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Background: Congenital hypothyroidism screening traditionally relies on detecting elevated thyroid-stimulating hormone levels, yet this approach may not detect a specific type of congenital hypothyroidism caused by iodotyrosine dehalogenase-1 (Dehal1) deficiency. The deficiency of this enzyme prevents the deiodination of mono-iodotyrosine (MIT) and di-iodotyrosine (DIT) in the process of iodine recycling. This underscores the potential use of iodotyrosine or its metabolites as non-invasive urinary biomarkers for early diagnosis of congenital hypothyroidism. However, the urinary metabolites of MIT/DIT have not yet been discovered. Thus, this study aimed to identify the urinary metabolites of iodotyrosine in experimental models., Method: Gas chromatography mass spectrometry was used to identify the urinary metabolites of iodotyrosine following intraperitoneal injection of MIT in rats. An isotope dilution mass spectrometric assay was developed for assessment of identified metabolites. Urine samples from Dehal1 knockout mice were used to confirm the results., Results: We identified novel iodotyrosine metabolites, 3-iodo-4-hydroxyphenylacetic acid (IHPA), and 3,5-diiodo-4-hydroxyphenylacetic acid (Di-IHPA) as the primary urinary metabolites of MIT and DIT respectively. The concentrations of urinary IHPA and Di-IHPA were significantly higher in Dehal1 knockout mice., Conclusion: Our findings suggest that IHPA is detected in larger quantities and may hold more clinical significance than previously identified biomarkers like MIT and DIT, making it a promising candidate for diagnosing congenital hypothyroidism or other conditions associated with iodine recycling inhibition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Reduced functional connectivity of physiological systems is associated with poor prognosis in critically ill patients. However, physiological network analysis is not commonly used in clinical practice and awaits quantitative evidence. Acute liver failure (ALF) is associated with multiorgan failure and mortality. Prognostication in ALF is highly important for clinical management but is currently dependent on models that do not consider the interaction between organ systems. This study aims to examine whether physiological network analysis can predict survival in patients with ALF. Data from 640 adult patients admitted to the ICU for paracetamol-induced ALF were extracted from the MIMIC-III database. Parenclitic network analysis was performed on the routine biomarkers using 28-day survivors as reference population and network clusters were identified for survivors and non-survivors using k-clique percolation method. Network analysis showed that liver function biomarkers were more clustered in survivors than in non-survivors. Arterial pH was also found to cluster with serum creatinine and bicarbonate in survivors compared with non-survivors, where it clustered with respiratory nodes indicating physiologically distinctive compensatory mechanism. Deviation along the pH-bicarbonate and pH-creatinine axes significantly predicts mortality independent of current prognostic indicators. These results demonstrate that network analysis can provide pathophysiologic insight and predict survival in critically ill patients with ALF., (© 2024. The Author(s).)

Decompensated liver disease is complicated by multi-organ failure and poor prognosis. The prognosis of patients with liver failure often dictates clinical management. Current prognostic models have focused on biomarkers considered as individual isolated units. Network physiology assesses the interactions among multiple physiological systems in health and disease irrespective of anatomical connectivity and defines the influence or dependence of one organ system on another. Indeed, recent applications of network mapping methods to patient data have shown improved prediction of response to therapy or prognosis in cirrhosis. Initially, different physical markers have been used to assess physiological coupling in cirrhosis including heart rate variability, heart rate turbulence, and skin temperature variability measures. Further, the parenclitic network analysis was recently applied showing that organ systems connectivity is impaired in patients with decompensated cirrhosis and can predict mortality in cirrhosis independent of current prognostic models while also providing valuable insights into the associated pathological pathways. Moreover, network mapping also predicts response to intravenous albumin in patients hospitalized with decompensated cirrhosis. Thus, this review highlights the importance of evaluating decompensated cirrhosis through the network physiologic prism. It emphasizes the limitations of current prognostic models and the values of network physiologic techniques in cirrhosis., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

