GPCRs in the round: SMA-like copolymers and SMALPs as a platform for investigating GPCRs.

G-protein-coupled receptors (GPCRs) are the largest family of membrane proteins, regulate a plethora of physiological responses and are the therapeutic target for 30-40% of clinically-prescribed drugs. They are integral membrane proteins deeply embedded in the plasma membrane where they activate intracellular signalling via coupling to G-proteins and ß-arrestin. GPCRs are in intimate association with the bilayer lipids and that lipid environment regulates the signalling functions of GPCRs. This complex lipid 'landscape' is both heterogeneous and dynamic. GPCR function is modulated by bulk membrane properties including membrane fluidity, microdomains, curvature, thickness and asymmetry but GPCRs are also regulated by specific lipid:GPCR binding, including cholesterol and anionic lipids. Understanding the molecular mechanisms whereby GPCR signalling is regulated by lipids is a very active area of research currently. A major advance in membrane protein research in recent years was the application of poly(styrene-co-maleic acid) (SMA) copolymers. These spontaneously generate SMA lipid particles (SMALPs) encapsulating membrane protein in a nano-scale disc of cell membrane, thereby removing the historical need for detergent and preserving lipid:GPCR interaction. The focus of this review is how GPCR-SMALPs are increasing our understanding of GPCR structure and function at the molecular level. Furthermore, an increasing number of 'second generation' SMA-like copolymers have been reported recently. These are reviewed from the context of increasing our understanding of GPCR molecular mechanisms. Moreover, their potential as a novel platform for downstream biophysical and structural analyses is assessed and looking ahead, the translational application of SMA-like copolymers to GPCR drug discovery programmes in the future is considered.

Prostate volume analysis in image registration for prostate cancer care: a verification study.

Combined magnetic resonance imaging (MRI) and positron emission tomography/computed tomography (PET/CT) may enhance diagnosis, aid surgical planning and intra-operative orientation for prostate biopsy and radical prostatectomy. Although PET-MRI may provide these benefits, PET-MRI machines are not widely available. Image fusion of Prostate specific membrane antigen PET/CT and MRI acquired separately may be a suitable clinical alternative. This study compares CT-MR registration algorithms for urological prostate cancer care. Paired whole-pelvis MR and CT scan data were used (n = 20). A manual prostate CTV contour was performed independently on each patients MR and CT image. A semi-automated rigid-, automated rigid- and automated non-rigid registration technique was applied to align the MR and CT data. Dice Similarity Index (DSI), 95% Hausdorff distance (95%HD) and average surface distance (ASD) measures were used to assess the closeness of the manual and registered contours. The automated non-rigid approach had a significantly improved performance compared to the automated rigid- and semi-automated rigid-registration, having better average scores and decreased spread for the DSI, 95%HD and ASD (all p < 0.001). Additionally, the automated rigid approach had similar significantly improved performance compared to the semi-automated rigid registration across all accuracy metrics observed (all p < 0.001). Overall, all registration techniques studied here demonstrated sufficient accuracy for exploring their clinical use. While the fully automated non-rigid registration algorithm in the present study provided the most accurate registration, the semi-automated rigid registration is a quick, feasible, and accessible method to perform image registration for prostate cancer care by urologists and radiation oncologists now.

Using Communities of Practice Theory to Understand the Crisis of Identity in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME).

OBJECTIVE: To explore the crisis of identity in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) through the lens of Communities of Practice. METHODS: A closed Facebook group was created to gather qualitative data from participants diagnosed with CFS/ME (n = 22). Data were analysed using a theoretical thematic analysis. RESULTS: The current research revealed the reality of enabling and disabling communities in the lived experience of CFS/ME and the role of participation in developing empowered identities. Learning how to be alongside CFS/ME aligned with participants' experiences of purpose and meaning. New identities may be developed which are not centrally defined by loss or stigma. DISCUSSION: Participation in supportive communities enables CFS/ME identities to emerge as a platform for positive change. Engaging with the CFS/ME virtual community may be a way for both families and health professionals to reflect on current practice.

