Progress towards World Health Organization HIV Infection 95-95-95 measures in Newfoundland and Labrador, Canada.

The HIV program in Newfoundland and Labrador (NL) provides care for all persons living with HIV (PLWH) in NL, yet progress toward UNAIDS 95-95-95 goals for diagnosis, linkage to care and viral suppression has not previously been documented. This analysis describes engagement in HIV care and virologic outcomes for the NL cohort in 2016 and 2019 and compares this data to the Canadian HIV Observational Cohort (CANOC). A retrospective review of the NL clinic included adults aged >18 years and descriptive statistics for demographics, risk factors, and clinical variables were assessed and compared using χ2 test or Fisher's Exact test (categorical) or Wilcoxon Sum Rank test (continuous). Engagement in care and virologic outcomes for the NL cohort were consistently high over the 2016 to 2019 period with > 98% on antiretroviral therapy (ART), and > 96% having a suppressed virus load. Engagement in care and virologic outcomes among PLWH in NL is high and compares favorably to a national cohort.

Selective regulation of chemosensitivity in glioblastoma by phosphatidylinositol 3-kinase beta.

Resistance to chemotherapies such as temozolomide is a major hurdle to effectively treat therapy-resistant glioblastoma. This challenge arises from the activation of phosphatidylinositol 3-kinase (PI3K), which makes it an appealing therapeutic target. However, non-selectively blocking PI3K kinases PI3Kα/ß/δ/γ has yielded undesired clinical outcomes. It is, therefore, imperative to investigate individual kinases in glioblastoma's chemosensitivity. Here, we report that PI3K kinases were unequally expressed in glioblastoma, with levels of PI3Kß being the highest. Patients deficient of O6-methylguanine-DNA-methyltransferase (MGMT) and expressing elevated levels of PI3Kß, defined as MGMT-deficient/PI3Kß-high, were less responsive to temozolomide and experienced poor prognosis. Consistently, MGMT-deficient/PI3Kß-high glioblastoma cells were resistant to temozolomide. Perturbation of PI3Kß, but not other kinases, sensitized MGMT-deficient/PI3Kß-high glioblastoma cells or tumors to temozolomide. Moreover, PI3Kß-selective inhibitors and temozolomide synergistically mitigated the growth of glioblastoma stem cells. Our results have demonstrated an essential role of PI3Kß in chemoresistance, making PI3Kß-selective blockade an effective chemosensitizer for glioblastoma.

Strategies to guide the successful implementation of deprescribing in community practice: Lessons learned from the front line.

Background: Sustainable implementation of new professional services into clinical practice can be difficult. In 2019, a population-wide initiative called SaferMedsNL was implemented across the province of Newfoundland and Labrador (NL), to promote appropriate medication use. Two evidence-based interventions were adapted to the context of NL to promote deprescribing of proton pump inhibitors and sedatives. The objective of this study was to identify and prioritize which actions supported the implementation of deprescribing in community practice for pharmacists, physicians and nurse practitioners across the province. Methods: Community pharmacists, physicians and nurse practitioners were invited to participate in virtual focus groups. Nominal Group Technique was used to elicit responses to the question: "What actions support the implementation of deprescribing into the daily workflow of your practice?" Participants prioritized actions within each group while thematic analysis permitted comparison across groups. Results: Five focus groups were held in fall 2020 involving pharmacists (n = 11), physicians (n = 7) and nurse practitioners (n = 4). Participants worked in rural (n = 10) and urban (n = 12) settings. The different groups agreed on what the top 5 actions were, with the top 5 receiving 68% of the scores: (1) providing patient education, (2) allocating time and resources, (3) building interprofessional collaboration and communication, (4) fostering patient relationships and (5) aligning with public awareness strategies. Conclusion: Pharmacists, physicians and nurse practitioners identified similar actions that supported implementing evidence-based deprescribing into routine clinical practice. Sharing these strategies may help others embed deprescribing into daily practice and assist the uptake of medication appropriateness initiatives by front-line providers. Can Pharm J (Ott) 2024;157:xx-xx.