It is critical to understand the impact of significant physiological changes during pregnancy on the extent of maternal and fetal drug exposure. Fostemsavir (FTR) is a prodrug of temsavir (TMR) and is approved in combination with other antiretrovirals for multi-drug-resistant human immunodeficiency virus (HIV) infections. This physiologically based pharmacokinetic model (PBPK) study was used to estimate TMR PK in pregnant populations during each trimester of pregnancy to inform FTR dosing. A PBPK model was developed and validated for TMR using PK data collected following intravenous TMR and oral FTR dosing (immediate-release and extended-release tablets) in healthy volunteers. Predicted TMR concentration-time profiles accurately predicted the reported clinical data and variability in healthy (dense data) and pregnant (sparse data) populations. Predicted versus observed TMR geometric mean (CV%) clearance following intravenous administration was 18.01 (29) versus 17 (21) (L/h). Predicted versus observed TMR AUC 0-inf (ng.h/mL) in healthy volunteers following FTR administration of the extended-release tablet were 9542 (66) versus 7339 (33). The validated TMR PBPK model was then applied to predict TMR PK in a population of pregnant individuals during each trimester. Simulations showed TMR AUC in pregnant individuals receiving FTR 600 mg twice daily was decreased by 25% and 38% in the second and third trimesters, respectively. However, TMR exposure remained within the range observed in nonpregnant adults with no need for dose adjustment. The current PBPK model can also be applied for the prediction of local tissue concentrations and drug-drug interactions in pregnancy., (© 2024 GlaxoSmithKline. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Background: Despite subsidised access to direct-acting antivirals (DAAs), hepatitis C (HCV) treatment uptake in Australia is declining. Interventions are needed to link people living with HCV to care and treatment. We implemented and measured effectiveness of a state-wide, health department-led, enhanced case management through the primary care practitioner for all HCV notifications, aiming to encourage and support treatment commencement., Methods: A randomised controlled trial compared enhanced case management, delivered by the health department to diagnosing clinicians, with standard of care using notifiable disease systems in Tasmania, Australia (2020-21). The intervention involved a nurse specialist contacting and providing support by telephone to primary care practitioners making an HCV notification. The primary outcome was the proportion of cases notified with chronic hepatitis C who commenced treatment within 12 weeks of notification. We allowed a 12-week extended follow-up period at the end of the study for participants with no outcomes., Results: Eighty-five primary care practitioners randomised to the intervention and 86 to standard of care arms notified 111 and 115 HCV cases, respectively. The proportion of cases notified with chronic hepatitis (HCV RNA detected) commencing treatment within 12 weeks was similar between study arms (41% vs 33%; p=0·51) and after extended study follow-up (65% vs 48%; p=0·18). RNA test completion was higher in the intervention than in standard of care arm (89% vs. 78%; p=0·03), while completing pre-treatment workup for chronic patients (65% vs. 64%; p=0·93) was similar., Conclusion: This was the first prospective randomised study of the utility of immediate HCV notification follow-up of primary care practitioners to enhance treatment uptake using disease notification surveillance data. We demonstrated improvement in HCV RNA testing and trend toward better engagement in care, but no significant increase in treatment uptake., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: JSD, MS, and MH report investigator-initiated research funding to their institution from Gilead Science, and AbbVie. JSD reports honoraria for speaking to his institution from Gilead Sciences and AbbVie, and MS reports consultant fees from Gilead Sciences. JR reports honoraria for speaking from Gilead Sciences and AbbVie. AP has received investigator-initiated grant funding from Gilead Sciences, MSD, and Abbvie and speaker fees from Gilead Sciences for unrelated work., (Copyright © 2023. Published by Elsevier B.V.)

Objective: To develop a lower limb prosthesis (LLP) sophistication classification system that categorizes prosthetic component prescriptions into "basic," "intermediate," and "advanced" and assess its content validity, reliability, and accuracy., Design: Classification development and validation study., Setting: The Veterans Affairs (VA) Corporate Data Warehouse database and National Prosthetics Patient Database were used to identify patients undergoing their first amputation at the transtibial or transfemoral level due to diabetes or peripheral artery disease and to identify the associated codes for each LLP., Participants: An expert panel of 6 nationally recognized certified prosthetists, a national expert in VA prosthetics data and coding, a physical medicine and rehabilitation physician, and an epidemiologist developed an LLP classification system ( PROClass ) using 30 transfemoral and transtibial lower limb amputees., Main Outcome Measures: The expert panel reviewed 20 consecutive participants meeting study criteria for the development of the PROClass system and a subsequent 30 consecutive cases for assessing the inter- and intra-rater reliability and accuracy., Results: The interrater and intrarater reliability was almost perfect with Gwet's AC1 values ranging from .82 to .96 for both expert panel members and research assistants. The accuracy of the research assistant's classifications to the "criterion standard" was excellent with Gwet's AC1 values ranging between .75 and .92., Conclusions: PROClass is a pragmatic, reliable, and accurate prosthetic classification system with strong face validity that will enable the classification of prosthetic components used for large data set research aimed at evaluating important clinical questions such as the effects of sophistication on patient outcomes., (© 2023 The Authors.)

