Interactome Analysis Identifies the Role of BZW2 in Promoting Endoplasmic Reticulum-Mitochondria Contact and Mitochondrial Metabolism.

Understanding the molecular functions of less-studied proteins is an important task of life science research. Despite reports of basic leucine zipper and W2 domain-containing protein 2 (BZW2) promoting cancer progression first emerging in 2017, little is known about its molecular function. Using a quantitative proteomic approach to identify its interacting proteins, we found that BZW2 interacts with both endoplasmic reticulum (ER) and mitochondrial proteins. We thus hypothesized that BZW2 localizes to and promotes the formation of ER-mitochondria contact sites and that such localization would promote calcium transport from ER to the mitochondria and promote ATP production. Indeed, we found that BZW2 localized to ER-mitochondria contact sites and that BZW2 knockdown decreased ER-mitochondria contact, mitochondrial calcium levels, and ATP production. These findings provide key insights into molecular functions of BZW2, the potential role of BZW2 in cancer progression, and highlight the utility of interactome data in understanding the function of less-studied proteins.

Risk of Multiple Sclerosis in People Living with HIV: An International Cohort Study.

OBJECTIVE: There has been interest in a possible negative association between HIV and multiple sclerosis (MS). We aimed to compare the risk of MS in a cohort of individuals living with HIV to that in the general population. METHODS: Population-based health data were accessed for 2 cohorts of HIV-positive persons from Sweden and British Columbia, Canada. Incident MS was identified using MS registries or a validated algorithm applied to administrative data. Individuals with HIV were followed from 1 year after the first clinical evidence of HIV or the first date of complete administrative health data (Canada = April 1, 1992 and Sweden = January 1, 2001) until the earliest of incident MS, emigration, death, or study end (Canada = March 31, 2020 and Sweden = December 31, 2018). The observed MS incidence rate in the HIV-positive cohort was compared to the expected age-, sex-, calendar year-, income-specific, and region of birth-specific rates in a randomly selected sample of >20% of each general population. The standardized incidence ratio (SIR) for MS following the first antiretroviral therapy exposure ("ART-exposed") was also calculated. RESULTS: The combined Sweden-Canada cohort included 29,163 (75% men) HIV-positive persons. During 242,248 person-years of follow-up, 14 incident MS cases were observed in the HIV-positive cohort, whereas 26.19 cases were expected. The SIR for MS in the HIV-positive population was 0.53 (95% confidence interval [CI] = 0.32-0.90). The SIR for MS following the first ART exposure was 0.55 (95% CI = 0.31-0.96). INTERPRETATION: This international population-based study demonstrated a lower risk of MS among HIV-positive individuals, and HIV-positive ART-exposed individuals. These findings provide support for further exploration into the relationship among HIV, ART, and MS. ANN NEUROL 2024;95:487-494.

Three biomarkers (HER2, PD-L1, and microsatellite status) in a large cohort of metastatic gastroesophageal adenocarcinomas: The MD Anderson Cancer Center experience.

Human epidermal growth factor receptor-2 (HER2), programmed death-ligand 1 (PD-L1), and microsatellite (MS) status are well-established biomarkers in gastroesophageal adenocarcinomas (GEAs). However, it is unclear how the combination of these biomarkers is associated with clinicopathological factors and prognosis. This retrospective study included baseline metastatic GEA patients who were tested for all three biomarkers (HER2, PD-L1, and MS status) at the MD Anderson Cancer Center between 2012 and 2022. Stratification was performed according to the combination of biomarker profiles: triple negative (TN), single positive (SP), and multiple positive (MP). Comparative analyses of clinicopathological factors and survival using combinations of biomarkers were performed. Among the 698 GEA patients analyzed, 251 (36.0%) were classified as TN, 334 (47.9%) as SP, and 113 (16.1%) as MP. The MP group showed a significant association with tumors located in the esophagus (p < .001), well to moderate differentiation (p < .001), and the absence of signet ring cells (p < .001). In the survival analysis, MP group had a significantly longer overall survival (OS) compared to the other groups (MP vs. TN, p < .001 and MP vs. SP, p < .001). Multivariate Cox regression analysis revealed that MP serves as an independent positive prognostic indicator for OS (hazard ratio = 0.63, p < .01). Our findings indicate that MP biomarkers are associated with a favorable prognosis in metastatic GEA. These results are reflective of clinical practice and offer valuable insights into how therapeutics and future biomarkers could influence therapy/prognosis.

