VA Tennessee Valley Healthcare System

Human milk oligosaccharides (HMOs) are complex sugars in breast milk that protect babies by preventing harmful bacteria from colonizing the gut. Our team extended the study of HMOs beyond the neonatal gut and characterized their antimicrobial activity against group B Streptococcus (GBS), a diplococcus responsible for invasive perinatal infection. To date, the mechanism of action of this antimicrobial activity has remained obscure. To address this key gap, we employed untargeted proteomics, which revealed downregulation of PcsB, an essential murein hydrolase required for cell division. Following successful purification of the active domain of PcsB, we found that this protein domain restores GBS growth in the presence of HMOs, thereby validating PcsB as an HMO protein-interacting partner. In silico docking and molecular dynamics simulations predicted that two fucosylated HMOs, lacto-N-fucopentaose I (LNFPI) and lacto-N-fucopentaose III (LNFPIII), bind to PcsB. In silico predictions were validated using microscale thermophoresis assays, which reported dissociation constants of 5 ± 1 mM for LNFPI and 263 ± 72 μM for LNFPIII. Lastly, to test the hypothesis that HMOs may directly modulate the enzymatic activity of the CHAP domain, we employed a turbidimetric assay with commercial PG as the substrate. This assay provided further evidence that HMOs inhibit CHAP. Together, these data suggest that HMOs inhibit GBS growth by binding PcsB at its catalytic site, disturbing essential cell wall separation and division.

Moral injury among healthcare workers is well recognised in extreme conditions such as the COVID-19 pandemic1 2 or critical care settings,3 but its impact on research personnel has received far less attention.4Exposure to moral injury is not part of standard training for research coordinators or investigators.This commentary reflects experiences from an ongoing clinical trial across multiple lowand middle-income countries (LMICs) in sub-Saharan Africa and offers 13 practical approaches by which to build ethical resilience and help mitigate moral injury.

Abstract Background Intravenous (IV) vancomycin management has dramatically changed since the 2020 American Journal of Health-System Pharmacist guidelines recommended area under the curve to minimum inhibitory concentration (AUC/MIC) as the optimal dosing strategy. This prompted widespread transitions in institutional dosing practices from trough-based to AUC/MIC-based strategies. The Veterans Affairs Tennessee Valley Healthcare System (VA TVHS) aimed to assess the extent of individual practice change and comfort with AUC/MIC vancomycin dosing at VA TVHS in the setting of increased evidence and available clinical tools but in the absence of an official institutional pharmacokinetic (PK) policy outlining IV vancomycin AUC/MIC dosing. Methods To assess current practice and readiness for change from trough to AUC/MIC-based vancomycin dosing, a 10-item multiple-choice electronic REDCap survey was emailed to all clinical pharmacists who dose IV vancomycin at our facility. This included clinical medicine, critical care, bone marrow transplant, emergency department, and Community Living Center pharmacists who practice at one of the two VA facilities incorporated in VA TVHS. Descriptive statistics were performed in REDCap. Results Among 34 pharmacists who received the survey link, 24 (71%) responded, of which 17 (70%) dose vancomycin daily. Dosing practice varied with 7 pharmacists (29%) reporting using trough-based, 3 (13%) using AUC/MIC, and 13 (54%) using a combination of both strategies. To determine maintenance dosing, 20 (83%) pharmacists use a PK online calculator as part of their approach, of which 18 (90%) used VancoPK.com©. While 13 (54%) were very or somewhat ready, 9 (38%) were completely or somewhat unready to completely transition to AUC/MIC dosing. Conclusion Our data show that at VA TVHS, without a clear institutional policy, adoption of best practice and comfort in vancomycin dosing practice were highly variable. This can lead to inconsistencies in patient care and a potential for adverse events. Future work will assess the time to change in practice and comfort level after policy implementation. Disclosures Milner Staub, MD, Gilead Sciences: Stocks/Bonds (Public Company)|Johnson & Johnson: Stocks/Bonds (Public Company)

Early data suggested higher sedative requirements for ventilated COVID+ patients, deviating from established guidelines. We assessed the relationship between sedative use and outcomes in mechanically ventilated Veterans during the COVID-19 pandemic. Retrospective Medication Use Evaluation. National Sample of 13 Distinct VA Medical Center Intensive Care Units. Critically ill Veteran patients requiring mechanically ventilation for ≥2 days. None. The proportion of patients receiving fentanyl, midazolam and propofol was higher during COVID years. Compared with pre-COVID, median fentanyl dose was higher during Years 1 and 2 (1575mcg [(IQR) 1000-1650] vs. 1900 [1250-3000] vs. 1910 [1150-3500]). Adjuvant antipsychotics use was relatively low but tended to increase over time (pre = 10.5% vs. Year 1 = 12.3% vs. Year 2 = 14.1%). Most patients started on antipsychotics in the ICU were continued on the drug after extubation. Mortality was higher during COVID years (pre = 26.9% vs. 1 = 36.8% and 2 = 35.9%). In stratified analyses by COVID status years 1-2 (n = 79, 27%), a higher proportion of COVID+ patients received fentanyl (96% vs. 84%) and propofol (90% vs. 77%) and at higher doses (fentanyl = 1650mcg vs. 2688mcg median cumulative dose; propofol maximum infusion rate = 30 mc/kg/min (20-50) vs. 40 (25-50)). Sedative doses were similar to pre-COVID among non-COVID patients. Anti-psychotics were more frequently continued post extubation among COVID+ (34.6% vs. non-COVID+=14.9%). COVID+ patients were also less likely to have awakening and breathing trials at 48 hours after intubation (18% vs. 46%). Sedative use and dosing increased during the first two years of COVID compared to pre-COVID, especially for COVID+ patients. The sustained elevated levels of fentanyl use in Year 2 suggests possible 'therapeutic creep' away from guideline-concordant practices for COVID+ patients. Antipsychotic prescription during intubation and following extubation was also more common among COVID + . These findings could inform development and implementation of safer sedation practices across VA ICUs during respiratory pandemics.