Introduction: The efficacy of targeted albumin therapy in the management of decompensatory events in cirrhosis is unclear, with different reports showing conflicting results. It is possible that only certain subgroups of patients may benefit from targeted albumin administration. However, extensive conventional subgroup analyses have not yet identified these subgroups. Albumin is an important regulator of physiological networks and may interact with homeostatic mechanism differently in patients according to the integrity of their physiological network. In this study, we aimed to assess the value of network mapping in predicting response to targeted albumin therapy in patients with cirrhosis., Methods: This is a substudy of the ATTIRE trial, a multicenter randomized trial conducted to assess the effect of targeted albumin therapy in cirrhosis. Baseline serum bilirubin, albumin, sodium, creatinine, CRP, white cell count (WCC), international normalized ratio, heart rate, and blood pressure of 777 patients followed up for 6 months were used for network mapping using parenclitic analysis. Parenclitic network analysis involves measuring the deviation of each patient from the existing network of physiological interactions in a reference population., Results: Overall network connectivity and deviations along the WCC-CRP axis predicted 6-month survival independent of age and model for end-stage liver disease in the standard care arm. Patients with lower deviation along the WCC-CRP axis showed lower survival in response to targeted albumin administration over a 6-month follow-up period. Likewise, patients with higher overall physiological connectivity survived significantly less than the standard care group after targeted albumin infusion., Discussion: The parenclitic network mapping can predict the survival of patients with cirrhosis and identify patient subgroups that do not benefit from targeted albumin therapy., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Objective: To develop and validate a patient-specific multivariable prediction model that uses variables readily available in the electronic medical record to predict 12-month mobility at the time of initial post-amputation prosthetic prescription. The prediction model is designed for patients who have undergone their initial transtibial (TT) or transfemoral (TF) amputation because of complications of diabetes and/or peripheral artery disease., Design: Multi-methodology cohort study that identified patients retrospectively through a large Veteran's Affairs (VA) dataset then prospectively collected their patient-reported mobility., Setting: The VA Corporate Data Warehouse, the National Prosthetics Patient Database, participant mailings, and phone calls., Participants: Three-hundred fifty-seven veterans who underwent an incident dysvascular TT or TF amputation and received a qualifying lower limb prosthesis between March 1, 2018, and November 30, 2020 (N=357)., Interventions: Not applicable., Main Outcome Measure: The Amputee Single Item Mobility Measure (AMPSIMM) was divided into a 4-category outcome to predict wheelchair mobility (0-2), and household (3), basic community (4), or advanced community ambulation (5-6)., Results: Multinomial logistic lasso regression, a machine learning methodology designed to select variables that most contribute to prediction while controlling for overfitting, led to a final model including 23 predictors of the 4-category AMPSIMM outcome that effectively discriminates household ambulation from basic community ambulation and from advanced community ambulation-levels of key clinical importance when estimating future prosthetic demands. The overall model performance was modest as it did not discriminate wheelchair from household mobility as effectively., Conclusions: The AMPREDICT PROsthetics model can assist providers in estimating individual patients' future mobility at the time of prosthetic prescription, thereby aiding in the formulation of appropriate mobility goals, as well as facilitating the prescription of a prosthetic device that is most appropriate for anticipated functional goals., (Copyright © 2023. Published by Elsevier Inc.)

Aim: To characterize cabotegravir population pharmacokinetics using data from phase 1, 2 and 3 studies and evaluate the association of intrinsic and extrinsic factors with pharmacokinetic variability., Methods: Analyses were implemented in NONMEM and R. Concentrations below the quantitation limit were modelled with likelihood-based approaches. Covariate relationships were evaluated using forward addition (P < .01) and backward elimination (P < .001) approaches. The impact of each covariate on trough and peak concentrations was evaluated through simulations. External validation was performed using prediction-corrected visual predictive checks., Results: The model-building dataset included 23 926 plasma concentrations from 1647 adult HIV-1-infected (72%) and uninfected (28%) subjects in 16 studies at seven dose levels (oral 10-60 mg, long-acting [LA] intramuscular injection 200-800 mg). A two-compartment model with first-order oral and LA absorption and elimination adequately described the data. Clearances and volumes were scaled to body weight. Estimated relative bioavailability of oral to LA was 75.6%. Race and age were not significant covariates. LA absorption rate constant (KA LA ) was 50.9% lower in females and 47.8% higher if the LA dose was given as two split injections. KA LA decreased with increasing BMI and decreasing needle length. Clearance was 17.4% higher in current smokers. The impact of any covariate was ≤32% on trough and peak concentrations following LA administration. The final model adequately predicted 5097 plasma concentrations from 647 subjects who were not included in the model-building dataset., Conclusions: A cabotegravir population pharmacokinetic model was developed that can be used to inform dosing strategies and future study design. No dose adjustment based on subject covariates is recommended., (© 2022 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 (" i nducer of ce ll d eath-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.