Noncanonical functions of telomerase and telomeres in viruses-associated cancer.

The cause of cancer is attributed to the uncontrolled growth and proliferation of cells resulting from genetic changes and alterations in cell behavior, a phenomenon known as epigenetics. Telomeres, protective caps on the ends of chromosomes, regulate both cellular aging and cancer formation. In most cancers, telomerase is upregulated, with the telomerase reverse transcriptase (TERT) enzyme and telomerase RNA component (TERC) RNA element contributing to the maintenance of telomere length. Additionally, it is noteworthy that two viruses, human papillomavirus (HPV) and Epstein-Barr virus (EBV), utilize telomerase for their replication or persistence in infected cells. Also, TERT and TERC may play major roles in cancer not related to telomere biology. They are involved in the regulation of gene expression, signal transduction pathways, cellular metabolism, or even immune response modulation. Furthermore, the crosstalk between TERT, TERC, RNA-binding proteins, and microRNAs contributes to a greater extent to cancer biology. To understand the multifaceted roles played by TERT and TERC in cancer and viral life cycles, and then to develop effective therapeutic strategies against these diseases, are fundamental for this goal. By investigating deeply, the complicated mechanisms and relationships between TERT and TERC, scientists will open the doors to new therapies. In its analysis, the review emphasizes the significance of gaining insight into the multifaceted roles that TERT and TERC play in cancer pathogenesis, as well as their involvement in the viral life cycle for designing effective anticancer therapy approaches.

Debamestrocel multimodal effects on biomarker pathways in amyotrophic lateral sclerosis are linked to clinical outcomes.

INTRODUCTION/AIMS: Biomarkers have shown promise in amyotrophic lateral sclerosis (ALS) research, but the quest for reliable biomarkers remains active. This study evaluates the effect of debamestrocel on cerebrospinal fluid (CSF) biomarkers, an exploratory endpoint. METHODS: A total of 196 participants randomly received debamestrocel or placebo. Seven CSF samples were to be collected from all participants. Forty-five biomarkers were analyzed in the overall study and by two subgroups characterized by the ALS Functional Rating Scale-Revised (ALSFRS-R). A prespecified model was employed to predict clinical outcomes leveraging biomarkers and disease characteristics. Causal inference was used to analyze relationships between neurofilament light chain (NfL) and ALSFRS-R. RESULTS: We observed significant changes with debamestrocel in 64% of the biomarkers studied, spanning pathways implicated in ALS pathology (63% neuroinflammation, 50% neurodegeneration, and 89% neuroprotection). Biomarker changes with debamestrocel show biological activity in trial participants, including those with advanced ALS. CSF biomarkers were predictive of clinical outcomes in debamestrocel-treated participants (baseline NfL, baseline latency-associated peptide/transforming growth factor beta1 [LAP/TGFß1], change galectin-1, all p < .01), with baseline NfL and LAP/TGFß1 remaining (p < .05) when disease characteristics (p < .005) were incorporated. Change from baseline to the last measurement showed debamestrocel-driven reductions in NfL were associated with less decline in ALSFRS-R. Debamestrocel significantly reduced NfL from baseline compared with placebo (11% vs. 1.6%, p = .037). DISCUSSION: Following debamestrocel treatment, many biomarkers showed increases (anti-inflammatory/neuroprotective) or decreases (inflammatory/neurodegenerative) suggesting a possible treatment effect. Neuroinflammatory and neuroprotective biomarkers were predictive of clinical response, suggesting a potential multimodal mechanism of action. These results offer preliminary insights that need to be confirmed.

Development and application of a multiple reaction monitoring method for the simultaneous quantification of sodium channels Nav 1.1, Nav 1.2, and Nav 1.6 in solubilized membrane proteins from stable HEK293 cell lines, rodents, and human brain tissues.

