPH oxidase complex are functionally and genetically separable, indicating that Rubicon functions in two ancient innate immune machineries, autophagy and phagocytosis, depending on the environmental stimulus. Rubicon may thus be pivotal to generating an optimal intracellular immune response against microbial infection.     

Engineering excellence in breakthrough biomedical technologies: bioengineering at the University of California, Riverside

The Department of Bioengineering at the University of California, Riverside (UCR), was established in 2006 and is the youngest department in the Bourns College of Engineering. It is an interdisciplinary research engine that builds strength from highly recognized experts in biochemistry, biophysics, biology, and engineering, focusing on common critical themes. The range of faculty research interests is notable for its diversity, from the basic cell biology through cell function to the physiology of the whole organism, each directed at breakthroughs in biomedical devices for measurement and therapy. The department forges future leaders in bioengineering, mirroring the field in being energetic, interdisciplinary, and fast moving at the frontiers of biomedical discoveries. Our educational programs combine a solid foundation in bio logical sciences and engineering, diverse communication skills, and training in the most advanced quantitative bioengineering research. Bioengineering at UCR also includes the Bioengineering Interdepartmental Graduate (BIG) program. With its slogan Start-Grow-Be-BIG, it is already recognized for its many accomplishments, including being third in the nation in 2011 for bioengineering students receiving National Science Foundation graduate research fellowships as well as being one of the most ethnically inclusive programs in the nation.     

An embedded longitudinal multi-faceted qualitative evaluation of a complex cluster randomized controlled trial aiming to reduce clinically important errors in medicines management in general practice

ABSTRACT: BACKGROUND: There is a need to shed light on the pathways through which complex interventions mediate their effects in order to enable critical reflection on their transferability. We sought to explore and understand key stakeholder accounts of the acceptability, likely impact and strategies for optimizing and rolling-out a successful pharmacist-led information technology-enabled (PINCER) intervention, which substantially reduced the risk of clinically important errors in medicines management in primary care. METHODS: Data were collected at two geographical locations in central England through a combination of one-to-one longitudinal semi-structured telephone interviews (one at the beginning of the trial and another when the trial was well underway), relevant documents, and focus group discussions following delivery of the PINCER intervention. Participants included PINCER pharmacists, general practice staff, researchers involved in the running of the trial, and primary care trust staff. PINCER pharmacists were interviewed at three different time-points during the delivery of the PINCER intervention. Analysis was thematic with diffusion of innovation theory providing a theoretical framework. RESULTS: We conducted 52 semi-structured telephone interviews and six focus group discussions with 30 additional participants. In addition, documentary data were collected from six pharmacist diaries, along with notes from four meetings of the PINCER pharmacists and feedback meetings from 34 practices. Key findings that helped to explain the success of the PINCER intervention included the perceived importance of focusing on prescribing errors to all stakeholders, and the credibility and appropriateness of a pharmacist-led intervention to address these shortcomings. Central to this was the face-to-face contact and relationship building between pharmacists and a range of practice staff, and pharmacists' explicitly designated role as a change agent. However, important concerns were identified about the likely sustainability of this new model of delivering care, in the absence of an appropriate support network for pharmacists and career development pathways. CONCLUSIONS: This embedded qualitative inquiry has helped to understand the complex organizational and social environment in which the trial was undertaken and the PINCER intervention was delivered. The longitudinal element has given insight into the dynamic changes and developments over time. Medication errors and ways to address these are high on stakeholders' agendas. Our results further indicate that pharmacists were, because of their professional standing and skill-set, able to engage with the complex general practice environment and able to identify and manage many clinically important errors in medicines management. The transferability of the PINCER intervention approach, both in relation to other prescribing errors and to other practices, is likely to be high.     

The blood-brain barrier significantly limits eflornithine entry into Trypanosoma brucei brucei infected mouse brain

Drugs to treat African trypanosomiasis are toxic, expensive and subject to parasite resistance. New drugs are urgently being sought. Although the existing drug, eflornithine, is assumed to reach the brain in high concentrations, little is known about how it crosses the healthy and infected blood-brain barrier. This information is essential for the design of drug combinations and new drugs. This study used novel combinations of animal models to address these omissions. Eflornithine crossed the healthy blood-CNS interfaces poorly, but this could be improved by co-administering suramin, but not nifurtimox, pentamidine or melarsoprol. Work using a murine model of sleeping sickness demonstrated that Trypanosoma brucei brucei crossed the blood-CNS interfaces, which remained functional, early in the course of infection. Concentrations of brain parasites increased during the infection and this resulted in detectable blood-brain barrier, but not choroid plexus, dysfunction at day 28 post-infection with resultant increases in eflornithine brain delivery. Barrier integrity was never restored and the animals died at day 37.9 +/- 1.2. This study indicates why an intensive treatment regimen of eflornithine is required (poor blood-brain barrier penetration) and suggests a possible remedy (combining eflornithine with suramin). The blood-brain barrier retains functionality until a late, possibly terminal stage, of trypanosoma infection.     