Introduction: By subsidising access to direct acting antivirals (DAAs) for all people living with hepatitis C (HCV) in 2016, Australia is positioned to eliminate HCV as a public health threat. However, uptake of DAAs has declined over recent years and new initiatives are needed to engage people living with HCV in care. Active follow-up of HCV notifications by the health department to the notifying general practitioner (GP) may increase treatment uptake. In this study, we explore the impact of using hepatitis C notifications systems to engage diagnosing GPs and improve patient access to treatment., Methods and Analysis: This study is a randomised controlled trial comparing enhanced case management of HCV notifications with standard of care. The intervention includes phone calls from a department of health (DoH) specialist HCV nurse to notifying GPs and offering HCV management support. The level of support requested by the GP was graded in complexity: level 1: HCV information only; level 2: follow-up testing advice; level 3: prescription support including linkage to specialist clinicians and level 4: direct patient contact. The study population includes all GPs in Tasmania who notified HCV diagnosis to the DoH between September 2020 and December 2021. The primary outcome is proportion of HCV cases who initiate DAAs after 12 weeks of HCV notification to the health department. Secondary outcomes are proportion of HCV notifications that complete HCV RNA testing, treatment workup and treatment completion. Multiple logistic regression modelling will explore factors associated with the primary and secondary outcomes. The sample size required to detect a significant difference for the primary outcome is 85 GPs in each arm with a two-sided alpha of 0.05% and 80% power., Ethics and Dissemination: The study was approved by University of Tasmania's Human Research Ethics Committee (Protocol ID: 18418) on 17 December 2019. Results of the project will be presented in scientific meetings and published in peer-reviewed journals., Trial Registration Number: NCT04510246., Trial Progression: The study commenced recruitment in September 2020 and end of study expected December 2021., Competing Interests: Competing interests: JD, MAS and MH report investigator-initiated research funding to their institution from Gilead Science, AbbVie and Merck. JD reports honoraria for speaking to his institution from Gilead Sciences and AbbVie and MAS reports consultant fees from Gilead Sciences. AEP has received investigator-initiated grant funding from Gilead Sciences, MSD and Abbvie and speaker fees from Gilead Sciences for unrelated work., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Liver cirrhosis involves multiple organ systems and has a high mortality. A network approach to complex diseases often reveals the collective system behaviours and intrinsic interactions between organ systems. However, mapping the functional connectivity for each individual patient has been challenging due to the lack of suitable analytical methods for assessment of physiological networks. In the present study we applied a parenclitic approach to assess the physiological network of each individual patient from routine clinical/laboratory data available. We aimed to assess the value of the parenclitic networks to predict survival in patients with cirrhosis. Methods: Parenclitic approach creates a network from the perspective of an individual subject in a population. In this study such an approach was used to measure the deviation of each individual patient from the existing network of physiological interactions in a reference population of patients with cirrhosis. 106 patients with cirrhosis were retrospectively enrolled and followed up for 12 months. Network construction and analysis were performed using data from seven clinical/laboratory variables (serum albumin, bilirubin, creatinine, ammonia, sodium, prothrombin time and hepatic encephalopathy) for calculation of parenclitic deviations. Cox regression was used for survival analysis. Result: Initial network analysis indicated that correlation between five clinical/laboratory variables can distinguish between survivors and non-survivors in this cohort. Parenclitic deviations along albumin-bilirubin (Hazard ratio = 1.063, p < 0.05) and albumin-prothrombin time (Hazard ratio = 1.138, p < 0.05) predicted 12-month survival independent of model for end-stage liver disease (MELD). Combination of MELD with the parenclitic measures could predict survival better than MELD alone. Conclusion: The parenclitic network approach can predict survival of patients with cirrhosis and provides pathophysiologic insight on network disruption in chronic liver disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Oyelade, Moore, Montagnese and Mani.)

Background: Early convalescent plasma transfusion may reduce mortality in patients with nonsevere coronavirus disease 2019 (COVID-19)., Methods: This study emulates a (hypothetical) target trial using observational data from a cohort of US veterans admitted to a Department of Veterans Affairs (VA) facility between 1 May and 17 November 2020 with nonsevere COVID-19. The intervention was convalescent plasma initiated within 2 days of eligibility. Thirty-day mortality was compared using cumulative incidence curves, risk differences, and hazard ratios estimated from pooled logistic models with inverse probability weighting to adjust for confounding., Results: Of 11 269 eligible person-trials contributed by 4755 patients, 402 trials were assigned to the convalescent plasma group. Forty and 671 deaths occurred within the plasma and nonplasma groups, respectively. The estimated 30-day mortality risk was 6.5% (95% confidence interval [CI], 4.0%-9.7%) in the plasma group and 6.2% (95% CI, 5.6%-7.0%) in the nonplasma group. The associated risk difference was 0.30% (95% CI, -2.30% to 3.60%) and the hazard ratio was 1.04 (95% CI, .64-1.62)., Conclusions: Our target trial emulation estimated no meaningful differences in 30-day mortality between nonsevere COVID-19 patients treated and untreated with convalescent plasma. Clinical Trials Registration. NCT04545047., (Published by Oxford University Press for the Infectious Diseases Society of America 2021.)

Background Testosterone treatment is common in men, although risks for major cardiovascular events are unclear. Methods and Results A study was conducted in US male veterans, aged ≥40 years, with low serum testosterone and multiple medical comorbidities and without history of myocardial infarction, stroke, venous thromboembolism, prostate cancer, or testosterone treatment in the prior year. For the primary outcome, we examined if testosterone treatment was associated with a composite cardiovascular outcome (incident myocardial infarction, ischemic stroke, or venous thromboembolism). Testosterone use was modeled as intramuscular or transdermal and as current use, former use, and no use. Current testosterone users were compared with former users to reduce confounding by indication. The cohort consisted of 204 857 men with a mean (SD) age of 60.9 (9.9) years and 4.7 (3.5) chronic medical conditions. During follow-up of 4.3 (2.8) years, 12 645 composite cardiovascular events occurred. In adjusted Cox regression analyses, current use of transdermal testosterone was not associated with risk for the composite cardiovascular outcome (hazard ratio [HR], 0.89; 95% CI, 0.76-1.05) in those without prevalent cardiovascular disease, and in those with prevalent cardiovascular disease was associated with lower risk (HR, 0.80; 95% CI, 0.70-0.91). In similar analyses, current use of intramuscular testosterone was not associated with risk for the composite cardiovascular outcome in men without or with prevalent cardiovascular disease (HR, 0.91; 95% CI, 0.80-1.04; HR, 0.98; 95% CI, 0.89-1.09, respectively). Conclusions In a large cohort of men without a history of myocardial infarction, stroke, or venous thromboembolism, testosterone treatment was not associated with increased risk for incident composite cardiovascular events.

