Association of personalized and tumor-informed ctDNA with patient survival outcomes in pancreatic adenocarcinoma.

INTRODUCTION: Personalized and tumor-informed circulating tumor DNA (ctDNA) testing is feasible and allows for molecular residual disease (MRD) identification in patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: In this retrospective analysis of commercial cases from multiple US institutions, personalized, tumor-informed, whole-exome sequenced, and germline-controlled ctDNA levels were quantified and analyzed in patients with PDAC. Plasma samples (n = 1329) from 299 clinically validated patients were collected at diagnosis, perioperatively (MRD-window; within 2-12 weeks after surgery, before therapy), and during surveillance (>12 weeks post-surgery if no ACT or starting 4 weeks post-ACT) from November 2019 to March 2023. RESULTS: Of the initially diagnosed patients with stages I-III PDAC who went for resection, the median follow-up time from surgery was 13 months (range 0.1-214). Positive ctDNA detection rates were 29% (29/100) and 29.6% (45/152) during the MRD and surveillance windows, respectively. Positive ctDNA detection was significantly associated with shorter DFS within the MRD window (median DFS of 6.37 months for ctDNA-positive vs 33.31 months for ctDNA-negative patients; HR: 5.45, P < .0001) as well as during the surveillance period (median DFS: 11.40 months for ctDNA-positive vs NR for ctDNA-negative; HR: 12.38, P < .0001). Additionally, DFS was significantly better with KRAS wildtype status followed by KRASG12R (HR: 0.99, P = .97), KRASG12D (HR: 1.42, P = .194), and worse with KRASG12V (HR: 2.19, P = .002) status. In multivariate analysis, ctDNA detection at surveillance was found to be the most significant prognostic factor for recurrence (HR: 24.28, P < .001). CONCLUSIONS: Perioperative tumor-informed ctDNA detection in PDAC is feasible across all stages and is associated with patient survival outcomes.

The current state of transplant advanced practice providers: results of the advanced practice provider practice survey.

Advanced practice providers (APPs) are trained, licensed health care providers. The American Society of Transplant APP community of practice developed an electronic survey to investigate transplant APP demographics, scope of practice, and academic activities. We defined the top of scope of practice as delivering health care to the fullest extent of APP education and training as allowed by state laws and regulations. From July 11, 2020, to August 31, 2020, 307 invitations were e-mailed and survey links were distributed electronically on the community of practice hub and social media. Two hundred fifty-three APPs responded. APPs practice in inpatient and outpatient settings. Among the respondent APPs, 11.5% assist in the operating room (OR), 46.3% of inpatient and 46.6% of outpatient APPs perform procedures, and 17.8% run specialized APP clinics. 26.2% feel they do not function at the top of their scope of practice and 29.7% were expected to function as a coordinator some or all of the time. Forty-three percent gave invited lectures, 41.5% have published, and 69.2% teach physician trainees. 74.7% and 35.1%, respectively, would like to participate in research and teach but are limited by time, opportunity, and experience. APPs should practice at the top of their scope of practice. Clinical workloads and lack of time limit the ability of APP to teach and contribute to evidence-based practice.

Utilizing open-source platforms to build and deploy interactive patient-reported quality of life tracking tools for monitoring protocol adherence.

PURPOSE: Tracking patient-reported outcomes (PROs) and quality-of-life response rates is essential for clinical trials. Historically, rates are monitored through scheduled reports, which can require gathering, merging, and cleaning data from multiple databases. At the end of this process, if gaps are found, new data are entered and the cycle repeats, leaving a trail of reports that are not up-to-date or immediately accessible to the investigator. The financial and person-hour cost of utilizing clinical research staff for this purpose is impractical. Online dashboards are continuously updated to monitor data, providing on-demand access to promote successful research. METHODS: Dashboard implementation utilizes R, an open-source statistical programming language, RMarkdown, a markup language, Flexdashboard, which creates structural elements, and Shiny, allowing investigators the ability to interact with data within the dashboard. By leveraging these four elements, powerful, cost-effective interactive dashboards can be built. RESULTS: Numerous dashboards have been utilized to identify potentially missing data and increase protocol adherence. Immediate patient consultation can occur to retrieve protocol-related forms, reducing research staff and patient burden while improving trial effectiveness. Dashboards can monitor PROs, enrollment, demographics, toxicity, and biomarker data, clinical outcomes, and implemented predictive models, creating a single hub for on-demand clinical trial monitoring. CONCLUSION: By employing a set of freely available tools, the burden of utilizing study staff to continuously monitor trials is greatly reduced. These tools allow users to rapidly build and deploy dynamic dashboards capable of meeting the research needs of any investigator while limiting missing data through simplified monitoring of protocol adherence.

