BLAP-Tags, TUBEs and DUB-Chips: Combined Novel Technologies will Advance Molecular Epithelial Physiology

The field of ubiquitylation and deubiquitylation of proteins in molecular physiology is growing at a rapid rate. Our understanding of molecular physiology of these processes may become limited by the advancement of technologies that scientists can employ. Therefore, it is important to approach physiological questions of ubiquitylation and deubiquitylation of proteins from a multiple methodological direction. Indeed, the role of ubiquitylation and deubiquitylation of proteins in cellular function has been implicated in the pathophysiology of human diseases including cancer, viral diseases, and neurodegenerative disorders. There are many modulators (activators and inhibitors) of ubiquitylation and deubiquitylation. Therefore, the link is being able to rapidly assess potential modulators of ubiquitylation and deubiquitylation and determine which specific modulators play a role(s) within a particular physiological setting. After the specific modulators have been identified, further experimentation is required to assess the downstream use as potential clinical targets for a particular disease. The first step is to identify the specific modulators. This perspective highlights a multi-prong technologies approach that uses three novel technologies (BLAP-tagged proteins, TUBES, and DUB-Chips) that can rapidly identify a number of potential candidates that modulate ubiquitylation and deubiquitylation of cellular proteins

Patient and Provider Perspectives on Medication Non-adherence Among Patients with Depression and/or Diabetes in Diverse Community Settings - A Qualitative Analysis.

BACKGROUND: Diabetes and depression affect a significant percentage of the world\u27s total population, and the management of these conditions is critical for reducing the global burden of disease. Medication adherence is crucial for improving diabetes and depression outcomes, and research is needed to elucidate barriers to medication adherence, including the intentionality of non-adherence, to intervene effectively. The purpose of this study was to explore the perspectives of patients and health care providers on intentional and unintentional medication adherence among patients with depression and diabetes through a series of focus groups conducted across clinical settings in a large urban area. METHODS: This qualitative study utilized a grounded theory approach to thematically analyze qualitative data using the framework method. Four focus groups in total were conducted, two with patients and two with providers, over a one-year period using a semi-structured facilitation instrument containing open-ended questions about experiences, perceptions and beliefs about medication adherence. RESULTS: Across the focus groups, communication difficulties between patients and providers resulting in medication non-adherence was a primary theme that emerged. Concerns about medication side effects and beliefs about medication effectiveness were identified as perceptual barriers related to intentional medication non-adherence. Practical barriers to medication adherence, including medication costs, forgetting to take medications and polypharmacy, emerged as themes related to unintentional medication non-adherence. CONCLUSION: The study findings contribute to a growing body of research suggesting health system changes are needed to improve provider education and implement multicomponent interventions to improve medication adherence among patients with depression and/or diabetes, both chronic illnesses accounting for significant disease burden globally

Plasma Membrane Insertion of KCa2.3 (SK3) is Dependent Upon the SNARE Proteins, Syntaxin-4 and SNAP23

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. We previously demonstrated endocytosis of KCa2.3 is caveolin-1-, dynamin II- and Rab5-dependent. KCa2.3 then enters Rab35/EPI64C- and RME-1-containing recycling endosomes and is returned to the plasma membrane (PM). Herein, we report on the mechanism by which KCa2.3 is inserted into the PM during recycling and following exit from the Golgi. We demonstrate KCa2.3 colocalizes with SNAP-23 and Syntaxin-4 in the PM of HEK and endothelial cells by confocal immunofluorescence microscopy. We further show KCa2.3 can be co-immunoprecipitated with SNAP-23 and Syntaxin-4. Overexpression of either Syntaxin-4 or SNAP-23 increased PM expression of KCa2.3, whereas shRNA-mediated knockdown of these SNARE proteins significantly decreased PM KCa2.3 expression, as assessed by cell surface biotinylation. Whole-cell patch clamp studies confirmed knockdown of SNAP-23 significantly decreased the apamin sensitive, KCa2.3 current. Using standard biotinylation/stripping methods, we demonstrate shRNA mediated knockdown of SNAP-23 inhibits recycling of KCa2.3 following endocytosis, whereas scrambled shRNA had no effect. Finally, using biotin ligase acceptor peptide (BLAP)-tagged KCa2.3, coupled with ER-resident biotin ligase (BirA), channels could be biotinylated in the ER after which we evaluated their rate of insertion into the PM following Golgi exit. We demonstrate knockdown of SNAP-23 significantly slows the rate of Golgi to PM delivery of KCa2.3. The inhibition of both recycling and PM delivery of newly synthesized KCa2.3 channels likely accounts for the decreased PM expression observed following knockdown of these SNARE proteins. In total, our results suggest insertion of KCa2.3 into the PM depends upon the SNARE proteins, Syntaxin-4 and SNAP-23

