Financial barriers and facilitators to secure firearm and medication storage among veterans with elevated suicide risk: a qualitative study.

OBJECTIVE: To explore financial barriers and facilitators to promoting secure firearm and medication storage among patients at risk for suicide. METHODS: Veterans seeking care in Veterans Affairs emergency care settings (N=28) participated in qualitative interviews on barriers and facilitators to adopting secure firearm and medication storage behaviours. Thematic analysis with inductive and iterative coding was used to identify themes pertaining to financial barriers and facilitators. Interviews were double-coded for reliability. RESULTS: We identified four themes-two related to financial barriers and two to financial facilitators. Barrier-related themes included: (1) the high cost of firearms and medications made owners less likely to dispose of medications, relinquish ownership of firearms or pursue out-of-home storage for firearms; (2) the high cost of out-of-home storage and preferred locking devices were barriers to secure storage. Facilitator-related themes included: (1) no-cost services or locking devices may help motivate secure firearm and medication storage and (2) preferences varied for no-cost locking devices versus coupons for devices. CONCLUSIONS: Addressing financial barriers and leveraging financial facilitators may motivate secure storage of lethal means, which could enhance suicide prevention efforts.

A community-based mentoring scheme for pregnant and parenting adolescents in Sierra Leone: Protocol for a hybrid pilot cluster randomised controlled trial.

BACKGROUND: Sierra Leone has a very high maternal mortality rate, and this burden falls heavily on adolescents, a particularly vulnerable group; this is usually driven by poverty, lack of education and employment opportunities. In 2017, a local grassroots organisation, Lifeline Nehemiah Projects, developed a community-based mentoring intervention '2YoungLives' (2YLs) for adolescent girls in Eastern Freetown. We aim to formally assess the feasibility and implementation of the 2YL mentorship scheme in new communities in Sierra Leone. METHODS: A hybrid type 2 pilot cluster randomised controlled trial of the 2YL mentoring scheme in urban and rural communities living around twelve peripheral health units (PHU) across five districts in Sierra Leone. Clusters will be matched into pairs and randomisation will be determined by computer-generated random numbers via a secure web-based system hosted by MedSciNet. All under-eighteen adolescents identified as pregnant in the community and/or the PHU are included. Feasibility (recruitment, retention, and attrition rates; data collection and completeness; sample calculation) and primary clinical outcome data (composite of maternal deaths, stillbirths, neonatal deaths) will be collected. A mixed-methods process evaluation will explore implementation outcomes, mechanisms of change, contextual factors, experiences of care, and health and wellbeing. A concurrent cost-consequence analysis will be undertaken. Main trial analysis will be pragmatic, by intention to treat, and a complementary per protocol analysis will also be included. DISCUSSION: Improving health and wellbeing for adolescent girls (including sexual and reproductive health) remains a top priority in Sierra Leone indicated by several government policies targeted to this group, in which maternal and infant mortality are still persistently high. Supporting these girls and facilitating their wellbeing is imperative, along with sensitisation of communities, strengthening of youth friendly services and collaboration with stakeholders at all levels (government, regional, community, family). We believe 2YL supports the global holistic agenda to integrate and implement interventions across health, education, and social systems in order to protect, nurture, and support the health and development potential of every adolescent girl, and thus become a model of good practice for adolescent pregnancy, to be adopted more widely in Sierra Leone and elsewhere. TRIAL REGISTRATION: ISRCTN registry ISRCTN32414369. Prospectively registered on 14/03/2022.

Patients' Perspectives on Medication-Related Suicide Prevention Interventions Delivered in Emergency Settings.