Relationship between bovine carotid artery grafts for hemodialysis access and human leukocyte antigen sensitization.

INTRODUCTION: Bovine carotid artery (BCA) Artegraft is a biologic graft that can be utilized as a conduit for permanent hemodialysis access and has been shown to outperform polytetrafluoroethylene grafts. However, concern regarding immunologic sensitization may limit the use of BCA in the transplant candidate. Panel reactive antibody (PRA) is an immunological test utilized in transplant recipient selection whereas increases in PRA limit access to transplantation. The purpose of our study was to determine whether BCA graft placement was adversely associated with increases in PRA. METHODS: Of patients listed for kidney transplant at our institution, we identified 10 patients who underwent BCA placement for hemodialysis access and a matched cohort of 10 patients who underwent native arteriovenous fistula (AVF) creation between 2014 and 2017. The PRA value nearest to the surgery date was compared to postsurgery PRA value for the BCA and AVF patients using a paired t test. Presurgery PRAs were also compared to the maximum PRA at 0 to 6, 6 to 12, 12 to 18, and 18 to 24 months postsurgery. FINDINGS: Prior to the dialysis access operation, the mean PRA was 14.1% ± 23.5% vs. 17.1% ± 29.0% (P = 0.76) and the median postsurgery follow-up time was 16 and 15 months for BCA and AVF cohorts, respectively. There were no statistically significant differences between presurgery and postsurgery PRA for BCA and AVF patients, regardless of time interval postsurgery. The difference in presurgery/postsurgery PRA change between cohorts was not statistically significant for PRAs closest to surgery (0.2% ± 40.6% vs. 1.0% ± 2.8%, P = 0.95, at a median 4 and 3 months postsurgery, respectively) or when using the maximum in any postsurgery interval. Prior to their dialysis access surgery, there were 16 sensitizing events in 5 patients in the BCA group compared to 10 events in 5 patients in the AVF group (P = 0.20). Only 1 of the 10 patients in the BCA group had a clinically relevant and sustained increase in PRA following their dialysis access operation vs. no patients in the AVF group (P > 0.99). However, this patient had a known sensitizing event (blood transfusion) between the BCA surgery and the postoperative PRA. Three of 10 patients in the BCA cohort vs. 5 of 10 patients in the AVF cohort went on to have successful kidney transplants (P = 0.65). DISCUSSION: The utilization of BCA for dialysis access was not associated with statistically significant changes in PRA. These data suggest that implantation of BCA will not affect access to organ transplantation.

Manganese Increases Lead Release to Drinking Water.

Lead and manganese are regulated in drinking water due to their neurotoxicity. These elements have been reported to co-occur in drinking water systems, in accordance with the metal-scavenging properties of MnO2. To the extent that manganese is a driver of lead release, controlling it during water treatment may reduce lead levels. We investigated transport of lead and manganese at the tap in a full-scale distribution system: consistent with a cotransport phenomenon, the two metals were detected in the same colloidal size fraction by size-exclusion chromatography with multielement detection. We also studied the effect of manganese on lead release using a model distribution system: increasing manganese from 4 to 215 µg L-1 nearly doubled lead release. This effect was attributed primarily to deposition corrosion of lead by oxidized phases of manganese, and we used 16S rRNA sequencing to identify bacteria that may be relevant to this process. We explored the deposition corrosion mechanism by coupling pure lead with either MnO2-coated lead or pure lead exposed to MnO2 in suspension; we observed galvanic currents in both cases. We attributed these to reduction of Mn(IV) under anaerobic conditions, and we attributed the additional current under aerobic conditions to oxygen reduction catalyzed by MnO2.

Cardiovascular disease in the kidney transplant recipient: epidemiology, diagnosis and management strategies.