Deciphering the structural role of histidine 83 for heme binding in hemophore HasA

Heme carrier HasA has a unique type of histidine/tyrosine heme iron ligation in which the iron ion is in a thermally driven two spin states equilibrium. We recently suggested that the H-bonding between Tyr75 and the invariantly conserved residue His83 modulates the strength of the iron-Tyr75 bond. To unravel the role of His83, we characterize the iron ligation and the electronic properties of both wild type and H83A mutant by a variety of spectroscopic techniques. Although His83 in wild type modulates the strength of the Tyr-iron bond, its removal causes detachment of the tyrosine ligand, thus giving rise to a series of pH-dependent equilibria among species with different axial ligation. The five coordinated species detected at physiological pH may represent a possible intermediate of the heme transfer mechanism to the receptor.     

DNA tape measurements of AraC

A new method for measuring distances between points in the AraC-DNA complex was developed and applied. It utilizes variable lengths of single-stranded DNA that connect double-stranded regions containing the two half-site binding sequences of AraC. These distances plus the protein interdomain linker distances are compatible with two classes of structure for the dimeric AraC gene regulatory protein. In one class, the N-terminal regulatory arm of one dimerization domain is capable of interacting with the DNA-binding domain on the same polypeptide chain for a cis interaction. In the other class, the possible arm-DNA-binding domain interaction is trans, where it adds to the dimerization interface.     

Design and performance of a pilot-scale constructed wetland treatment system for natural gas storage produced water

To test the hypothesis that water produced from natural gas storage wells could be treated effectively by constructed wetland treatment systems, a modular pilot-scale system was designed, built, and used for treating gas storage produced waters. Four simulated waters representing the range of contaminant concentrations typical of actual produced waters were treated, and the system's performance was monitored. Freshwater wetland cells planted with Schoenoplectus californicus and Typha latifolia were used to treat fresh and brackish waters. Saline and hypersaline waters were treated by saltwater wetland cells planted with Spartina alterniflora and by reverse osmosis. Effective removal of cadmium, copper, lead, and zinc was achieved by the pilot-scale system. Results suggest that use of specifically designed constructed wetland treatment systems provides a flexible and effective approach for treating gas storage produced waters over a wide range of compositions.     

The Strengths and Difficulties Questionnaire as a Predictor of Parent-Reported Diagnosis of Autism Spectrum Disorder and Attention Deficit Hyperactivity Disorder

The Strengths and Difficulties Questionnaire (SDQ) is widely used as an international standardised instrument measuring child behaviour. The primary aim of our study was to examine whether behavioral symptoms measured by SDQ were elevated among children with autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD) relative to the rest of the population, and to examine the predictive value of the SDQ for outcome of parent-reported clinical diagnosis of ASD/ADHD. A secondary aim was to examine the extent of overlap in symptoms between children diagnosed with these two disorders, as measured by the SDQ subscales. A cross-sectional secondary analysis of data from the Millennium Birth Cohort (n = 19,519), was conducted. Data were weighted to be representative of the UK population as a whole. ADHD or ASD identified by a medical doctor or health professional were reported by parents in 2008 and this was the case definition of diagnosis; (ADHD n = 173, ASD n = 209, excluding twins and triplets). Study children''s ages ranged from 6.3–8.2 years; (mean 7.2 years). Logistic regression was used to examine the association between the parent-reported clinical diagnosis of ASD/ADHD and teacher and parent-reported SDQ subscales. All SDQ subscales were strongly associated with both ASD and ADHD. There was substantial co-occurrence of behavioral difficulties between children diagnosed with ASD and those diagnosed with ADHD. After adjustment for other subscales, the final model for ADHD, contained hyperactivity/inattention and impact symptoms only and had a sensitivity of 91% and specificity of 90%; (AUC) = 0.94 (95% CI, 0.90–0.97). The final model for ASD was composed of all subscales except the ‘peer problems’ scales, indicating of the complexity of behavioural difficulties that may accompany ASD. A threshold of 0.03 produced model sensitivity and specificity of 79% and 93% respectively; AUC = 0.90 (95% CI, 0.86–0.95). The results support changes to DSM-5 removing exclusivity clauses.     

Fine specificity of cellular immune responses in humans to human cytomegalovirus immediate-early 1 protein.

Cell-mediated immunity is important in maintaining the virus-host equilibrium in persistent human cytomegalovirus (HCMV) infection. The HCMV 72-kDa major immediate early 1 protein (IE1) is a target for CD8+ cytotoxic T cells in humans, as is the equivalent 89-kDa protein in mouse. Less is known about responses against this protein by CD4+ T cells, which may be important as direct effector cells or helper cells for antibody and CD8+ responses. Proliferative-T-cell responses to HCMV IE1 were studied in normal seropositive subjects. Peripheral blood mononuclear cells from 85% of seropositive subjects proliferated in response to HCMV from infected fibroblasts, and of these, 73% responded to recombinant baculovirus IE1. Responding cells were predominantly CD3+ CD4+. IE1 antigen preparations, including baculovirus recombinant protein, transfected rat cell nuclei, and synthetic peptides, induced IE1-specific T-cell lines which cross-reacted between the preparations. The fine specificity of these IE1-specific T-cell lines was studied by using overlapping synthetic peptides encompassing the entire sequence of the IE1 protein. The regions of the IE1 molecule recognized were identified and these varied between individuals, possibly reflecting differences in major histocompatibility complex (MHC) class II haplotype. In one subject, the peptide specificities of proliferative and MHC class I-restricted cytotoxic determinants on IE1 were spatially distinct. Thus, no single immunodominant T-cell determinant within HCMV IE1 was identified, suggesting that multiple peptides or a region of the 72-kDa IE1 protein would be required to induce specific T-cell responses in humans.