Background: Patients undergoing amputation of the lower extremity for the complications of peripheral artery disease and/or diabetes are at risk of treatment failure and the need for reamputation at a higher level. The aim of this study was to develop a patient-specific reamputation risk prediction model., Methods: Patients with incident unilateral transmetatarsal, transtibial or transfemoral amputation between 2004 and 2014 secondary to diabetes and/or peripheral artery disease, and who survived 12 months after amputation, were identified using Veterans Health Administration databases. Procedure codes and natural language processing were used to define subsequent ipsilateral reamputation at the same or higher level. Stepdown logistic regression was used to develop the prediction model. It was then evaluated for calibration and discrimination by evaluating the goodness of fit, area under the receiver operating characteristic curve (AUC) and discrimination slope., Results: Some 5260 patients were identified, of whom 1283 (24·4 per cent) underwent ipsilateral reamputation in the 12 months after initial amputation. Crude reamputation risks were 40·3, 25·9 and 9·7 per cent in the transmetatarsal, transtibial and transfemoral groups respectively. The final prediction model included 11 predictors (amputation level, sex, smoking, alcohol, rest pain, use of outpatient anticoagulants, diabetes, chronic obstructive pulmonary disease, white blood cell count, kidney failure and previous revascularization), along with four interaction terms. Evaluation of the prediction characteristics indicated good model calibration with goodness-of-fit testing, good discrimination (AUC 0·72) and a discrimination slope of 11·2 per cent., Conclusion: A prediction model was developed to calculate individual risk of primary healing failure and the need for reamputation surgery at each amputation level. This model may assist clinical decision-making regarding amputation-level selection., (© 2019 BJS Society Ltd Published by John Wiley & Sons Ltd.)

Little is known about the population genetics of water balance. A recent meta-genome-wide association study on plasma sodium concentration identified novel loci of high biological plausibility, yet heritability of the phenotype has never been convincingly shown in European ancestry. The present study linked the Vietnam Era Twin Registry with the Department of Veterans Affairs VistA patient care clinical database. Participants ( n = 2,370, 59.6% monozygotic twins and 40.4% dizygotic twins) had a median of seven (interquartile range: 3-14) plasma sodium determinations between October 1999 and March 2017. Heritability of the mean plasma sodium concentration among all twins was 0.41 (95% confidence interval: 0.35-0.46) and 0.49 (95% confidence interval: 0.43-0.54) after exclusion of 514 twins with only a single plasma sodium determination. Heritability among Caucasian ( n = 1,958) and African-American ( n = 268) twins was 0.41 (95% confidence interval: 0.34-0.47) and 0.36 (95% confidence interval: 0.17-0.52), respectively. Exclusion of data from twins who had been prescribed medications known to impact systemic water balance had no effect. The ability of the present study to newly detect substantial heritability across multiple racial groups was potentially a function of the cohort size and relatedness, exclusion of sodium determinations confounded by elevated plasma glucose and/or reduced glomerular filtration rate, transformation of plasma sodium for the independent osmotic effect of plasma glucose, and use of multiple laboratory determinations per individual over a period of years. Individual-level plasma sodium concentration exhibited longitudinal stability (i.e., individuality); the degree to which individual-level means differed from the population mean was substantial, irrespective of the number of determinations. In aggregate, these data establish the heritability of plasma sodium concentration in European ancestry and corroborate its individuality.

Background: Patients who undergo lower extremity amputation secondary to the complications of diabetes or peripheral artery disease have poor long-term survival. Providing patients and surgeons with individual-patient, rather than population, survival estimates provides them with important information to make individualized treatment decisions., Methods: Patients with peripheral artery disease and/or diabetes undergoing their first unilateral transmetatarsal, transtibial or transfemoral amputation were identified in the Veterans Affairs Surgical Quality Improvement Program (VASQIP) database. Stepdown logistic regression was used to develop a 1-year mortality risk prediction model from a list of 33 candidate predictors using data from three of five Department of Veterans Affairs national geographical regions. External geographical validation was performed using data from the remaining two regions. Calibration and discrimination were assessed in the development and validation samples., Results: The development sample included 5028 patients and the validation sample 2140. The final mortality prediction model (AMPREDICT-Mortality) included amputation level, age, BMI, race, functional status, congestive heart failure, dialysis, blood urea nitrogen level, and white blood cell and platelet counts. The model fit in the validation sample was good. The area under the receiver operating characteristic (ROC) curve for the validation sample was 0·76 and Cox calibration regression indicated excellent calibration (slope 0·96, 95 per cent c.i. 0·85 to 1·06; intercept 0·02, 95 per cent c.i. -0·12 to 0·17). Given the external validation characteristics, the development and validation samples were combined, giving a total sample of 7168., Conclusion: The AMPREDICT-Mortality prediction model is a validated parsimonious model that can be used to inform the 1-year mortality risk following non-traumatic lower extremity amputation of patients with peripheral artery disease or diabetes., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA.)