Kidney transplantation (KT) is the optimal therapy for end-stage kidney disease (ESKD), resulting in significant improvement in survival as well as quality of life when compared with maintenance dialysis. The burden of cardiovascular disease (CVD) in ESKD is reduced after KT; however, it still remains the leading cause of premature patient and allograft loss, as well as a source of significant morbidity and healthcare costs. All major phenotypes of CVD including coronary artery disease, heart failure, valvular heart disease, arrhythmias and pulmonary hypertension are represented in the KT recipient population. Pre-existing risk factors for CVD in the KT recipient are amplified by superimposed cardio-metabolic derangements after transplantation such as the metabolic effects of immunosuppressive regimens, obesity, posttransplant diabetes, hypertension, dyslipidemia and allograft dysfunction. This review summarizes the major risk factors for CVD in KT recipients and describes the individual phenotypes of overt CVD in this population. It highlights gaps in the existing literature to emphasize the need for future studies in those areas and optimize cardiovascular outcomes after KT. Finally, it outlines the need for a joint 'cardio-nephrology' clinical care model to ensure continuity, multidisciplinary collaboration and implementation of best clinical practices toward reducing CVD after KT.

MgF3- and AlF4- transition state analogue complexes of yeast phosphoglycerate kinase.

The phospho-transfer mechanism of yeast phosphoglycerate kinase (PGK) has been probed through formation of trifluoromagnesate (MgF3-) and tetrafluoroaluminate (AlF4-) transition state analogue complexes and analyzed using 19F, 1H waterLOGSY and 1H chemical shift perturbation NMR spectroscopy. We observed the first 19F NMR spectroscopic evidence for the formation of metal fluoride transition state analogues of yeast PGK and also observed significant changes to proton chemical shifts of PGK in the presence, but not in the absence, of fluoride upon titration of ligands, providing indirect evidence of the formation of a closed ternary transition state. WaterLOGSY NMR spectroscopy experiments using an uncompetitive model were used in an attempt to measure ligand binding affinities within the transition state analogue complexes.

Synthesis of α-Deoxymono and Difluorohexopyranosyl 1-Phosphates and Kinetic Evaluation with Thymidylyl- and Guanidylyltransferases.

Eight fluorinated isosteric α-d-glucopyranosyl 1-phosphate (Glc 1P) analogues have been synthesized. A promiscuity investigation of the thymidylyltransferase Cps2L and the guanidylyltansferase GDP-ManPP with these analogues showed that all were accepted by either enzyme, with the exception of 1,6-diphosphate 6. Kinetic parameters were determined for these analogues using a continuous coupled assay. These data demonstrated the broad substrate promiscuity of Cps2L, with kcat/Km changes for monofluoro substitution at C-2, C-4, and C-6 and difluoro substitution at C-2 within two orders of magnitude. In contrast, the kinetic analysis of GDP-ManPP was only possible with three out of eight analogues. The pKa2 values of analogues (1-3) were determined by proton decoupled 31P and 19F NMR titration experiments. Counterintuitively, the axial fluoro substituent in 3 did not change chemical shift upon titration, and there was no significant increase in acidity for the difluoro analogue over the monofluoro analogues. No strong Brønsted linear free-energy correlations were observed among all five substrates (1-3, Glc 1P, and Man 1P) for either enzyme-catalyzed reactions. However, Brønsted correlations were observed among selected substrates, indicating that the acidity of the nucleophilic phosphate and the configuration of the hexose each plays a significant role in determining the substrate specificity.

JadX is a Disparate Natural Product Binding Protein.

We report that JadX, a protein of previously undetermined function coded for in the jadomycin biosynthetic gene cluster of Streptomyces venezuelae ISP5230, affects both chloramphenicol and jadomycin production levels in blocked mutants. Characterization of recombinant JadX through protein-ligand interactions by chemical shift perturbation and WaterLOGSY NMR spectroscopy resulted in the observation of binding between JadX and a series of jadomycins and between JadX and chloramphenicol, another natural product produced by S. venezuelae ISP5230. These results suggest JadX to be an unusual class of natural product binding protein involved in binding structurally disparate natural products. The ability for JadX to bind two different natural products in vitro and the ability to affect production of these secondary metabolites in vivo suggest a potential role in regulation or signaling. This is the first example of functional characterization of these JadX-like proteins, and provides insight into a previously unobserved regulatory process.

Investigations into the binding of jadomycin DS to human topoisomerase IIß by WaterLOGSY NMR spectroscopy.

The jadomycins are a family of secondary metabolites produced by S. venezuelae ISP5230. Specific jadomycins have been shown to possess a variety of anticancer, antifungal, and antibacterial properties, with different molecular mechanisms of action. Herein we demonstrate qualitative and quantitative direct binding between the validated anticancer target human topoisomerase IIß and jadomycin DS using WaterLOGSY NMR spectroscopy. Additionally, we report for the first time, that jadomycin DS also binds a variety of other proteins, likely in a non-specific manner. Such interactions may rationalize the potential polypharmacology of jadomycin DS.