Canadian Resources on Cannabis Use and Fertility, Pregnancy, and Lactation: Scoping Review.

BACKGROUND: Cannabis use among reproductive-aged Canadians is increasing, but our understanding of its impacts on fertility, pregnancy, and breast milk is still evolving. Despite the availability of many web-based resources, informed decision-making and patient counseling are challenging for expectant families and providers alike. OBJECTIVE: We aimed to conduct a scoping review of publicly available web-based Canadian resources to provide information on the effects of cannabis on fertility, pregnancy, and breast milk. METHODS: Following PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews), we systematically searched 8 databases between January 1, 2010, and November 30, 2020, and web pages of 71 Canadian obstetrical, government, and public health organizations. We included English resources discussing the effects of cannabis on fertility, pregnancy, breastfeeding, or the exposed fetus and infant. Epidemiological characteristics, readability, and content information were extracted and summarized. RESULTS: A total of 183 resources met our inclusion criteria. Resources included content for public audiences (163/183, 89.1%) and health care providers (HCPs; 31/183, 16.9%). The resources were authored by national-level (46/183, 25.1%), provincial or territorial (65/183, 35.5%), and regional (72/183, 39.3%) organizations. All provinces and territories had at least one resource attributed to them. The majority (125/183, 68.3%) were written at a >10 grade reading level, and a few (7/183, 3.8%) were available in languages other than English or French. The breadth of content on fertility (55/183, 30.1%), pregnancy (173/183, 94.5%), and breast milk or breastfeeding (133/183, 72.7%) varied across resources. Common themes included citing a need for more research into the effects of cannabis on reproductive health and recommending that patients avoid or discontinue cannabis use. Although resources for providers were consistent in recommending patient counseling, resources targeting the public were less likely to encourage seeking advice from HCPs (23/163, 14.1%). CONCLUSIONS: Canadian resources consistently identify that there is no known safe amount of cannabis that can be consumed in the context of fertility, pregnancy, and breastfeeding. Areas of improvement include increasing readability and language accessibility and encouraging bidirectional communication between HCPs and patients. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.1136/bmjopen-2020-045006.

Phosphoregulation accommodates Type III secretion and assembly of a tether of ER-Chlamydia inclusion membrane contact sites.

Membrane contact sites (MCS) are crucial for nonvesicular trafficking-based interorganelle communication. Endoplasmic reticulum (ER)-organelle tethering occurs in part through the interaction of the ER resident protein VAP with FFAT motif-containing proteins. FFAT motifs are characterized by a seven amino acidic core surrounded by acid tracks. We have previously shown that the human intracellular bacterial pathogen Chlamydia trachomatis establishes MCS between its vacuole (the inclusion) and the ER through expression of a bacterial tether, IncV, displaying molecular mimicry of eukaryotic FFAT motif cores. Here, we show that multiple layers of host cell kinase-mediated phosphorylation events govern the assembly of the IncV-VAP tethering complex and the formation of ER-Inclusion MCS. Via a C-terminal region containing three CK2 phosphorylation motifs, IncV recruits CK2 to the inclusion leading to IncV hyperphosphorylation of the noncanonical FFAT motif core and serine-rich tracts immediately upstream of IncV FFAT motif cores. Phosphorylatable serine tracts, rather than genetically encoded acidic tracts, accommodate Type III-mediated translocation of IncV to the inclusion membrane, while achieving full mimicry of FFAT motifs. Thus, regulatory components and post-translational modifications are integral to MCS biology, and intracellular pathogens such as C. trachomatis have evolved complex molecular mimicry of these eukaryotic features.

Webinar and survey on quality management principles within the Australian and New Zealand ACPSEM Workforce.