RATIONALE: Nav 1.1, 1.2, and 1.6 are transmembrane proteins acting as voltage-gated sodium channels implicated in various forms of epilepsy. There is a need for knowing their actual concentration in target tissues during drug development. METHODS: Unique peptides for Nav 1.1, Nav 1.2, and Nav 1.6 were selected as quantotropic peptides for each protein and used for their quantification in membranes from stably transfected HEK293 cells and rodent and human brain samples using ultra-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. RESULTS: Nav 1.1, 1.2, and 1.6 protein expressions in three stably individually transfected HEK293 cell lines were found to be 2.1 ± 0.2, 6.4 ± 1.2, and 4.0 ± 0.6 fmol/µg membrane protein, respectively. In brains, Nav 1.2 showed the highest expression, with approximately three times higher (P < 0.003) in rodents than in humans at 3.05 ± 0.57, with 3.35 ± 0.56 in mouse and rat brains and 1.09 ± 0.27 fmol/µg in human brain. Both Nav 1.1 and 1.6 expressions were much lower in the brains, with approximately 40% less expression in human Nav 1.1 than rodent Nav 1.1 at 0.49 ± 0.1 (mouse), 0.43 ± 0.3 (rat), and 0.28 ± 0.04 (humans); whereas Nav 1.6 had approximately 60% less expression in humans than rodents at 0.27 ± 0.09 (mouse), 0.26 ± 0.06 (rat), and 0.11 ± 0.02 (humans) fmol/µg membrane proteins. CONCLUSIONS: Multiple reaction monitoring was used to quantify sodium channels Nav 1.1, 1.2, and 1.6 expressed in stably transfected HEK293 cells and brain tissues from mice, rats, and humans. We found significant differences in the expression of these channels in mouse, rat, and human brains. Nav expression ranking among the three species was Nav 1.2 ≫ Nav 1.1 > Nav 1.6, with the human brain expressing much lower concentrations overall compared to rodent brain.

Identification of HPV oncogene and host cell differentiation associated cellular heterogeneity in cervical cancer via single-cell transcriptomic analysis.

Human Papillomaviruses (HPVs) are associated with around 5%-10% of human cancer, notably nearly 99% of cervical cancer. The mechanisms HPV interacts with stratified epithelium (differentiated layers) during the viral life cycle, and oncogenesis remain unclear. In this study, we used single-cell transcriptome analysis to study viral gene and host cell differentiation-associated heterogeneity of HPV-positive cervical cancer tissue. We examined the HPV16 genes-E1, E6, and E7, and found they expressed differently across nine epithelial clusters. We found that three epithelial clusters had the highest proportion of HPV-positive cells (33.6%, 37.5%, and 32.4%, respectively), while two exhibited the lowest proportions (7.21% and 5.63%, respectively). Notably, the cluster with the most HPV-positive cells deviated significantly from normal epithelial layer markers, exhibiting functional heterogeneity and altered epithelial structuring, indicating that significant molecular heterogeneity existed in cancer tissues and that these cells exhibited unique/different gene signatures compared with normal epithelial cells. These HPV-positive cells, compared to HPV-negative, showed different gene expressions related to the extracellular matrix, cell adhesion, proliferation, and apoptosis. Further, the viral oncogenes E6 and E7 appeared to modify epithelial function via distinct pathways, thus contributing to cervical cancer progression. We investigated the HPV and host transcripts from a novel viewpoint focusing on layer heterogeneity. Our results indicated varied HPV expression across epithelial clusters and epithelial heterogeneity associated with viral oncogenes, contributing biological insights to this critical field of study.

BRD4 as a potential target for human papillomaviruses associated cancer.

Around 99% of cervical cancer and 5%-10% of human cancer are associated with human papillomaviruses (HPV). Notably, the life-cycle of HPV begins by low-level infection of the basal cells of the stratified epithelium, where the viral genomes are replicated and passed on to the daughter proliferating basal cells. The production of new viral particles remains restricted to eventually differentiated cells. HPVs support their persistent infectious cycle by hijacking pivotal pathways and cellular processes. Bromodomain-containing protein 4 (BRD4) is one of the essential cellular factors involved in multiple stages of viral transcription and replication. In this review, we demonstrate the role of BRD4 in the multiple stages of HPV infectious cycle. Also, we provide an overview of the intense research about the cellular functions of BRD4, the mechanism of action of bromodomain and extra terminal inhibitors, and how it could lead to the development of antiviral/anticancer therapies.