OBJECTIVE: Encouraging patients at risk for suicide to reduce access to potentially lethal medications and drugs is a key component of evidence-based suicide prevention. However, little research has been done to inform interventions for reducing intentional self-harm. METHODS: Semistructured interviews were conducted with 28 U.S. veterans who sought emergency care from the Veterans Health Administration between 2021 and 2023 to explore veterans' perspectives on medication-related interventions, including opinions on intervention components (e.g., medication return envelopes). Matrix analysis was used to aggregate data into categories, which were predefined by using constructs from the health belief model (e.g., perceived benefits). RESULTS: The participating veterans generally endorsed interventions as acceptable and were particularly supportive of distributing medication return envelopes. However, they often conceptualized these efforts as steps to prevent unintentional overdose or theft-not necessarily to prevent suicide-and rarely indicated that such interventions were appropriate for themselves. Across the interviews, participants identified important facilitators to care, such as ensuring that interventions were convenient and accounted for the perceived cost of disposing medications. Perspectives on engaging family or friends in interventions were mixed. The importance of the interventions was more readily acknowledged among participants with previous opioid use exposure-perspectives that appeared to stem from lived experiences. CONCLUSIONS: This study contributes important foundational knowledge that can be used to inform research and clinical initiatives aimed at preventing medication- and drug-related suicides.

Women Veterans' perspectives, experiences, and preferences for firearm lethal means counseling discussions.

AIMS: Firearms have become an increasingly common method of suicide among women Veterans, yet this population has rarely been a focus in firearm suicide prevention research. Limited knowledge is available regarding the preferences, experiences, or needs of women Veterans with respect to firearm lethal means counseling (LMC), an evidence-based suicide prevention strategy. Understanding is necessary to optimize delivery for this population. METHOD: Our sample included forty women Veterans with lifetime suicidal ideation or suicide attempt(s) and firearm access following military separation, all enrolled in the Veterans Health Administration. Participants were interviewed regarding their perspectives, experiences, and preferences for firearm LMC. Data were analyzed using a mixed inductive-deductive thematic analysis. RESULTS: Women Veterans' firearm and firearm LMC perspectives were shaped by their military service histories and identity, military sexual trauma, spouses/partners, children, rurality, and experiences with suicidal ideation and attempts. Half reported they had not engaged in firearm LMC previously. For those who had, positive aspects included a trusting, caring relationship, direct communication of rationale for questions, and discussion of exceptions to confidentiality. Negative aspects included conversations that felt impersonal, not sufficiently comprehensive, and Veterans' fears regarding implications of disclosure, which impeded conversations. Women Veterans' preferences for future firearm LMC encompassed providers communicating why such conversations are important, how they should be framed (e.g., around safety and genuine concern), what they should entail (e.g., discussing concerns regarding disclosure), whom should initiate (e.g., trusted caring provider) and where they should occur (e.g., safe spaces, women-specific groups comprised of peers). DISCUSSION: This study is the first to examine women Veterans' experiences with, and preferences for, firearm LMC. Detailed inquiry of the nuances of how, where, why, and by whom firearms are stored and used may help to facilitate firearm LMC with women Veterans.

Women Veterans' experiences discussing household firearms with their intimate partners: collaborative, devalued, and deferential relational types.

BACKGROUND: Rates of firearm suicide have increased among women Veterans. Discussing firearm access and reducing access to lethal means of suicide when suicide risk is heightened are central tenets of suicide prevention, as is tailoring suicide prevention strategies to specific populations. While research has begun to explore how to optimize firearm lethal means safety counseling with women Veterans, there is limited knowledge of women Veterans' perspectives on including their intimate partners in such efforts. This gap is notable since many women Veterans have access to firearms owned by other household members. Understanding women Veterans' experiences and perspectives regarding including their partners in firearm lethal means safety conversations can provide important information for tailoring firearm lethal means safety counseling for women Veterans. METHODS: Qualitative interviews were conducted with 40 women Veterans with current or prior household firearm access. Interview questions focused on the roles of women Veterans' partners in household firearm access and storage, as well as women Veterans' perspectives regarding including intimate partners in firearm lethal means safety counseling. Inductive thematic analysis was performed. RESULTS: Three relational types characterized how household firearms were discussed between women Veterans and their partners: collaborative, devalued, and deferential. These types were distinguished via women Veterans' agency in decision-making related to household firearms, partners' receptivity to women Veterans' mental health or trauma histories, and willingness (or lack thereof) of partners to change household firearm access and storage considering such histories. Intimate partner violence was common in the devalued relational subtype. CONCLUSIONS: Findings extend knowledge regarding the context of women Veterans' household firearm access, including relational dynamics between women Veterans and their partners. The acceptability, feasibility, challenges, and facilitators of including women Veterans' partners in firearm lethal means safety efforts likely vary for each relational type. For example, in dyads with a collaborative dynamic, incorporating partners may create opportunities for increased firearm safety, whereas including partners in devalued dynamics may present unique challenges. Research is warranted to determine optimal methods of navigating firearm lethal means safety counseling in the presence of each relational dynamic.