Healthcare relies upon the accurate and safe delivery of patient care. This is only achievable when systems are developed to ensure high quality, robust outcomes, for instance quality management systems. The concept of quality management can take on a different meaning depending on the context in which it is found. To add complication, the amount of education required for quality management will vary depending on one's exposure to the implementation of quality systems. In part to address these issues, the Australasian College of Physical Scientists and Engineers in Medicine (ACPSEM) Queensland Branch held a quality management webinar for members and non-members across Australia and New Zealand. The purpose of the webinar was to educate and facilitate discussion regarding the application of quality management principles for the ACPSEM profession. In conjunction, a pre- and post-webinar survey was conducted to gain an insight into existing knowledge and attitudes within the professions governed by the ACPSEM and students undertaking related studies. This paper authored by the webinar speakers reintroduces the quality management principles that were discussed in webinar, exemplifies the importance of quality management skills within the ACPSEM professions and presents the results of the surveys, promoting the need for more educational resources on quality management tools.

Clinical validation of a next-generation sequencing-based multi-cancer early detection "liquid biopsy" blood test in over 1,000 dogs using an independent testing set: The CANcer Detection in Dogs (CANDiD) study.

Cancer is the leading cause of death in dogs, yet there are no established screening paradigms for early detection. Liquid biopsy methods that interrogate cancer-derived genomic alterations in cell-free DNA in blood are being adopted for multi-cancer early detection in human medicine and are now available for veterinary use. The CANcer Detection in Dogs (CANDiD) study is an international, multi-center clinical study designed to validate the performance of a novel multi-cancer early detection "liquid biopsy" test developed for noninvasive detection and characterization of cancer in dogs using next-generation sequencing (NGS) of blood-derived DNA; study results are reported here. In total, 1,358 cancer-diagnosed and presumably cancer-free dogs were enrolled in the study, representing the range of breeds, weights, ages, and cancer types seen in routine clinical practice; 1,100 subjects met inclusion criteria for analysis and were used in the validation of the test. Overall, the liquid biopsy test demonstrated a 54.7% (95% CI: 49.3-60.0%) sensitivity and a 98.5% (95% CI: 97.0-99.3%) specificity. For three of the most aggressive canine cancers (lymphoma, hemangiosarcoma, osteosarcoma), the detection rate was 85.4% (95% CI: 78.4-90.9%); and for eight of the most common canine cancers (lymphoma, hemangiosarcoma, osteosarcoma, soft tissue sarcoma, mast cell tumor, mammary gland carcinoma, anal sac adenocarcinoma, malignant melanoma), the detection rate was 61.9% (95% CI: 55.3-68.1%). The test detected cancer signal in patients representing 30 distinct cancer types and provided a Cancer Signal Origin prediction for a subset of patients with hematological malignancies. Furthermore, the test accurately detected cancer signal in four presumably cancer-free subjects before the onset of clinical signs, further supporting the utility of liquid biopsy as an early detection test. Taken together, these findings demonstrate that NGS-based liquid biopsy can offer a novel option for noninvasive multi-cancer detection in dogs.

Development of 3D Printed Biodegradable Mesh with Antimicrobial Properties for Pelvic Organ Prolapse.