First-Generation Palestinian Refugees in Jordan: Experiences of Occupational Disruption From an Occupational Justice Perspective.

IMPORTANCE: Only a limited amount of research has investigated the impact of prolonged refugee status of Palestinian refugees who have been displaced for more than 70 yr. OBJECTIVE: To explore lived experiences of Palestinian refugees in Jordan and understand their occupational disruption. DESIGN: Thematic analysis guided by descriptive phenomenology with one-on-one and group interviews. SETTING: An AlBaqa'a community-based rehabilitation center or participants' homes. PARTICIPANTS: First-generation Palestinian refugees who fled Palestine and live in Jordan. RESULTS: Fifteen Palestinians, mainly widowed women in their 70s, participated in this study. Ten completed interviews, and five participated in two group interviews. Four themes emerged: (1) Palestinian pride, (2) trauma leaving one's home country, (3) challenges of living in a host country, and (4) internalized prejudice. CONCLUSIONS AND RELEVANCE: After 70 yr, prolonged refugeeism has led to occupational disruption and negative implications for occupational justice, especially in the absence of social justice. The area most negatively affected was social participation; however, participants still had a great sense of pride about their homeland and their heritage. What This Article Adds: This foundational research explores the occupational injustices of the protracted refugee status of first-generation Palestinians in Jordan and identifies meaningful interventions to promote the alleviation of occupational disruption.

The role of microbiome in pancreatic cancer.

Recent studies of the human microbiome have offered new insights into how the microbiome can impact cancer development and treatment. Specifically, in pancreatic ductal adenocarcinoma (PDAC), the microbiota has been shown to modulate PDAC risk, contribute to tumorigenesis, impact the tumor microenvironment, and alter treatment response. These findings provide rationale for further investigations into leveraging the microbiome to develop new strategies to diagnose and treat PDAC patients. There is growing evidence that microbiome analyses have the potential to become easily performed, non-invasive diagnostic, prognostic, and predictive biomarkers in pancreatic cancer. More excitingly, there is now emerging interest in developing interventions based on the modulation of microbiota. Fecal microbiota transplantation, probiotics, dietary changes, and antibiotics are all potential strategies to augment the efficacy of current therapeutics and reduce toxicities. While there are still challenges to overcome, this is a rapidly growing field that holds promise for translation into clinical practice and provides a new approach to improving patient outcomes.

Novel Roles of the N1 Loop and N4 Alpha-Helical Region of the Nipah Virus Fusion Glycoprotein in Modulating Early and Late Steps of the Membrane Fusion Cascade.

Nipah virus (NiV) is a zoonotic bat henipavirus in the family Paramyxoviridae NiV is deadly to humans, infecting host cells by direct fusion of the viral and host cell plasma membranes. This membrane fusion process is coordinated by the receptor-binding attachment (G) and fusion (F) glycoproteins. Upon G-receptor binding, F fuses membranes via a cascade that sequentially involves F-triggering, fusion pore formation, and viral or genome entry into cells. Using NiV as an important paramyxoviral model, we identified two novel regions in F that modulate the membrane fusion cascade. For paramyxoviruses and other viral families with class I fusion proteins, the heptad repeat 1 (HR1) and HR2 regions in the fusion protein prefusion conformation bind to form a six-helix bundle in the postfusion conformation. Here, structural comparisons between the F prefusion and postfusion conformations revealed that a short loop region (N1) undergoes dramatic spatial reorganization and a short alpha helix (N4) undergoes secondary structural changes. The roles of the N1 and N4 regions during the membrane fusion cascade, however, remain unknown for henipaviruses and paramyxoviruses. By performing alanine scanning mutagenesis and various functional analyses, we report that specific residues within these regions alter various steps in the membrane fusion cascade. While the N1 region affects early F-triggering, the N4 region affects F-triggering, F thermostability, and extensive fusion pore expansion during syncytium formation, also uncovering a link between F-G interactions and F-triggering. These novel mechanistic roles expand our understanding of henipaviral and paramyxoviral F-triggering, viral entry, and cell-cell fusion (syncytia), a pathognomonic feature of paramyxoviral infections.IMPORTANCE Henipaviruses infect bats, agriculturally important animals, and humans, with high mortality rates approaching ∼75% in humans. Known human outbreaks have been concentrated in Southeast Asia and Australia. Furthermore, about 20 new henipaviral species have been recently discovered in bats, with geographical spans in Asia, Africa, and South America. The development of antiviral therapeutics requires a thorough understanding of the mechanism of viral entry into host cells. In this study, we discovered novel roles of two regions within the fusion protein of the deadly henipavirus NiV. Such roles were in allowing viral entry into host cells and cell-cell fusion, a pathological hallmark of this and other paramyxoviruses. These novel roles were in the previously undescribed N1 and N4 regions within the fusion protein, modulating early and late steps of these important processes of viral infection and henipaviral disease. Notably, this knowledge may apply to other henipaviruses and more broadly to other paramyxoviruses.