Access to high quality photoaffinity probe molecules is often constrained by synthetic limitations related to diazirine installation. A survey of recently published photoaffinity probe syntheses identified the Suzuki-Miyaura (S-M) coupling reaction, ubiquitous in drug discovery, as being underutilized to incorporate diazirines. To test whether advances in modern cross-coupling catalysis might enable efficient S-M couplings tolerant of the diazirine moiety, a fragment-based screening approach was employed. A model S-M coupling reaction was screened under various conditions in the presence of an aromatic diazirine fragment. This screen identified reaction conditions that gave good yields of S-M coupling product while minimally perturbing the diazirine reporter fragment. These conditions were found to be highly scalable and exhibited broad scope when applied to a chemistry informer library of 24 pharmaceutically relevant aryl boron pinacol esters. Furthermore, these conditions were used to synthesize a known diazirine-containing probe molecule with improved synthetic efficiency., Competing Interests: The authors declare no competing financial interest.

Purpose: Testosterone treatment of men with low testosterone is common and, although relatively short-term, has raised concern regarding an increased risk of prostate cancer (CaP). We investigated the association between modest-duration testosterone treatment and incident aggressive CaP., Materials and Methods: Retrospective inception cohort study of male Veterans aged 40 to 89 years with a laboratory-defined low testosterone measurement from 2002 to 2011 and recent prostate specific antigen (PSA) testing; excluding those with recent testosterone treatment, prostate or breast cancer, high PSA or prior prostate biopsy. Histologically-confirmed incident aggressive prostate cancer or any prostate cancer were the primary and secondary outcomes, respectively., Results: Of the 147,593 men included, 58,617 were treated with testosterone. 313 aggressive CaPs were diagnosed, 190 among untreated men (incidence rate (IR) 0.57 per 1000 person years, 95% CI 0.49-0.65) and 123 among treated men (IR 0.58 per 1000 person years; 95% CI 0.48-0.69). After adjusting for age, race, hospitalization during year prior to cohort entry, geography, BMI, medical comorbidities, repeated testosterone and PSA testing, testosterone treatment was not associated with incident aggressive CaP (HR 0.89; 95% CI 0.70-1.13) or any CaP (HR 0.90; 95% CI 0.81-1.01). No association between cumulative testosterone dose or formulation and CaP was observed., Conclusions: Among men with low testosterone levels and normal PSA, testosterone treatment was not associated with an increased risk of aggressive or any CaP. The clinical risks and benefits of testosterone treatment can only be fully addressed by large, longer-term randomized controlled trials., Competing Interests: TJW is a consultant for and receives grant support from Boston Scientific (2014 to 2018); AMM is a consultant for AbbVie and Aytu, and receives grant support from AbbVie (2009-2018) and GlaxoSmithKline (2004-2018). This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

Objective: To assess changes in metabolic risk factors and cancer-related growth factors associated with short-term abstinence from alcohol., Design: Prospective, observational study., Setting: Single tertiary centre., Participants: Healthy subjects were recruited based on intention to: (1) abstain from alcohol for 1 month (abstinence group), or (2) continue to drink alcohol (control group). Inclusion criteria were baseline alcohol consumption >64 g/week (men) or >48 g/week (women). Exclusion criteria were known liver disease or alcohol dependence., Primary and Secondary Outcome Measures: The primary outcome was change in insulin resistance (homeostatic model assessment (HOMA) score). Secondary outcomes were changes in weight, blood pressure (BP), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and liver function tests. Primary and secondary outcomes were adjusted for changes in diet, exercise and cigarette smoking., Results: The abstinence group comprised 94 participants (mean age 45.5 years, SD ±1.2) and the control group 47 participants (mean age 48.7 years, SD ±1.8). Baseline alcohol consumption in the abstinence group was 258.2 g/week, SD ±9.4, and in the control group 233.8 g, SD ±19.0. Significant reductions from baseline in the abstinence group (all p<0.001) were found in: HOMA score (-25.9%, IQR -48.6% to +0.3%), systolic BP (-6.6%, IQR -11.8% to 0.0%), diastolic BP (-6.3%, IQR -14.1% to +1.3%), weight (-1.5%, IQR -2.9% to -0.4%), VEGF (-41.8%, IQR -64.9% to -17.9%) and EGF (-73.9%, IQR -86.1% to -36.4%). None of these changes were associated with changes in diet, exercise or cigarette smoking. No significant changes from baseline in primary or secondary outcomes were noted in the control group., Conclusion: These findings demonstrate that abstinence from alcohol in moderate-heavy drinkers improves insulin resistance, weight, BP and cancer-related growth factors. These data support an independent association of alcohol consumption with cancer risk, and suggest an increased risk of metabolic diseases such as type 2 diabetes and fatty liver disease., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Background: For patients with type 2 diabetes and chronic kidney disease (CKD), high-quality evidence about the relative benefits and harms of oral glucose-lowering drugs is limited., Objective: To evaluate whether mortality risk differs after the initiation of monotherapy with either metformin or a sulfonylurea in Veterans with type 2 diabetes and CKD., Design: Observational, national cohort study in the Veterans Health Administration (VHA)., Participants: Veterans who received care from the VHA for at least 1 year prior to initiating monotherapy treatment for type 2 diabetes with either metformin or a sulfonylurea between 2004 and 2009., Main Measures: Metformin and sulfonylurea use was assessed from VHA electronic pharmacy records. The CKD-EPI equation was used to estimate glomerular filtration rate (eGFR). The outcome of death from January 1, 2004, through December 31, 2009, was assessed from VHA Vital Status files., Key Results: Among 175,296 new users of metformin or a sulfonylurea monotherapy, 5121 deaths were observed. In primary analyses adjusted for all measured potential confounding factors, metformin monotherapy was associated with a lower mortality hazard ratio (HR) compared with sulfonylurea monotherapy across all ranges of eGFR evaluated (HR ranging from 0.59 to 0.80). A secondary analysis of mortality risk differences favored metformin across all eGFR ranges; the greatest risk difference was observed in the eGFR category 30-44 mL/min/1.73m 2 (12.1 fewer deaths/1000 person-years, 95% CI 5.2-19.0)., Conclusions: Initiation of metformin versus a sulfonylurea among individuals with type 2 diabetes and CKD was associated with a substantial reduction in mortality, in terms of both relative and absolute risk reduction. The largest absolute risk reduction was observed among individuals with moderately-severely reduced eGFR (30-44 mL/min/1.73m 2 ).