Establishing an Antimicrobial Stewardship Program in Sierra Leone: A Report of the Experience of a Low-Income Country in West Africa.

Antimicrobial Resistance (AMR) is a growing global health challenge that threatens to undo gains in human and animal health. Prevention and control of AMR requires functional antimicrobial stewardship (AMS) program, which is complex and often difficult to implement in low- and middle-income countries. We aimed to describe the processes of establishing and implementing an AMS program at Connaught Hospital in Sierra Leone. The project involved the setting up of an AMS program, capacity building and performing a global point prevalence survey (GPPS) at Sierra Leone's national referral hospital. Connaught Hospital established a multidisciplinary AMS subcommittee in 2021 to provide AMS services such as awareness campaigns, education and training and review of guidelines. We performed a GPPS on 175 patients, of whom more than half (98, 56.0%) were prescribed an antibiotic: 63 (69.2%) in the surgical wards and 53 (51.2%) in the medical wards. Ceftriaxone (60, 34.3%) and metronidazole (53, 30.3%) were the most common antibiotics prescribed to patients. In conclusion, it is feasible to establish and implement an AMS program in low-income countries, where most hospitalized patients were prescribed an antibiotic.

Women's experiences of a pregnancy whilst attending a specialist antenatal service for pregnancies after stillbirth or neonatal death: a qualitative interview study.

Aim: Pregnancy after the death of a baby is associated with numerous, varied psychological challenges for pregnant women. This study aimed to explore women's experiences of pregnancy whilst attending a specialist antenatal service for pregnancies after a perinatal death.Methods: Semi-structured interviews with twenty women in a subsequent pregnancy after a perinatal death were conducted and analyzed taking an inductive thematic analysis approach.Results: All women expressed a heightened "awareness of risk". Two subthemes demonstrated how increased awareness of risk affected their experience and their desire regarding antenatal and postnatal support. Women talked about stillbirth being a "quiet, unspoken subject" causing them internal conflict as they had an awareness of pregnancy complications that other people did not. Navigating subsequent pregnancies relied on them "expecting the worst and hoping for the best" in terms of pregnancy outcomes. Women viewed specialist antenatal care in pregnancy after perinatal loss favorably, as it enabled them to receive tailored care that met their needs stemming from their increased awareness of and personal expectations of risk.Conclusion: Women's experiences can be used to develop models of care but further studies are required to determine to identify which components are most valued.

Measures of anxiety, depression and stress in the antenatal and perinatal period following a stillbirth or neonatal death: a multicentre cohort study.