Altered microRNA expression links IL6 and TNF-induced inflammaging with myeloid malignancy in humans and mice.

Aging is associated with significant changes in the hematopoietic system, including increased inflammation, impaired hematopoietic stem cell (HSC) function, and increased incidence of myeloid malignancy. Inflammation of aging ("inflammaging") has been proposed as a driver of age-related changes in HSC function and myeloid malignancy, but mechanisms linking these phenomena remain poorly defined. We identified loss of miR-146a as driving aging-associated inflammation in AML patients. miR-146a expression declined in old wild-type mice, and loss of miR-146a promoted premature HSC aging and inflammation in young miR-146a-null mice, preceding development of aging-associated myeloid malignancy. Using single-cell assays of HSC quiescence, stemness, differentiation potential, and epigenetic state to probe HSC function and population structure, we found that loss of miR-146a depleted a subpopulation of primitive, quiescent HSCs. DNA methylation and transcriptome profiling implicated NF-κB, IL6, and TNF as potential drivers of HSC dysfunction, activating an inflammatory signaling relay promoting IL6 and TNF secretion from mature miR-146a-/- myeloid and lymphoid cells. Reducing inflammation by targeting Il6 or Tnf was sufficient to restore single-cell measures of miR-146a-/- HSC function and subpopulation structure and reduced the incidence of hematological malignancy in miR-146a-/- mice. miR-146a-/- HSCs exhibited enhanced sensitivity to IL6 stimulation, indicating that loss of miR-146a affects HSC function via both cell-extrinsic inflammatory signals and increased cell-intrinsic sensitivity to inflammation. Thus, loss of miR-146a regulates cell-extrinsic and -intrinsic mechanisms linking HSC inflammaging to the development of myeloid malignancy.

Third Helical Domain of the Nipah Virus Fusion Glycoprotein Modulates both Early and Late Steps in the Membrane Fusion Cascade.

Medically important paramyxoviruses, such as measles, mumps, parainfluenza, Nipah, and Hendra viruses, infect host cells by directing fusion of the viral and cellular plasma membranes. Upon infection, paramyxoviruses cause a second type of membrane fusion, cell-cell fusion (syncytium formation), which is linked to pathogenicity. Host cell receptor binding causes conformational changes in the attachment glycoprotein (HN, H, or G) that trigger a conformational cascade in the fusion (F) glycoprotein that mediates membrane fusion. F, a class I fusion protein, contains the archetypal heptad repeat regions 1 (HR1) and 2 (HR2). It is well established that binding of HR1 and HR2 is key to fusing viral and cellular membranes. In this study, we uncovered a novel fusion-modulatory role of a third structurally conserved helical region (HR3) in F. Based on its location within the F structure, and structural differences between its prefusion and postfusion conformations, we hypothesized that the HR3 modulates triggering of the F conformational cascade (still requiring G). We used the deadly Nipah virus (NiV) as an important paramyxoviral model to perform alanine scan mutagenesis and a series of multidisciplinary structural/functional analyses that dissect the various states of the membrane fusion cascade. Remarkably, we found that specific residues within the HR3 modulate not only early F-triggering but also late extensive fusion pore expansion steps in the membrane fusion cascade. Our results characterize these novel fusion-modulatory roles of the F HR3, improving our understanding of the membrane fusion process for NiV and likely for the related Henipavirus genus and possibly Paramyxoviridae family members.IMPORTANCE The Paramyxoviridae family includes important human and animal pathogens, such as measles, mumps, and parainfluenza viruses and the deadly henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviruses infect the respiratory tract and the central nervous system (CNS) and can be highly infectious. Most paramyxoviruses have a limited host range. However, the biosafety level 4 NiV and HeV are highly pathogenic and have a wide mammalian host range. Nipah viral infections result in acute respiratory syndrome and severe encephalitis in humans, leading to 40 to 100% mortality rates. The lack of licensed vaccines or therapeutic approaches against NiV and other important paramyxoviruses underscores the need to understand viral entry mechanisms. In this study, we uncovered a novel role of a third helical region (HR3) of the NiV fusion glycoprotein in the membrane fusion process that leads to viral entry. This discovery sets HR3 as a new candidate target for antiviral strategies for NiV and likely for related viruses.