Clostridioides difficile is a pathogenic bacterium and a leading cause of antibiotic-associated diarrhea. Symptoms of the infection arise because of the production of large clostridial toxins that disrupt the intestinal barrier and cause an acute host inflammatory response. Epidemic C. difficile strains also produce the C. difficile transferase toxin (CDT), a binary toxin consisting of separate enzymatically active (CDTa) and cell-binding (CDTb) components. However, the role of CDT during C. difficile pathogenesis remains poorly understood. We created a CDTb nanobody (Nb) clone library and identified and purified five clones with promising CDTb-binding properties. Studies using the Carterra LSAXT platform revealed high-affinity binding interactions between the Nbs and three distinct CDTb epitopes. Functionally, these Nbs potently neutralize cellular cytopathic effects of CDT at equimolar concentrations in vitro. We further identified two distinct neutralization mechanisms—inhibition of CDTb heptamer formation and inhibition of cell surface binding, both of which are crucial for CDTa delivery into the host cell. These Nbs were used in a sandwich ELISA assay to monitor CDTb levels between 1- and 7-day post R20291 infection in the cecal material of infected mice. Notably, levels of CDTb spiked during days 3 and 4, with monomers constituting the majority of CDTb. We anticipate that these reagents will allow researchers to further expand toxin intervention and monitoring strategies to obtain a deeper understanding of the CDT mechanism of action.

Low health literacy (HL) is associated with poor health outcomes. As cancer care becomes increasingly complex, HL plays a critical role in empowering patients to successfully navigate care. Although HL has been shown to play a role in colorectal cancer (CRC) screening, its impact on oncologic treatment is less well defined. This study evaluated the association between HL and receipt of guideline-concordant CRC treatment and subsequent survival outcomes. We conducted a retrospective analysis of patients with CRC from 2010 to 2022 at a tertiary referral center. HL level was assessed using the Brief Health Literacy Screen (BHLS). Receipt of CRC treatment was evaluated based on the NCCN Guidelines according to cancer stage and type. Multivariable logistic regression and Cox proportional hazard models were used to assess associations with treatment and survival. Among 2,111 patients with CRC, mean BHLS score was 12.9. Higher HL was associated with an increased likelihood of receiving guideline-directed treatment in unadjusted (88.2% vs 72.0%; P<.0001) and adjusted analyses (odds ratio [OR], 1.13; 95% CI, 1.09-1.19). The relationship between HL and receipt of NCCN-concordant treatment was most prominent in patients with higher-stage disease, with an adjusted OR [aOR] of 1.12 (95% CI, 1.04-1.22) for stage II; 1.08 (95% CI, 1.002-1.17) for stage III; and 1.29 (95% CI, 1.17-1.43) for stage IV. Higher HL was also associated with lower all-cause mortality (aHR, 0.95; 95% CI, 0.92-0.97), particularly in stage 0-I (aOR, 0.87; 95% CI, 0.81-0.93) and stage III CRC (aOR, 0.94; 95% CI, 0.89-0.99). In patients with CRC, lower HL was independently associated with decreased odds of receiving guideline recommended treatment and poorer survival. As a modifiable risk factor, addressing HL may enhance CRC treatment and outcomes.

BackgroundThe presence of amyloid pathology can have a profound effect on the surrounding cellular neighborhood. While this impact has been mainly investigated for amyloid plaques in the context of Alzheimer's disease (AD), other forms of amyloid deposits can also be found in the brain and in other organs. In the pancreas, amyloid deposits consist of islet amyloid polypeptide (IAPP) and are a hallmark of type 2 diabetes (T2D). Notably, T2D has been associated with an increased risk of developing AD, and as such T2D is a common comorbidity of AD. It has therefore been suggested that these diseases may share pathophysiological processes. To advance our understanding in this respect, we compared the cellular and transcriptomic responses related to the proximity of amyloid pathology across the AD brain and T2D pancreas.MethodXenium single‐cell spatial transcriptomic profiling was applied to tissue sections from a human post‐mortem AD brain (150,060 cells) and a T2D pancreas (256,907 cells). Spatial transcriptomics images were integrated with amyloid histopathology images to determine the proximity of individual cells to amyloid deposits. Together with cell type predictions, this enabled the investigation and cross‐organ comparison of amyloid‐associated changes in cell type composition and gene expression changes.ResultWith respect to cell type composition, in the brain a higher proportion of microglia could be observed close to amyloid pathology, while in the pancreas this was mirrored by a higher proportion of macrophages as well as a higher proportion of activated stellate cells. Cell type specific differential gene expression analysis based on amyloid proximity revealed many cell types with altered gene expression, including astrocytes, microglia, oligodendrocytes and endothelial cells in the brain and acinar, alpha and activated stellate cells in the pancreas. Comparison across organs revealed 16 shared genes differentially expressed with proximity to amyloid deposits, including CAV1, CXCR4, MS4A6A, SNCG, and SOX2.ConclusionHere we spatially investigate the impact of amyloid deposits on the cellular and transcriptomic microenvironment in the brain and pancreas. Our analysis revealed a common set of amyloid proximity related genes, providing insight into potentially shared pathological pathways underlying AD and T2D.