BACKGROUND: The grief associated with the death of a baby is enduring, however most women embark on another pregnancy, many in less than a year following their loss. Symptoms of anxiety and depression are reported to be increased in pregnancies after perinatal death, although effect on maternal stress is less clear. Variation between individual studies may result from differences in gestation at sampling, the questionnaire used and the type of antecedent perinatal death. We aimed to describe quantitative measures of anxiety, depression, stress and quality of life at different timepoints in pregnancies after perinatal death and in the early postnatal period. METHODS: Women recruited from three sites in the North-West of England. Women were asked to participate if a previous pregnancy had ended in a perinatal death. Participants completed validated measures of psychological state (Cambridge Worry Score, Edinburgh Postnatal Depression Score (EPDS), Generalized Anxiety Disorder 7-item score) and health status (EQ-5D-5L™ and EQ5D-Visual Analogue Scale) at three time points, approximately 15 weeks' and 32 weeks' gestation and 6 weeks postnatally. A sample of hair was taken at approximately 36 weeks' gestation for measurement of hair cortisol in a subgroup of women. The hair sample was divided into samples from each trimester and cortisol measured by ELISA. RESULTS: In total 112 women participated in the study. Measures of anxiety and depressive symptoms decreased from the highest levels at 15 weeks' gestation to 6-weeks postnatal (for example mean GAD-7: 15 weeks 8.2 ± 5.5, 6 weeks postnatal 4.4 ± 5.0, p<0.001). Hair cortisol levels fell in a similar profile to anxiety and depression symptoms (p<0.05). In contrast, the median EQ-5D index, measuring health status was 0.768 at 15 weeks' gestation (Interquartile range (IQR) 0.684-0.879), 0.696 at 32 weeks' (IQR 0.637-0.768) and 0.89 (0.760-1.00) at 6 weeks postnatal. There was a negative relationship between EPDS and perceived health status. CONCLUSIONS: This study demonstrated heightened anxiety and depressive symptoms and elevated cortisol levels in women in pregnancies after a stillbirth or neonatal death which decrease as pregnancy progresses. Further studies are needed to determine optimal care for women to address these negative psychological consequences.

Drinking Patterns of Post-Deployment Veterans: The Role of Personality, Negative Urgency, and Posttraumatic Stress.

Rates of posttraumatic stress disorder (PTSD) and alcohol misuse are known to be high among post-deployment Veterans. Previous research has found that personality factors may be relevant predictors of post-deployment drinking, yet results have been inconsistent and may be influenced by the selection of drinking outcome. This study aimed to examine relations between PTSD, negative urgency, and the five factor models of personality with multiple alcohol consumption patterns, including maximum drinks in a day, number of binge drinking episodes, at-risk drinking, and average weekly drinks in a sample of 397 Operation Enduring Freedom/Operation Iraqi Freedom/Operation New Dawn (OEF/OIF/OND) Veterans. The pattern of results suggested that the association between personality, PTSD, and drinking may depend on which drinking outcome is selected. For example, maximum drinks in a day was significantly associated with younger age, male gender, low agreeableness, and an interaction between negative urgency and PTSD, whereas number of binge drinking days was significantly associated with younger age, extraversion, low agreeableness, and negative urgency. This study highlights the heterogeneity of drinking patterns among Veterans and the need for careful consideration and transparency of outcomes selection in alcohol research.

A Methodological Approach for Evaluating the Enterprise Community Healthy Start Program in Rural Georgia: An Analysis Using Linked PRAMS, Birth Records and Program Data.

INTRODUCTION: Community Healthy Start program evaluations are often limited by a lack of robust data and rigorous study designs. This study describes an enhanced methodological approach using local program data linked with existing population-level datasets for external comparison to evaluate the Enterprise Community Healthy Start (ECHS) program in two rural Georgia counties and presents results from the evaluation. METHODS: ECHS program data were linked to birth records and the Pregnancy Risk Assessment Monitoring System (PRAMS) for 869 women who delivered a live birth in Burke and McDuffie counties from 2010 to 2011. Multivariate logistic regressions with and without propensity score methods modeled the association between ECHS participation and maternal health indicators and pregnancy outcomes. RESULTS: 107 ECHS participants and 726 non-participants responded to PRAMS and met eligibility criteria. Compared with non-participants, ECHS participants were younger, completed fewer years of education, and were more likely to be non-Hispanic Black, unmarried, insured with Medicaid, participating in WIC, and having an unintended pregnancy. Models with and without propensity score weighting derived similar results: there was a positive association between ECHS participation and receiving adequate or adequate plus prenatal care (p < 0.05); no statistically significant associations were observed between ECHS participation and any other health behaviors, health care access and utilization measures or pregnancy outcomes. DISCUSSION: Rigorous evaluation of a local Healthy Start program using linked PRAMS and birth records with a population-based external comparison group and propensity score methods is an enhanced and feasible approach that can be applied in other local and state jurisdictions.