Plasma microRNA Interindividual Variability in Healthy Individuals, Pregnant Women, and an Individual with a Stably Altered Neuroendocrine Phenotype.

BACKGROUND: Extracellular RNAs (exRNAs) in biofluids are amenable to quantitative analysis and proposed as noninvasive biomarkers for monitoring organ function. Cell-lineage-specific microRNAs (miRNAs) are present in plasma as soluble ribonucleoproteins or enclosed in exRNA carriers and transported through the vasculature. However, more extensive studies of healthy individuals are needed to gain insights into the variability of plasma miRNA abundance and composition. METHODS: The exRNA composition of platelet-depleted plasma collected twice from 236 healthy individuals was characterized by small RNA sequencing. Plasma of pregnant women featuring dramatically increased placental miRNAs and samples from subject P12 with noticeably increased epithelial- and neuroendocrine-origin miRNAs were included for comparison. The miRNA content of 10 000g and 100 000g pellet fractions of plasma generated by ultracentrifugation was also determined. Data analysis methods included Pearson correlation, differential gene expression, and unsupervised clustering. RESULTS: The abundance changes for more variable miRNAs in plasma of normal individuals correlated between coexpressed cell-lineage-specific miRNAs of the liver, neuroendocrine organs, epithelial cells, and muscle. ExRNA of pellet fractions contained <2% of total plasma miRNA with modest enrichment of lineage-specific and variable miRNAs compared to supernatant. The abundance fold changes of miRNAs observed in pregnancy and P12 compared to normal exceeded interquartile variability of healthy individuals. The neuroendocrine miRNA signature of P12 persisted for more than 4 years and was absent in other individuals. CONCLUSIONS: This study defines the framework and effect size for screening of extensive plasma collections for miRNA phenotypes and biomarker discovery.

Human plasma and serum extracellular small RNA reference profiles and their clinical utility.

Circulating extracellular RNAs (exRNAs) have the potential to serve as biomarkers for a wide range of medical conditions. However, limitations in existing exRNA isolation methods and a lack of knowledge on parameters affecting exRNA variability in human samples may hinder their successful discovery and clinical implementation. Using combinations of denaturants, reducing agents, proteolysis, and revised organic extraction, we developed an automated, high-throughput approach for recovery of exRNAs and exDNA from the same biofluid sample. We applied this method to characterize exRNAs from 312 plasma and serum samples collected from 13 healthy volunteers at 12 time points over a 2-month period. Small RNA cDNA library sequencing identified nearly twofold increased epithelial-, muscle-, and neuroendocrine-cell-specific miRNAs in females, while fasting and hormonal cycle showed little effect. External standardization helped to detect quantitative differences in erythrocyte and platelet-specific miRNA contributions and in miRNA concentrations between biofluids. It also helped to identify a study participant with a unique exRNA phenotype featuring a miRNA signature of up to 20-fold elevated endocrine-cell-specific miRNAs and twofold elevated total miRNA concentrations stable for over 1 year. Collectively, these results demonstrate an efficient and quantitative method to discern exRNA phenotypes and suggest that plasma and serum RNA profiles are stable over months and can be routinely monitored in long-term clinical studies.

Nipah and Hendra Virus Glycoproteins Induce Comparable Homologous but Distinct Heterologous Fusion Phenotypes.