Whole genome sequencing of Plasmodium falciparum from dried blood spots using selective whole genome amplification

BACKGROUND: Translating genomic technologies into healthcare applications for the malaria parasite Plasmodium falciparum has been limited by the technical and logistical difficulties of obtaining high quality clinical samples from the field. Sampling by dried blood spot (DBS) finger-pricks can be performed safely and efficiently with minimal resource and storage requirements compared with venous blood (VB). Here, the use of selective whole genome amplification (sWGA) to sequence the P. falciparum genome from clinical DBS samples was evaluated, and the results compared with current methods that use leucodepleted VB. METHODS: Parasite DNA with high (>95%) human DNA contamination was selectively amplified by Phi29 polymerase using short oligonucleotide probes of 8-12 mers as primers. These primers were selected on the basis of their differential frequency of binding the desired (P. falciparum DNA) and contaminating (human) genomes. RESULTS: Using sWGA method, clinical samples from 156 malaria patients, including 120 paired samples for head-to-head comparison of DBS and leucodepleted VB were sequenced. Greater than 18-fold enrichment of P. falciparum DNA was achieved from DBS extracts. The parasitaemia threshold to achieve >5× coverage for 50% of the genome was 0.03% (40 parasites per 200 white blood cells). Over 99% SNP concordance between VB and DBS samples was achieved after excluding missing calls. CONCLUSION: The sWGA methods described here provide a reliable and scalable way of generating P. falciparum genome sequence data from DBS samples. The current data indicate that it will be possible to get good quality sequence on most if not all drug resistance loci from the majority of symptomatic malaria patients. This technique overcomes a major limiting factor in P. falciparum genome sequencing from field samples, and paves the way for large-scale epidemiological applications

A structured framework for improving outbreak investigation audits

Outbreak investigation is a core function of public health agencies. Suboptimal outbreak investigation endangers both public health and agency reputations. While audits of clinical medical and nursing practice are conducted as part of continuous quality improvement, public health agencies rarely make systematic use of structured audits to ensure best practice for outbreak responses, and there is limited guidance or policy to guide outbreak audit. A framework for prioritising which outbreak investigations to audit, an approach for conducting a successful audit, and a template for audit trigger questions was developed and trialled in four foodborne outbreaks and a respiratory disease outbreak in Australia. The following issues were identified across several structured audits: the need for clear definitions of roles and responsibilities both within and between agencies, improved communication between agencies and with external stakeholders involved in outbreaks, and the need for development of performance standards in outbreak investigations - particularly in relation to timeliness of response. Participants considered the audit process and methodology to be clear, useful, and non-threatening. Most audits can be conducted within two to three hours, however, some participants felt this limited the scope of the audit. The framework was acceptable to participants, provided an opportunity for clarifying perceptions and enhancing partnership approaches, and provided useful recommendations for approaching future outbreaks. Future challenges include incorporating feedback from broader stakeholder groups, for example those of affected cases, institutions and businesses; assessing the quality of a specific audit; developing training for both participants and facilitators; and building a central capacity to support jurisdictions embarking on an audit. The incorporation of measurable performance criteria or sharing of benchmark performance criteria will assist in the standardisation of outbreak investigation audit and further quality improvement

Women out, children out : the effect of female labor on portuguese preschool enrollment rates

This article tests whether Portuguese female activity rates have increased preschool enrollment rates. Particularly during the last 20 years, Portuguese women have assumed new roles in the marketplace and have become active workers outside of the home environment. This change has encouraged more sensible decisions with respect to preschool enrollment. Using cointegration techniques, we concluded that female activity rates and real income per capita caused a long-term increase in preschool enrollment rates. Although the percentage of agricultural gross value added to the gross domestic product and the number of preschool institutes were also found to be significant in the estimated vector error correction model, their causal relationship with preschool enrollment was only short term.COMPETE; QREN; FEDER; Fundação para a Ciência e a Tecnologia (FCT