Associations of subjective and objective stress responses with interpersonal trauma, PTSD, stress-induced drinking, and drinking to cope in young adults.

OBJECTIVE: To understand how interpersonal trauma (IPT), stress response, and drinking to cope converge to predict stress-induced drinking, a risk factor for alcohol use disorder. METHOD: Young adults with no substance use disorder were classified into three trauma history groups: (a) IPT with PTSD (n = 27), (b) IPT without PTSD (n = 35), and (c) Control (no trauma-history/no PTSD; n = 36). Participants completed a baseline assessment, including a structured clinical interview, to confirm PTSD diagnosis, followed by the Trier Social Stressor Task (TSST) and an alcohol use task. Subjective units of distress and blood serum cortisol were collected at standardized timepoints throughout the tasks. RESULTS: In all three groups (PTSD, IPT, control), males consumed more alcohol in the lab than females. Participants in the PTSD group had significantly higher drinking to cope motives, which were associated with greater subjective reactivity; however, neither drinking to cope motives nor subjective reactivity to the TSST predicted post-stressor alcohol consumption for those with PTSD. CONCLUSIONS: The interplay among trauma history, stress, and drinking among young adults is nuanced; additional lab-based studies are needed to further clarify the nuanced connection between trauma history, acute stress reactions, and alcohol use. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Sleep Quality Moderates the Relationship between Anxiety Sensitivity and PTSD Symptoms in Combat-exposed Veterans.

Objective/Background: Posttraumatic stress disorder (PTSD) and related conditions (e.g., depression) are common in Operation Enduring Freedom, Operation Iraqi Freedom, and Operation New Dawn (OEF/OIF/OND) veterans. High anxiety sensitivity (AS), defined as fear of anxiety and anxiety-related consequences, is related to greater PTSD and depressive symptoms; however, few studies have identified possible modifiers of these associations. The current study examined the moderating role of sleep quality in the associations between AS and PTSD and depressive symptoms. Participants: Participants were 155 OEF/OIF/OND community veterans ages 21-40 (12.3% women). Methods: Participants completed a semi-structured clinical interview for DSM-IV PTSD symptoms (Clinician Administered PTSD Scale; CAPS) and self-report measures of anxiety sensitivity (Anxiety Sensitivity Index), sleep quality (Pittsburgh Sleep Quality Index global score; PSQI), and depressive symptoms (Beck Depression Inventory-II; BDI-II). Results: Results of hierarchical linear regression models indicated that the main effects of AS and global PSQI score were significantly associated with greater PTSD and depressive symptoms (both with sleep items removed), above and beyond the covariates of trauma load and military rank. Sleep quality moderated the relationship between AS and PTSD symptoms (but not depressive symptoms), such that greater AS was associated with greater PTSD symptoms for individuals with good sleep quality, but not poor sleep quality. Conclusions: Sleep quality and AS account for unique variance in PTSD and depressive symptoms in combat-exposed veterans. AS may be less relevant to understanding risk for PTSD among combat-exposed veterans experiencing poor sleep quality.

Plant-based CO2 drawdown and storage as SiC.