To address the increasing demand for safe and effective treatment options for pelvic organ prolapse (POP) due to the worldwide ban of the traditional polypropylene meshes, this study introduced degradable polycaprolactone (PCL)/polyethylene glycol (PEG) composite meshes fabricated with melt-electrowriting (MEW). Two PCL/PEG mesh groups: 90:10 and 75:25 (PCL:PEG, wt%) were fabricated and characterized for their degradation rate and mechanical properties, with PCL meshes used as a control. The PCL/PEG composites showed controllable degradation rates by adjusting the PEG content and produced mechanical properties, such as maximal forces, that were higher than PCL alone. The antibacterial properties of the meshes were elicited by coating them with a commonly used antibiotic: azithromycin. Two dosage levels were used for the coating: 0.5 mg and 1 mg per mesh, and both dosage levels were found to be effective in suppressing the growth of S. aureus bacteria. The biocompatibility of the meshes was assessed using human immortalized adipose derived mesenchymal stem cells (hMSC). In vitro assays were used to assess the cell viability (LIVE/DEAD assay), cell metabolic activity (alamarBlue assay) and cell morphology on the meshes (fluorescent and electron microscopy). The cell attachment was found to decrease with increased PEG content. The freshly drug-coated meshes showed signs of cytotoxicity during the cell study process. However, when pre-released for 14 days in phosphate buffered saline, the initial delay in cell attachment on the drug-coated mesh groups showed full recovery at the 14-day cell culture time point. These results indicated that the PCL/PEG meshes with antibiotics coating will be an effective anti-infectious device when first implanted into the patients, and, after about 2 weeks of drug release, the mesh will be supporting cell attachment and proliferation. These meshes demonstrated a potential effective treatment option for POP that may circumvent the issues related to the traditional polypropylene meshes.

Childhood Poverty, Adverse Childhood Experiences, and Adult Health Outcomes.

This study aimed to consider childhood poverty in relation to a count measure of adverse childhood experiences (ACEs) as a predictor of adult health outcomes and to determine whether associations are sensitive to how childhood poverty is operationalized. A sample of 10,784 adult residents was derived using data 2014-2015 Wisconsin annual Behavioral Risk Factor Survey data, derived from the Centers for Disease Control and Prevention (CDC) Behavioral Risk Factor Surveillance System (BRFSS). Adult health outcomes (health risk behaviors, general health problems, chronic health problems, and depression) were predicted using a more conservative and severe indicator of childhood poverty, and authors tested whether observed associations were attenuated by the inclusion of an ACE count variable. Findings showed that severe indicators of childhood poverty are associated with general and chronic health problems as well as adult depression. These associations are attenuated, but remain intact, when ACEs are included in regression models. Using the CDC BRFSS data for Wisconsin, the study showed that associations between childhood poverty and adult health are sensitive to the way in which childhood poverty is operationalized. The relationship between childhood poverty and other ACEs is complex and thus warrants treating the former as a distinct childhood adversity rather than an item in an ACE summary score.

Canadian educational resources about cannabis use and fertility, pregnancy and breast feeding: a scoping review protocol.

INTRODUCTION: Cannabis use in Canada is becoming more prevalent across all demographic groups due to increases in accessibility and lowered perceptions of harm. These patterns are mirrored among women of reproductive age, including women who are pregnant. Given increasing evidence for detrimental short- and long-term impacts of cannabis exposure on fetal, newborn and child outcomes, there is a need for high-quality, accessible resources providing reliable guidance and recommendations on this topic for both the public and healthcare providers. We will conduct a scoping review to identify and characterise all publicly available online educational resources discussing cannabis use related to fertility, pregnancy and breastfeeding developed by Canadian organisations. METHODS AND ANALYSIS: Using Arksey and O'Malley's scoping review methodology as a guide, we will search Medline (Ovid), Medline in Process (Ovid), Embase (Ovid), ERIC (Ovid), CINAHL (EBSCOhost) and Education Source (EBSCOhost). We will also conduct a grey literature search targeting the websites of national and independent Canadian obstetrical societies and networks, and government and public health offices that provide recommendations or guidance to individuals and their healthcare providers seeking information on cannabis use related to fertility, pregnancy or breastfeeding. ETHICS AND DISSEMINATION: Research ethics approval is not required for scoping review studies. We anticipate that this review's findings will be disseminated through traditional channels, including preprint and peer-reviewed publications and presentations at academic conferences. In addition, the resources and guidelines identified in the study will be gathered and made available online on a single comprehensive public repository. PROTOCOL REGISTRATION NUMBER: osf.io/p24y5.

Teaching Object Play to Young Children With Disabilities: A Systematic Review of Methods and Rigor.