Background: Early revascularization of ischemic organs is key to improving outcomes, yet consequent reperfusion injury may be harmful. Reperfusion injury is largely attributed to excess mitochondrial production of reactive oxygen species (ROS). Sulfide inhibits mitochondria and reduces ROS production. Ammonium tetrathiomolybdate (ATTM), a copper chelator, releases sulfide in a controlled and novel manner, and may offer potential therapeutic utility., Methods and Findings: In vitro, ATTM releases sulfide in a time-, pH-, temperature-, and thiol-dependent manner. Controlled sulfide release from ATTM reduces metabolism (measured as oxygen consumption) both in vivo in awake rats and ex vivo in skeletal muscle tissue, with a superior safety profile compared to standard sulfide generators. Given intravenously at reperfusion/resuscitation to rats, ATTM significantly reduced infarct size following either myocardial or cerebral ischemia, and conferred survival benefit following severe hemorrhage. Mechanistic studies (in vitro anoxia/reoxygenation) demonstrated a mitochondrial site of action (decreased MitoSOX fluorescence), where the majority of damaging ROS is produced., Conclusions: The inorganic thiometallate ATTM represents a new class of sulfide-releasing drugs. Our findings provide impetus for further investigation of this compound as a novel adjunct therapy for reperfusion injury.

HIV integrase strand transfer inhibitors (InSTIs) represent an important class of antiviral therapeutics with proven efficacy and excellent tolerability for the treatment of HIV infections. In 2007, Raltegravir became the first marketed strand transfer inhibitor pioneering the way to a first-line therapy for treatment-naïve patients. Challenges with this class of therapeutics remain, including frequency of the dosing regimen and the genetic barrier to resistance. To address these issues, research towards next-generation integrase inhibitors has focused on imparting potency against RAL-resistent mutants and improving pharmacokinetic profiles. Herein, we detail medicinal chemistry efforts on a novel class of 2-pyridinone aminal InSTIs, inpsired by MK-0536, which led to the discovery of important lead molecules for our program. Systematic optimization carried out at the amide and aminal positions on the periphery of the core provided the necessary balance of antiviral activity and physiochemical properties. These efforts led to a novel aminal lead compound with the desired virological profile and preclinical pharmacokinetic profile to support a once-daily human dose prediction., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose: To investigate whether weight change in the first year after initiating an oral hypoglycemic agent (OHA) for type 2 diabetes treatment is associated with mortality in a national cohort., Procedures: We prospectively followed Veterans Health Administration patients with type 2 diabetes initiating treatment with an OHA and not receiving any other diabetes pharmacotherapy for at least one year. Information on OHAs, weight, co-morbidities, other medications, demographics, and laboratory measurements was obtained from electronic medical records. Logistic regression was used to estimate 5-year mortality odds by weight change during the first year after OHA treatment initiation., Findings: Patients (mean age 65years, 97% male, mean BMI 32.3kg/m 2 ) initiating OHA monotherapy between 2003 and 2008 totaled 145,198 (metformin n=89,111, glipizide n=27,100, glyburide n=25,226, rosiglitazone n=3,761). Most patients (65%) maintained a stable weight (change ⩽5% from baseline) during the first year after OHA initiation. Those losing >5% of baseline weight had a significantly higher odds of death over the subsequent 5-years ranging from 1.64 to 2.13 depending on OHA type. In the metformin group, weight gain >5% of baseline was also associated with higher odds of 5-year mortality. The same results were obtained after conducting three sensitivity analyses that excluded patients for the following reasons: weight loss in the one year prior to OHA initiation, weight change >100lbs, or weight change >50lbs., Conclusions: Weight loss was associated with higher odds of 5-year mortality among patients initiating an OHA, as was weight gain for metformin only., (Published by Elsevier B.V.)