Since the 1950's the Earth's natural carbon cycle has not sufficiently sequestrated excess atmospheric CO2 contributed by human activities. CO2 levels rose above 400 ppm in 2013 and are forecasted to exceed 500 ppm by 2070, a level last experienced during the Paleogene period 25-65 MYA. While humanity benefits from the extraction and combustion of carbon from Earth's crust, we have overlooked the impact on global climate change. Here, we present a strategy to mine atmospheric carbon to mitigate CO2 emissions and create economically lucrative green products. We employ an artificial carbon cycle where agricultural plants capture CO2 and the carbon is transformed into silicon carbide (SiC), a valuable commercial material. By carefully quantifying the process we show that 14% of plant-sequestered carbon is stored as SiC and estimate the scale needed for this process to have a global impact.

Piloting of the Just Say Know prevention program: a psychoeducational approach to translating the neuroscience of addiction to youth.

BACKGROUND: Substance use during adolescence can have a number of negative consequences and interfere with normal brain development. Given limited time and resources, brief group- and school-based prevention programs are an efficient strategy for educating youth about the effects of substance use on health outcomes. OBJECTIVES: To determine if a science-based, interactive substance prevention program could improve student knowledge and influence students' attitudes toward future substance use behaviors. METHODS: The Just Say Know program was given to 1,594 middle and high school students. The facilitator engaged students in an interactive, hour-long session covering brain basics and effects of substance use. Students completed an eight-item pre- and post-knowledge-based test to measure learning outcomes along with feedback questions about youths' attitudes toward substance use and the program. RESULTS: After the program, 94% of students reported that it provided helpful information; 92% reported it may influence their approach to substance use, with 76% specifying that they would delay or cut back on substance use. Knowledge-based test performance increased by 78%, with high schoolers displaying significantly higher scores than middle schoolers, but both showing similar improvements in scores. Students who reported higher levels of friends' substance use had smaller improvements from pre- to posttest. CONCLUSION: Results suggest Just Say Know, a scientifically-based prevention program, is effective in increasing adolescents' program based-knowledge, has the potential to affect youths' attitudes toward substance use, and is well-received. These findings provide preliminary evidence that a cost-effective, neuroscience-informed group prevention program might reduce or delay adolescents' future substance use.

Site-Specific Protein Photochemical Covalent Attachment to Carbon Nanotube Side Walls and Its Electronic Impact on Single Molecule Function.

Functional integration of proteins with carbon-based nanomaterials such as nanotubes holds great promise in emerging electronic and optoelectronic applications. Control over protein attachment poses a major challenge for consistent and useful device fabrication, especially when utilizing single/few molecule properties. Here, we exploit genetically encoded phenyl azide photochemistry to define the direct covalent attachment of four different proteins, including the fluorescent protein GFP and a ß-lactamase binding protein (BBP), to carbon nanotube side walls. AFM showed that on attachment BBP could still recognize and bind additional protein components. Single molecule fluorescence revealed that on attachment to SWCNTs function was retained and there was feedback to GFP in terms of fluorescence intensity and improved resistance to photobleaching; GFP is fluorescent for much longer on attachment. The site of attachment proved important in terms of electronic impact on GFP function, with the attachment site furthest from the chromophore having the larger effect on fluorescence. Our approach provides a versatile and general method for generating intimate protein-CNT hybrid bioconjugates. It can be potentially applied to any protein of choice; the attachment position and thus interface characteristics with the CNT can easily be changed by simply placing the phenyl azide chemistry at different residues by gene mutagenesis. Thus, our approach will allow consistent construction and modulate functional coupling through changing the protein attachment position.

Development of a new fusion-enhanced oncolytic immunotherapy platform based on herpes simplex virus type 1.