CCK Receptor Inhibition Reduces Pancreatic Tumor Fibrosis and Promotes Nanoparticle Delivery.

The poor prognosis for pancreatic ductal adenocarcinoma (PDAC) patients is due in part to the highly fibrotic nature of the tumors that impedes delivery of therapeutics, including nanoparticles (NPs). Our prior studies demonstrated that proglumide, a cholecystokinin receptor (CCKR) antagonist, reduced fibrosis pervading PanIN lesions in mice. Here, we further detail how the reduced fibrosis elicited by proglumide achieves the normalization of the desmoplastic tumor microenvironment (TME) and improves nanoparticle uptake. One week following the orthotopic injection of PDAC cells, mice were randomized to normal or proglumide-treated water for 3-6 weeks. Tumors were analyzed ex vivo for fibrosis, vascularity, stellate cell activation, vascular patency, and nanoparticle distribution. The histological staining and three-dimensional imaging of tumors each indicated a reduction in stromal collagen in proglumide-treated mice. Proglumide treatment increased tumor vascularity and decreased the activation of cancer-associated fibroblasts (CAFs). Additionally, PANC-1 cells with the shRNA-mediated knockdown of the CCK2 receptor showed an even greater reduction in collagen, indicating the CCK2 receptors on tumor cells contribute to the desmoplastic TME. Proglumide-mediated reduction in fibrosis also led to functional changes in the TME as evidenced by the enhanced intra-tumoral distribution of small (<12 nm) Rhodamine-loaded nanoparticles. The documented in vivo, tumor cell-intrinsic anti-fibrotic effects of CCK2R blockade in both an immunocompetent syngeneic murine PDAC model as well as a human PDAC xenograft model demonstrates that CCK2R antagonists, such as proglumide, can improve the delivery of nano-encapsulated therapeutics or imaging agents to pancreatic tumors.

Expression of concern: Microchip-based structure determination of low-molecular weight proteins using cryo-electron microscopy.

Expression of concern for 'Microchip-based structure determination of low-molecular weight proteins using cryo-electron microscopy' by Michael A. Casasanta et al., Nanoscale, 2021, 13, 7285-7293, https://doi.org/10.1039/D1NR00388G.

Elucidating structural variability in p53 conformers using combinatorial refinement strategies and molecular dynamics.

Low molecular weight proteins and protein assemblies can now be investigated using cryo-electron microscopy (EM) as a complement to traditional structural biology techniques. It is important, however, to not lose sight of the dynamic information inherent in macromolecules that give rise to their exquisite functionality. As computational methods continue to advance the field of biomedical imaging, so must strategies to resolve the minute details of disease-related entities. Here, we employed combinatorial modeling approaches to assess flexible properties among low molecular weight proteins (~100 kDa or less). Through a blend of rigid body refinement and simulated annealing, we determined new hidden conformations for wild type p53 monomer and dimer forms. Structures for both states converged to yield new conformers, each revealing good stereochemistry and dynamic information about the protein. Based on these insights, we identified fluid parts of p53 that complement the stable central core of the protein responsible for engaging DNA. Molecular dynamics simulations corroborated the modeling results and helped pinpoint the more flexible residues in wild type p53. Overall, the new computational methods may be used to shed light on other small protein features in a vast ensemble of structural data that cannot be easily delineated by other algorithms.

The Glioblastoma Landscape: Hallmarks of Disease, Therapeutic Resistance, and Treatment Opportunities.