In this systematic review, we examined the rigor and outcomes across 27 object play intervention studies using single-case research methodology. We focused on studies including children age 5 years or younger and examined several descriptive characteristics including materials, instructional packages, and settings. We also analyzed the facilitation and measurement of generalized play and several methodological features including quality, rigor, and visual analysis procedures. Overall, the identified studies demonstrated positive outcomes, although quality and rigor limited interpretations of the outcomes. Previous reviews also have noted strong outcomes and weak to moderate quality for single-case studies. Our results should be interpreted with caution given previous reviews of play intervention studies identified strong outcomes and quality from group-design studies. Additional replications testing robust interventions using single-case research with strong methodological rigor are warranted.

In vitro evaluation of negative pressure generated during application of negative suction volumes by use of various syringes with and without thoracostomy tubes.

OBJECTIVE: To determine the amount of negative pressure generated by syringes of various sizes with and without an attached thoracostomy tube and whether composition of thoracostomy tubes altered the negative pressure generated. SAMPLE: Syringes ranging from 1 to 60 mL and 4 thoracostomy tubes of various compositions (1 red rubber catheter, 1 polyvinyl tube, and 2 silicone tubes). PROCEDURES: A syringe or syringe with attached thoracostomy tube was connected to a pneumatic transducer. Each syringe was used to aspirate a volume of air 10 times. Negative pressure generated was measured and compared among the various syringe sizes and various thoracostomy tubes. RESULTS: The negative pressure generated decreased as size of the syringe increased for a fixed volume across syringes. Addition of a thoracostomy tube further decreased the amount of negative pressure. The red rubber catheter resulted in the least amount of negative pressure, followed by the polyvinyl tube and then the silicone tubes. There was no significant difference in negative pressure between the 2 silicone tubes. The smallest amount of negative pressure generated was -74 to -83 mm Hg. CONCLUSIONS AND CLINICAL RELEVANCE: Limited data are available on the negative pressure generated during intermittent evacuation of the thoracic cavity. For the present study, use of a syringe of ≥ 20 mL and application of 1 mL of negative suction volume resulted in in vitro pressures much more negative than the currently recommended pressure of -14.71 mm Hg for continuous suction. Additional in vitro or cadaveric studies are needed.

Therapeutic Alliance Between Dietitians and Patients With Head and Neck Cancer: The Effect of Training in a Health Behavior Change Intervention.

BACKGROUND: The relationship between a clinician and their client-the "therapeutic alliance" is a robust predictor of outcome in healthcare settings; yet, few interventions to improve alliance have been tested. Motivational interviewing is a client-centered approach that embodies many principles and strategies consistent with a strong therapeutic alliance. PURPOSE: To examine whether alliance is enhanced by training dietitians to deliver a motivational interviewing informed health behavior change intervention ("Eating as Treatment"; EAT) as part of routine consultations with patients with head and neck cancer. The predictive ability of motivational interviewing techniques was also assessed. METHODS: A secondary analysis of the EAT stepped-wedge cluster-randomized controlled trial was conducted. Patients with head and neck cancer undergoing radiotherapy (n = 307) were treated by radiotherapy dietitians (n = 29) during the control (Treatment as Usual) or intervention (EAT) phase. Alliance was rated during the first and final weeks of radiotherapy, and again 4 and 12 weeks post-radiotherapy. Dietetic sessions were audiotaped. Week one sessions were objectively rated for dietitians' use of motivational interviewing techniques. RESULTS: Generalized linear-mixed effects regressions found no effect of EAT on dietitian-rated alliance (p = .237). After excluding outliers, patient-rated alliance was 0.29 points lower after EAT training (p = .016). Post hoc analyses revealed lower patient ratings on perceived support and dietitian confidence. Hierarchical multiple regressions found that no specific motivational interviewing techniques predicted patient-rated alliance. Dietitian acknowledgment of patient challenges was related to dietitian-rated alliance (ß =.15, p =.035). CONCLUSIONS: Patient and dietitian ratings of alliance were high after EAT training, but not significantly improved. Further research is needed to better understand the differential impact of intervention training and delivery on patient and clinician ratings of therapeutic alliance. CLINICAL TRIAL INFORMATION: Trial registration number ACTRN12613000320752.