BACKGROUND: Oncolytic viruses preferentially replicate in tumors as compared to normal tissue and promote immunogenic cell death and induction of host systemic anti-tumor immunity. HSV-1 was chosen for further development as an oncolytic immunotherapy in this study as it is highly lytic, infects human tumor cells broadly, kills mainly by necrosis and is a potent activator of both innate and adaptive immunity. HSV-1 also has a large capacity for the insertion of additional, potentially therapeutic, exogenous genes. Finally, HSV-1 has a proven safety and efficacy profile in patients with cancer, talimogene laherparepvec (T-VEC), an oncolytic HSV-1 which expresses GM-CSF, being the only oncolytic immunotherapy approach that has received FDA approval. As the clinical efficacy of oncolytic immunotherapy has been shown to be further enhanced by combination with immune checkpoint inhibitors, developing improved oncolytic platforms which can synergize with other existing immunotherapies is a high priority. In this study we sought to further optimize HSV-1 based oncolytic immunotherapy through multiple approaches to maximize: (i) the extent of tumor cell killing, augmenting the release of tumor antigens and danger-associated molecular pattern (DAMP) factors; (ii) the immunogenicity of tumor cell death; and (iii) the resulting systemic anti-tumor immune response. METHODS: To sample the wide diversity amongst clinical strains of HSV-1, twenty nine new clinical strains isolated from cold sores from otherwise healthy volunteers were screened across a panel of human tumor cell lines to identify the strain with the most potent tumor cell killing ability, which was then used for further development. Following deletion of the genes encoding ICP34.5 and ICP47 to provide tumor selectivity, the extent of cell killing and the immunogenicity of cell death was enhanced through insertion of a gene encoding a truncated, constitutively highly fusogenic form of the envelope glycoprotein of gibbon ape leukemia virus (GALV-GP-R-). A number of further armed derivatives of this virus were then constructed intended to further enhance the anti-tumor immune response which was generated following fusion-enhanced, oncolytic virus replication-mediated cell death. These viruses expressed GMCSF, an anti-CTLA-4 antibody-like molecule, CD40L, OX40L and/or 4-1BB, each of which is expected to act predominantly at the site and time of immune response initiation. Expression of these proteins was confirmed by ELISA and/or western blotting. Immunogenic cell death was assessed by measuring the levels of HMGB1 and ATP from cell free supernatants from treated cells, and by measuring the surface expression of calreticulin. GALV-GP-R- mediated cell to cell fusion and killing was tested in a range of tumor cell lines in vitro. Finally, the in vivo therapeutic potential of these viruses was tested using human A549 (lung cancer) and MDA-MB-231(breast cancer) tumor nude mouse xenograft models and systemic anti-tumor effects tested using dual flank syngeneic 4434 (melanoma), A20 (lymphoma) mouse tumor models alone and in combination with a murine anti-PD1 antibody, and 9 L (gliosarcoma) tumors in rats. RESULTS: The twenty nine clinical strains of HSV-1 isolated and tested demonstrated a broad range of tumor cell killing abilities allowing the most potent strain to be identified which was then used for further development. Oncolytic ability was demonstrated to be further augmented by the expression of GALV-GP-R- in a range of tumor cell lines in vitro and in mouse xenograft models in nude mice. The expression of GALV-GP-R- was also demonstrated to lead to enhanced immunogenic cell death in vitro as confirmed by the increased release of HMGB1 and ATP and increased levels of calreticulin on the cell surface. Experiments using the rat 9 L syngeneic tumor model demonstrated that GALV-GP-R- expression increased abscopal uninjected (anenestic) tumor responses and data using mouse 4434 tumors demonstrated that virus treatment increased CD8+ T cell levels both in the injected and uninjected tumor, and also led to increased expression of PD-L1. A combination study using varying doses of a virus expressing GALV-GP-R- and mGM-CSF and an anti-murine PD1 antibody showed enhanced anti-tumor effects with the combination which was most evident at low virus doses, and also lead to immunological memory. Finally, treatment of mice with derivatives of this virus which additionally expressed anti-mCTLA-4, mCD40L, m4-1BBL, or mOX40L demonstrated enhanced activity, particularly in uninjected tumors. CONCLUSION: The new HSV-1 based platform described provides a potent and versatile approach to developing new oncolytic immunotherapies for clinical use. Each of the modifications employed was demonstrated to aid in optimizing the potential of the virus to both directly kill tumors and to lead to systemic therapeutic benefit. For clinical use, these viruses are expected to be most effective in combination with other anti-cancer agents, in particular PD1/L1-targeted immune checkpoint blockade. The first virus from this program (expressing GALV-GP-R- and hGM-CSF) has entered clinical development alone and in combination with anti-PD1 therapy in a number of tumor types (NCT03767348).