Malignant brain tumors are aggressive and difficult to treat. Glioblastoma is the most common and lethal form of primary brain tumor, often found in patients with no genetic predisposition. The median life expectancy for individuals diagnosed with this condition is 6 months to 2 years and there is no known cure. New paradigms in cancer biology implicate a small subset of tumor cells in initiating and sustaining these incurable brain tumors. Here, we discuss the heterogenous nature of glioblastoma and theories behind its capacity for therapy resistance and recurrence. Within the cancer landscape, cancer stem cells are thought to be both tumor initiators and major contributors to tumor heterogeneity and therapy evasion and such cells have been identified in glioblastoma. At the cellular level, disruptions in the delicate balance between differentiation and self-renewal spur transformation and support tumor growth. While rapidly dividing cells are more sensitive to elimination by traditional treatments, glioblastoma stem cells evade these measures through slow division and reversible exit from the cell cycle. At the molecular level, glioblastoma tumor cells exploit several signaling pathways to evade conventional therapies through improved DNA repair mechanisms and a flexible state of senescence. We examine these common evasion techniques while discussing potential molecular approaches to better target these deadly tumors. Equally important, the presented information encourages the idea of augmenting conventional treatments with novel glioblastoma stem cell-directed therapies, as eliminating these harmful progenitors holds great potential to modulate tumor recurrence.

Rotavirus core shell protein sites that regulate intra-particle polymerase activity.

IMPORTANCE: Rotaviruses are important causes of severe gastroenteritis in young children. A characteristic feature of rotaviruses is that they copy ribonucleic acid (RNA) inside of the viral particle. In fact, the viral polymerase (VP1) only functions when it is connected to the viral inner core shell protein (VP2). Here, we employed a biochemical assay to identify which sites of VP2 are critical for regulating VP1 activity. Specifically, we engineered VP2 proteins to contain amino acid changes at structurally defined sites and assayed them for their capacity to support VP1 function in a test tube. Through this work, we were able to identify several VP2 residues that appeared to regulate the activity of the polymerase, positively and negatively. These results are important because they help explain how rotavirus synthesizes its RNA while inside of particles and they identify targets for the future rational design of drugs to prevent rotavirus disease.

Delineating Conformational Variability in Small Protein Structures Using Combinatorial Refinement Strategies.

As small protein assemblies and even small proteins are becoming more amenable to cryo-Electron Microscopy (EM) structural studies, it is important to consider the complementary dynamic information present in the data. Current computational strategies are limited in their ability to resolve minute differences among low molecular weight entities. Here, we demonstrate a new combinatorial approach to delineate flexible conformations among small proteins using real-space refinement applications. We performed a meta-analysis of structural data for the SARS CoV-2 Nucleocapsid (N) protein using a combination of rigid-body refinement and simulated annealing methods. For the N protein monomer, we determined three new flexible conformers with good stereochemistry and quantitative comparisons provided new evidence of their dynamic properties. A similar analysis performed for the N protein dimer showed only minor structural differences among the flexible models. These results suggested a more stable view of the N protein dimer than the monomer structure. Taken together, the new computational strategies can delineate conformational changes in low molecular weight proteins that may go unnoticed by conventional assessments. The results also suggest that small proteins may be further stabilized for structural studies through the use of solution components that limit the movement of external flexible regions.

Liquid Transmission Electron Microscopy for Probing Collagen Biomineralization.

Collagen biomineralization is fundamental to hard tissue assembly. While studied extensively, collagen mineralization processes are not fully understood, with the majority of theories derived from electron microscopy (EM) under static, dehydrated, or frozen conditions, unlike the liquid phase environment where mineralization occurs. Herein, novel liquid transmission EM (TEM) strategies are presented, in which collagen mineralization was explored in liquid for the first time via TEM. Custom thin-film enclosures were employed to visualize the mineralization of reconstituted collagen fibrils in a calcium phosphate and polyaspartic acid solution to promote intrafibrillar mineralization. TEM highlighted that at early time points precursor mineral particles attached to collagen and progressed to crystalline mineral platelets aligned with fibrils at later time points. This aligns with observations from other techniques and validates the liquid TEM approach. This work provides a new liquid imaging approach for exploring collagen biomineralization, advancing toward understanding disease pathogenesis and remineralization strategies for hard tissues.

Structural Insights of the SARS-CoV-2 Nucleocapsid Protein: Implications for the Inner-workings of Rapid Antigen Tests.