Nipah and Hendra viruses (NiV and HeV) exhibit high lethality in humans and are biosafety level 4 (BSL-4) paramyxoviruses in the growing genus Henipavirus The attachment (G) and fusion (F) envelope glycoproteins are both required for viral entry into cells and for cell-cell fusion, which is pathognomonic of henipaviral infections. Here, we compared the fusogenic capacities between homologous and heterologous pairs of NiV and HeV glycoproteins. Importantly, to accurately measure their fusogenic capacities, as these depend on glycoprotein cell surface expression (CSE) levels, we inserted identical extracellular tags to both fusion (FLAG tags) or both attachment (hemagglutinin [HA] tags) glycoproteins. Importantly, these tags were placed in extracellular sites where they did not affect glycoprotein expression or function. NiV and HeV glycoproteins induced comparable levels of homologous HEK293T cell-cell fusion. Surprisingly, however, while the heterologous NiV F/HeV G (NF/HG) combination yielded a hypofusogenic phenotype, the heterologous HeV F/NiV G (HF/NG) combination yielded a hyperfusogenic phenotype. Pseudotyped viral entry levels primarily corroborated the fusogenic phenotypes of the glycoprotein pairs analyzed. Furthermore, we constructed G and F chimeras that allowed us to map the overall regions in G and F that contributed to these hyperfusogenic or hypofusogenic phenotypes. Importantly, the fusogenic phenotypes of the glycoprotein combinations negatively correlated with the avidities of F-G interactions, supporting the F/G dissociation model of henipavirus-induced membrane fusion, even in the context of heterologous glycoprotein pairs.IMPORTANCE The NiV and HeV henipaviruses are BSL-4 pathogens transmitted from bats. NiV and HeV often lead to human death and animal diseases. The formation of multinucleated cells (syncytia) is a hallmark of henipaviral infections and is caused by fusion of cells coordinated by interactions of the viral attachment (G) and fusion (F) glycoproteins. We found via various assays that viral entry and syncytium formation depend on the viral origin of the glycoproteins, with HeV F and NiV G promoting higher membrane fusion levels than their counterparts. This is important knowledge, since both viruses use the same bat vector species and potential coinfections of these or subsequent hosts may alter the outcome of disease.

Assessing the impact of food insecurity on HIV medication adherence in the context of an integrated care programme for people living with HIV in Vancouver, Canada.

OBJECTIVE: Food insecurity, or self-reports of inadequate food access due to limited financial resources, remains prevalent among people living with HIV (PLHIV). We examined the impact of food insecurity on combination antiretroviral therapy (cART) adherence within an integrated care programme that provides services to PLHIV, including two meals per day. DESIGN: Adjusted OR (aOR) were estimated by generalized estimating equations, quantifying the relationship between food insecurity (exposure) and cART adherence (outcome) with multivariable logistic regression. SETTING: We drew on survey data collected between February 2014 and March 2016 from the Dr. Peter Centre Study based in Vancouver, Canada. PARTICIPANTS: The study included 116 PLHIV at baseline, with ninety-nine participants completing a 12-month follow-up interview. The median (quartile 1-quartile 3) age was 46 (39-52) years at baseline and 87 % (n 101) were biologically male at birth. RESULTS: At baseline, 74 % (n 86) of participants were food insecure (≥2 affirmative responses on Health Canada's Household Food Security Survey Module) and 67 % (n 78) were adherent to cART ≥95 % of the time. In the adjusted regression analysis, food insecurity was associated with suboptimal cART adherence (aOR = 0·47, 95 % CI 0·24, 0·93). CONCLUSIONS: While food provision may reduce some health-related harms, there remains a relationship between this prevalent experience and suboptimal cART adherence in this integrated care programme. Future studies that elucidate strategies to mitigate food insecurity and its effects on cART adherence among PLHIV in this setting and in other similar environments are necessary.

Formation of a statewide community pharmacy practice-based research network: Pharmacist opinions on research participation and engagement.