Polyphosphate-containing bisubstrate analogues as inhibitors of a bacterial cell wall thymidylyltransferase.

A series of polyphosphate containing sugar nucleotide analogues were synthesized and evaluated as bisubstrate inhibitors of α-D-glucose 1-phosphate thymidylyltransferase Cps2L, the first enzyme in Streptococcus pneumoniael-rhamnose biosynthesis, and a novel antibacterial target. WaterLOGSY NMR spectroscopy demonstrated binding of bisubstrate analogues to Cps2L and a spectrophotometric coupled assay was used to determine apparent Ki values.

On the mechanism of phosphoenolpyruvate synthetase (PEPs) and its inhibition by sodium fluoride: potential magnesium and aluminum fluoride complexes of phosphoryl transfer.

Phosphoenolpyruvate synthase (PEPs) catalyzes the conversion of pyruvate to phosphoenolpyruvate (PEP) using a two-step mechanism invoking a phosphorylated-His intermediate. Formation of PEP is an initial step in gluconeogenesis, and PEPs is essential for growth of Escherichia coli on 3-carbon sources such as pyruvate. The production of PEPs has also been linked to bacterial virulence and antibiotic resistance. As such, PEPs is of interest as a target for antibiotic development, and initial investigations of PEPs have indicated inhibition by sodium fluoride. Similar inhibition has been observed in a variety of phospho-transfer enzymes through the formation of metal fluoride complexes within the active site. Herein we quantify the inhibitory capacity of sodium fluoride through a coupled spectrophotometric assay. The observed inhibition provides indirect evidence for the formation of a MgF3(-) complex within the enzyme active site and insight into the phospho-transfer mechanism of PEPs. The effect of AlCl3 on PEPs enzyme activity was also assessed and found to decrease substrate binding and turnover.

Synthesis and evaluation of l-rhamnose 1C-phosphonates as nucleotidylyltransferase inhibitors.

We report the synthesis of a series of phosphonates and ketosephosphonates possessing an L-rhamnose scaffold with varying degrees of fluorination. These compounds were evaluated as potential inhibitors of α-D-glucose 1-phosphate thymidylyltransferase (Cps2L), the first enzyme in Streptococcus pneumoniae L-rhamnose biosynthesis, and a novel antibiotic target. Enzyme-substrate and enzyme-inhibitor binding experiments were performed using water-ligand observed binding via gradient spectroscopy (WaterLOGSY) NMR for known sugar nucleotide substrates and selected phosphonate analogues. IC50 values were measured and Ki values were calculated for inhibitors. New insights were gained into the binding promiscuity of enzymes within the prokaryotic L-rhamnose biosynthetic pathway (Cps2L, RmlB-D) and into the mechanism of inhibition for the most potent inhibitor in the series, L-rhamnose 1C-phosphonate.

Regulation of D-alanylation of lipoteichoic acid in Streptococcus gordonii.

d-Alanyl esters on lipoteichoic acid (LTA) are involved in adhesion, biofilm formation, resistance to cationic antimicrobial peptides, and immune stimulation. There is evidence that bacteria can modulate the level of d-alanyl esters on LTA in response to challenge, but the mechanism of regulation appears to be different among bacteria. In this study, expression of the dlt operon responsible for d-alanylation of LTA was examined in the commensal bacterium Streptococcus gordonii. dlt expression was assessed using the dlt promoter-lacZ reporter gene assay, LTA d-alanine content measurements and dlt mRNA quantification. The results showed that dlt expression was growth phase-dependent, with the greatest expression at the mid-exponential phase of growth. In contrast to Staphylococcus aureus, dlt expression in Strep. gordonii was not affected by the exogenous addition of Mg(2+) or K(+). Interestingly, dlt expression was upregulated under acidic conditions or when cells were stressed with polymyxin B, indicating that cell envelope stress may be a signal for dlt expression. In view of these results, mutants defective in the cell envelope stress LiaSR two-component regulatory system were constructed. The liaS and liaR mutants showed an increase in dlt expression over the parent strain at neutral pH. The mutants failed to respond to low pH and polymyxin B stress; dlt expression remained the same in the presence or absence of these stresses. These results suggest that dlt expression in Strep. gordonii is regulated by the LiaSR regulatory system in response to environmental signals such as pH and polymyxin B. The regulation appears to be complex, involving both repression and activation mechanisms.