Making Contact: VAP Targeting by Intracellular Pathogens.

In naïve cells, the endoplasmic reticulum (ER) and the ER-resident Vesicle-associated membrane protein-Associated Proteins (VAP) are common components of sites of membrane contacts that mediate the nonvesicular transfer of lipids between organelles. There is increasing recognition that the hijacking of VAP by intracellular pathogens is a novel mechanism of host-pathogen interaction. Here, we summarize our recent findings showing that the Chlamydia inclusion membrane protein IncV tethers the ER to the inclusion membrane by binding to VAP via the molecular mimicry of two eukaryotic FFAT motifs. We extend the discussion to other microorganisms that have evolved similar mechanisms.

In Vitro Evaluation of Evacuated Blood Collection Tubes as a Closed-Suction Surgical Drain Reservoir.

The initial negative pressures of evacuated blood collection tubes (EBCT) and their in vitro performance as a rigid closed-suction surgical drain (CSSD) reservoir has not been evaluated in the scientific literature despite being described in both human and veterinary texts and journals. The initial negative pressures of EBCT sized 3, 6, 10, and 15 mL were measured and the stability of the system monitored. The pressure-to-volume curve as either air or water was added and maximal filling volumes were measured. Evacuated blood collection tubes beyond the manufacture's expiration date were evaluated for initial negative pressures and maximal filling volumes. Initial negative pressure ranged from -214 mm Hg to -528 mm Hg for EBCT within the manufacturer's expiration date. Different pressure-to-volume curves were found for air versus water. Optimal negative pressures of CSSD are debated in the literature. Drain purpose and type of exudates are factors that should be considered when deciding which EBCT size to implement. Evacuated blood collection tubes have a range of negative pressures and pressure-to-volume curves similar to previously evaluated CSSD rigid reservoirs. Proper drain management and using EBCT within labeled expiration date are important to ensure that expected negative pressures are generated.

IncV, a FFAT motif-containing Chlamydia protein, tethers the endoplasmic reticulum to the pathogen-containing vacuole.

Membrane contact sites (MCS) are zones of contact between the membranes of two organelles. At MCS, specific proteins tether the organelles in close proximity and mediate the nonvesicular trafficking of lipids and ions between the two organelles. The endoplasmic reticulum (ER) integral membrane protein VAP is a common component of MCS involved in both tethering and lipid transfer by binding directly to proteins containing a FFAT [two phenylalanines (FF) in an acidic tract (AT)] motif. In addition to maintaining cell homeostasis, MCS formation recently emerged as a mechanism by which intracellular pathogens hijack cellular resources and establish their replication niche. Here, we investigated the mechanism by which the Chlamydia-containing vacuole, termed the inclusion, establishes direct contact with the ER. We show that the Chlamydia protein IncV, which is inserted into the inclusion membrane, displays one canonical and one noncanonical FFAT motif that cooperatively mediated the interaction of IncV with VAP. IncV overexpression was sufficient to bring the ER in close proximity of IncV-containing membranes. Although IncV deletion partially decreased VAP association with the inclusion, it did not suppress the formation of ER-inclusion MCS, suggesting the existence of redundant mechanisms in MCS formation. We propose a model in which IncV acts as one of the primary tethers that contribute to the formation of ER-inclusion MCS. Our results highlight a previously unidentified mechanism of bacterial pathogenesis and support the notion that cooperation of two FFAT motifs may be a common feature of VAP-mediated MCS formation. Chlamydia-host cell interaction therefore constitutes a unique system to decipher the molecular mechanisms underlying MCS formation.