Substrate Specificity and Engineering of Mevalonate 5-Phosphate Decarboxylase.

A bifurcation of the mevalonate (MVA) pathway was recently discovered in bacteria of the Chloroflexi phylum. In this alternative route for the biosynthesis of isopentenylpyrophosphate (IPP), the penultimate step is the decarboxylation of (R)-mevalonate 5-phosphate ((R)-MVAP) to isopentenyl phosphate (IP), which is followed by the ATP-dependent phosphorylation of IP to IPP catalyzed by isopentenyl phosphate kinase (IPK). Notably, the decarboxylation reaction is catalyzed by mevalonate 5-phosphate decarboxylase (MPD), which shares considerable sequence similarity with mevalonate diphosphate decarboxylase (MDD) of the classical MVA pathway. We show that an enzyme originally annotated as an MDD from the Chloroflexi bacterium Anaerolinea thermophila possesses equal catalytic efficiency for (R)-MVAP and (R)-mevalonate 5-diphosphate ((R)-MVAPP). Further, the molecular basis for this dual specificity is revealed by near atomic-resolution X-ray crystal structures of A. thermophila MPD/MDD bound to (R)-MVAP or (R)-MVAPP. These findings, when combined with sequence and structural comparisons of this bacterial enzyme, functional MDDs, and several putative MPDs, delineate key active-site residues that confer substrate specificity and functionally distinguish MPD and MDD enzyme classes. Extensive sequence analyses identified functional MPDs in the halobacteria class of archaea that had been annotated as MDDs. Finally, no eukaryotic MPD candidates were identified, suggesting the absence of the alternative MVA (altMVA) pathway in all eukaryotes, including, paradoxically, plants, which universally encode a structural and functional homologue of IPK. Additionally, we have developed a viable engineered strain of Saccharomyces cerevisiae as an in vivo metabolic model and a synthetic biology platform for enzyme engineering and terpene biosynthesis in which the classical MVA pathway has been replaced with the altMVA pathway.

Carbohydrate deficient transferrin (CDT) predicts heavy drinking in adolescents with alcohol dependence.

A biomarker that could indicate problematic drinking in adolescents and/or reflect changes in heavy drinking would be a valuable addition to prevention, treatment, and research efforts. Carbohydrate deficient transferrin (CDT) is a valid biomarker of heavy drinking in adults; however, it is not well examined in adolescents. Adolescents with alcohol dependence (AD) (n = 21; 9 females) and non-dependent controls (ND) (n = 6; 3 females), ages 14-20, were interviewed to determine drinks per drinking day, peak number of drinks, and percent days heavy drinking (≥4 standard drinks/day). Blood samples from participants were assayed for percent of transferrin that was carbohydrate deficient (%CDT). Analyses compared groups on drinking and %CDT, examined the relationship between %CDT and indices of drinking, and provided preliminary estimates of the test validity of %CDT for heavy drinking in adolescents. Among adolescents with AD, %CDT was significantly and strongly correlated (r = .54) with percent heavy drinking days, and this relationship was consistent for both males and females. AD adolescents did not differ from ND on mean %CDT levels, despite significantly greater alcohol use. Indicators of test validity showed that %CDT had low sensitivity (33%) but adequate specificity (83%) for heavy drinking. Results provide proof of concept that %CDT is a potentially valuable tool to use in alcohol treatment and research in adolescents. Even if %CDT does not discriminate between adolescents with and without alcohol dependence, it could be an effective monitoring tool to indicate changes over time in binge drinking. Improved %CDT measurement methods might enhance its utility.