The nucleocapsid (N) protein is an abundant component of SARS-CoV-2 and a key analyte for lateral-flow rapid antigen tests. Here, we present new structural insights for the SARS-CoV-2 N protein using cryo-electron microscopy (EM) and molecular modeling tools. Epitope mapping based on structural data supported host-immune interactions in the C-terminal portion of the protein, while other regions revealed protein-protein interaction sites. Complementary modeling results suggested that N protein structures from known variants of concern (VOC) are nearly 100% conserved at specific antibody-binding sites. Collectively, these results suggest that rapid tests that target the nucleocapsid C-terminal domain should have similar accuracy across all VOCs. In addition, our combined structural modeling workflow may guide the design of immune therapies to counter viral processes as we plan for future variants and pandemics.

Target users' acceptance of a pharmacist-led prescribing service for pre-exposure prophylaxis (PrEP) for human immunodeficiency virus (HIV).

Background: Pre-exposure prophylaxis (PrEP) for human immunodeficiency virus (HIV) is a highly effective way to reduce virus transmission. There have been increasing calls to improve access to PrEP in Canada. One way to improve access is by having more prescribers available. The objective of this study was to determine target users' acceptance of a PrEP-prescribing service by pharmacists in Nova Scotia. Methods: A triangulation, mixed-methods study was conducted consisting of an online survey and qualitative interviews underpinned by the Theoretical Framework of Acceptability (TFA) constructs (affective attitude, burden, ethicality, intervention coherence, opportunity cost, perceived effectiveness and self-efficacy). Participants were those eligible for PrEP in Nova Scotia (men who have sex with men or transgender women, persons who inject drugs and HIV-negative individuals in serodiscordant relationships). Descriptive statistics and ordinal logistic regression were used to analyze survey data. Interview data were deductively coded according to each TFA construct and then inductively coded to determine themes within each construct. Results: A total of 148 responses were captured by the survey, and 15 participants were interviewed. Participants supported pharmacists' prescribing PrEP across all TFA constructs from both survey and interview data. Identified concerns related to pharmacists' abilities to order and view lab results, pharmacists' knowledge and skills for sexual health and the potential for experiencing stigma within pharmacy settings. Conclusion: A pharmacist-led PrEP-prescribing service is acceptable to eligible populations in Nova Scotia. The feasibility of PrEP prescribing by pharmacists should be pursued as an intervention to increase access to PrEP.

Community pharmacists' acceptance of prescribing pre-exposure prophylaxis (PrEP) for human immunodeficiency virus (HIV).

Background: Pre-exposure prophylaxis (PrEP) for human immunodeficiency virus (HIV) prevention is highly effective. Pharmacists can increase PrEP accessibility through pharmacist prescribing. This study aimed to determine pharmacists' acceptance of a pharmacist PrEP prescribing service in Nova Scotia. Methods: A triangulation mixed methods study consisting of an online survey and qualitative interviews was conducted with Nova Scotia community pharmacists. The survey questionnaire and qualitative interview guide were underpinned by the 7 constructs of the Theoretical Framework of Acceptability (affective attitude, burden, ethicality, opportunity costs, intervention coherence, perceived effectiveness and self-efficacy). Survey data were analyzed descriptively and with ordinal logistic regression to determine associations between variables. Interview transcripts were deductively coded according to the same constructs and then inductively coded to identify themes within each construct. Results: A total of 214 community pharmacists completed the survey, and 19 completed the interview. Pharmacists were positive about PrEP prescribing in the constructs of affective attitude (improved access), ethicality (benefits communities), intervention coherence (practice alignment) and self-efficacy (role). Pharmacists expressed concerns about burden (increased workload), opportunity costs (time to provide the service) and perceived effectiveness (education/training, public awareness, laboratory test ordering and reimbursement). Conclusion: A PrEP prescribing service has mixed acceptability to Nova Scotia pharmacists yet represents a model of service delivery to increase PrEP access to underserved populations. Future service development must consider pharmacists' workload, education and training as well as factors relating to laboratory test ordering and reimbursement.

Barriers and enablers to testing for hepatitis C virus infection in people who inject drugs - a scoping review of the qualitative evidence.