OBJECTIVE: To gather the insights and opinions of pharmacist stakeholders to inform the creation of a community pharmacy practice-based research network (PBRN) in Pennsylvania. DESIGN: A stakeholder advisory board of pharmacists, patients, and researchers was established to guide this research. This was a qualitative study using a semistructured interview guide. SETTING AND PARTICIPANTS: Community pharmacists from the Pennsylvania Pharmacist Care Network. OUTCOME MEASURES: Themes were identified that describe pharmacist insights and opinions on research participation and preferences for engagement in the PBRN. RESULTS: A total of 16 pharmacists participated in the study. The pharmacists believed that participating in research would help demonstrate their value and commitment to improving patients' health. Enhancing patient-pharmacist relationships and driving innovation were additional benefits that were reported. The pharmacists believed that they could effectively leverage their relationships with patients to engage them in research opportunities. The pharmacists reported that they would like to share research ideas and successful research practices with other members of the PBRN. CONCLUSION: Gathering pharmacists' opinions on participating in research was an important step in developing a community pharmacy PBRN that meets stakeholder needs. The results of this study can help others who seek to form community pharmacy PBRNs that facilitate stakeholder-driven research.

On the feasibility of quantifying sodium channel Nav 1.6 protein in mouse brain using targeted ultra-high-performance/electrospray ionization multiple reaction monitoring mass spectrometry.

RATIONALE: Nav 1.6 is a transmembrane voltage gated sodium channel implicated in various forms of epilepsy. Modulation of its activity in epilepsy animal models can be accomplished using inhibitors which may result in changes in its expression. There is a need to generate reliable quantitative measurements of Nav 1.6 expression in animal models. This research explores the feasibility of quantifying Nav 1.6 expression in mouse brains using targeted multiple reaction monitoring (MRM) mass spectrometry. METHODS: A combination of in silico tryptic Nav 1.6 peptides and MRM transitions were used to select target peptides. This was followed by a simple proteomic work-up including plasma membrane isolation, trypsin-based proteolysis and ultra-high-performance/electrospray ionization tandem mass spectrometry (UHPLC/ESI-MS/MS) to detect the presence of Nav 1.6 in induced HEK293 cells. The unique Nav 1.6 peptide, DSLFIPR, was selected as probe for quantifying Nav 1.6 levels in brains from C57BL/6J wild-type mice as well as two kinds of mutants including Scn8aN1768D/+ and heterozygous null Scn8a+/- mice using isotope dilution targeted mass spectrometry. RESULTS: The feasibility of using targeted MRM for quantifying Nav 1.6 expression in mice brains was demonstrated. Expression of Nav 1.6 in brains (hippocampi) from wild-type and mutant Scn8aN1768D/+ mice were found to be around 0.40 fmol/µg. Mutant null Scn8a+/- heterozygous mice, on the other hand, showed levels of 0.22 fmol/µg as expected based on this particular mutation which only generates 50% of the expression in wild-type mice. Nav 1.6-overexpressed HEK293 cells showed 3.7 fmol/µg of Nav 1.6 expression, suitable for screening new compounds for Nav 1.6 blocking activity. CONCLUSIONS: The results of the present feasibility study support the use of DSLFIPIR for quantification of Nav1.6 in brain tissues using UHPL/ESI-MS/MS.

A narrative review and update on management following negative prostate biopsy.

PURPOSE OF REVIEW: Prostate cancer has traditionally been diagnosed using systematic transrectal ultrasound-guided biopsy. However, given the inherent nature of sampling, a negative biopsy does not exclude clinically significant prostate cancer (csPCa), and continued controversy exists in the optimal management following initial biopsy. Numerous avenues for evaluation include multiparametric MRI (mpMRI), use of molecular biomarkers, repeat biopsy, and observation. RECENT FINDINGS: mpMRI has shown promise in guiding further biopsy management: for individuals with identified target lesions, increased accuracy and detection using combination targeted and systematic sampling has been repeatedly demonstrated in the literature as an effective strategy. For those with negative MRIs and/or negative biomarker (blood, urinary, tissue) studies, increasing evidence has suggested that these individuals may be able to avoid biopsy altogether, albeit at a small risk of missing csPCa. Observation should be based on an individual's risk of csPCa versus their competing health risks, and saturation biopsy reserved for rare cases with high clinical suspicion. SUMMARY: Management following an initial negative prostate biopsy requires careful discussion with the patient, their risk tolerance, and threshold for intervention. Although subject to availability, mpMRI and molecular biomarkers may better risk stratify patients, identify target lesions, and in certain cases, spare biopsy altogether.