BACKGROUND: Injection drug use is the primary mode of transmission of hepatitis C virus (HCV) infection in the developed world and guidelines recommend screening individuals with current or history of injection drug use for HCV; however, the majority of those living with HCV in Canada are not aware of their positive status. This low level of HCV status awareness suggests that screening is not effective with current testing strategies. The aim of this review is to determine what barriers and enablers people who inject drugs (PWID) experience surrounding testing for HCV to help inform the development of an engaging testing strategy. METHODS: Comprehensive literature searches were conducted using Medline, Embase and CINAHL in February 2021. Included studies investigated the barriers and enablers to testing for HCV in PWID and the experiences of PWID in testing for HCV. Studies were included if they were qualitative or mixed-methods design, involved people with current injection drug use or those with a history of injecting drugs, and were written in the English language. Studies were compared and common themes were coded and analyzed. RESULTS: The literature search resulted in 1554 citations and ultimately nine studies were included. Common barriers included self-perception of low risk for HCV, fear of diagnosis, stigma associated with IV drug use and HCV, antipathy in relation to mainstream health care services, limited knowledge about HCV, lack of rapport with provider, lack of motivation or competing priority of drug use, and limited awareness of new treatment options. Common enablers to testing included increasing awareness of HCV testing and treatment and providing positive narratives around HCV care, positive rapport with provider, accessible testing options and individualized care. CONCLUSION: While there has been some qualitative research on barriers and enablers to testing for HCV in PWID more research is needed to focus on this research question as a primary objective in order to provide more understanding from the participant's perspective.

Estimated cost-effectiveness of point-of-care testing in community pharmacies vs. self-testing and standard laboratory testing for HIV.

OBJECTIVE: Point-of-care-testing (POCT) for HIV at community pharmacies can enhance care linkage compared with self-tests and increase testing uptake relative to standard lab testing. While the higher test uptake may increase testing costs, timely diagnosis and treatment can reduce downstream HIV treatment costs and improve health outcomes. This study provides the first evidence on the cost-effectiveness of pharmacist-led POCT vs. HIV self-testing and standard lab testing. DESIGN: Dynamic transmission model. METHODS: We compared three HIV testing strategies: POCT at community pharmacies; self-testing using HIV self-test kits; and standard lab testing. Analyses were conducted from the Canadian health system perspective over a 30-year time horizon for all individuals aged 15-64 years in Canada. Costs were measured in 2021 Canadian dollars and effectiveness was captured using quality-adjusted life-years (QALYs). RESULTS: Compared with standard lab testing, POCT at community pharmacies would save $885 million in testing costs over 30 years. Though antiretroviral treatment costs would increase by $190 million with POCT as more persons living with HIV are identified and treated, these additional costs would be partly offset by their lower downstream healthcare utilization (savings of $150 million). POCT at community pharmacies would also yield over 5000 additional QALYs. Compared with HIV self-testing, POCT at community pharmacies would generate both higher costs and higher QALYs and would be cost-effective with an incremental cost-effectiveness ratio of $47 475 per QALY gained. CONCLUSIONS: Offering POCT at community pharmacies can generate substantial cost savings and improve health outcomes compared with standard lab testing. It would also be cost-effective vs. HIV self-testing.

Complete Models of p53 Better Inform the Impact of Hotspot Mutations.

Mutations in tumor suppressor genes often lead to cancerous phenotypes. Current treatments leverage signaling pathways that are often compromised by disease-derived deficiencies in tumor suppressors. P53 falls into this category as genetic mutations lead to physical changes in the protein that impact multiple cellular pathways. Here, we show the first complete structural models of mutated p53 to reveal how hotspot mutations physically deviate from the wild-type protein. We employed a recently determined structure for the p53 monomer to map seven frequent clinical mutations using computational modeling approaches. Results showed that missense mutations often changed the conformational structure of p53 in the DNA-binding site along with its electrostatic surface charges. We posit these changes may amplify the toxic effects of these hotspot mutations by destabilizing an important zinc ion coordination region in p53 to impede proper DNA interactions. These results highlight the imperative need for new studies on patient-derived proteins that may assist in redesigning structure-informed targeted therapies.