The reactivity of a representative set of 17 organozinc pivalates with 18 polyfunctional druglike electrophiles (informers) in Negishi cross-coupling reactions was evaluated by high-throughput experimentation protocols. The high-fidelity scaleup of successful reactions in parallel enabled the isolation of sufficient material for biological testing, thus demonstrating the high value of these new solid zinc reagents in a drug-discovery setting and potentially for many other applications in chemistry. Principal component analysis (PCA) clearly defined the independent roles of the zincates and the informers toward druggable-space coverage., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Electronic health data are routinely used to conduct studies of cardiovascular disease in the setting of the Veterans Health Administration (VA). Previous studies have estimated the positive predictive value (PPV) of International Classification of Disease, Ninth Revision (ICD-9) codes for acute myocardial infarction (MI), but the sensitivity of these codes for all true events and the accuracy of coding algorithms for prevalent disease status at baseline are largely unknown., Methods: We randomly sampled 180 Veterans from the VA Puget Sound Health Care System who initiated diabetes treatment. The full electronic medical record was reviewed to identify prevalent conditions at baseline and acute MI events during follow-up. The accuracy of various coding algorithms was assessed., Results: Algorithms for previous acute events at baseline had high PPV (previous MI: 97%; previous stroke: 81%) but low sensitivity (previous MI: 38%; previous stroke: 52%). Algorithms for chronic conditions at baseline had high PPV (heart failure: 72%; coronary heart disease [CHD]: 85%) and high sensitivity (heart failure: 90%, CHD: 84%). For current smoking status at baseline, ICD-9 codes with pharmacy data had a PPV of 77% and sensitivity of 73%. The coding algorithm for acute MI events during follow-up had high PPV (80%) and sensitivity (89%)., Conclusions: ICD-9 codes for acute MI events during follow-up had high PPV and sensitivity. The sensitivity of ICD-9 codes for previous acute events at baseline was low, but a composite variable for baseline CHD had good accuracy., (Copyright © 2015 John Wiley & Sons, Ltd.)

During inflammation, leukocyte-derived eosinophil peroxidase catalyses the formation of hypobromous acid, which can brominate tyrosine residues in proteins to form bromotyrosine. Since eosinophils are involved in the pathogenesis of allergic reactions, such as asthma, urinary bromotyrosine level has been used for the assessment of children with asthma. However, little is known about the metabolism and disposition of bromotyrosine in vivo. The aim of this study was to identify the major urinary metabolites formed during bromotyrosine metabolism and to develop mass spectrometric methods for their quantitation. Deuterium-labeled bromotyrosine was synthesized by deuterium exchange. [D3]bromotyrosine (500 nmole) was injected intraperitoneally into Sprague-Dawley rats and urine was collected for 24h in a metabolic cage. (13)C-labeled derivatives of bromotyrosine and its major urinary metabolite were synthesized and used as internal standards for quantitation. Following solid phase extraction, urine samples were derivatized to the pentafluorobenzyl ester, and analyzed using isotope dilution gas chromatography and negative-ion chemical ionization mass spectrometry. A novel brominated metabolite, 3-bromo-4-hydroxyphenylacetic acid (bromo-HPA), was identified as the major brominated metabolite of bromotyrosine. Bromo-HPA only accounted for 0.43 ± 0.04% of infused [D3]bromotyrosine and 0.12 ± 0.02% of infused [D3]bromotyrosine was excreted in the urine unchanged. However, ~1.3% (6.66 ± 1.33 nmole) of infused [D3]bromotyrosine was excreted in the urine as the de-brominated metabolite, [D3]4-hydroxyphenylacetic acid, which is also a urinary metabolite of tyrosine in mammals. We also tested whether or not iodotyrosine dehalogenase can catalyse de-bromination of bromotyrosine and showed that iodotyrosine dehalogenase is able to de-brominate free bromotyrosine in vitro. We identified bromo-HPA as the main brominated urinary metabolite of bromotyrosine in rats. However, de-halogenation of bromotyrosine is the major metabolic pathway to eliminate free brominated tyrosine in vivo., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The search for new molecular constructs that resemble the critical two-metal binding pharmacophore required for HIV integrase strand transfer inhibition represents a vibrant area of research within drug discovery. Here we present the discovery of a new class of HIV integrase strand transfer inhibitors based on the 2-pyridinone core of MK-0536. These efforts led to the identification of two lead compounds with excellent antiviral activity and preclinical pharmacokinetic profiles to support a once-daily human dose prediction. Dose escalating PK studies in dog revealed significant issues with limited oral absorption and required an innovative prodrug strategy to enhance the high-dose plasma exposures of the parent molecules.

Low serum testosterone (T) is common and increasingly prevalent with increased age. Recent studies report an 'epidemic' of T prescribing and concern about unnecessary T treatment. We investigated the number of men tested for T, the prevalence of low serum T levels, and initiation of T treatment among those with low T levels in men treated at Veterans Affairs (VA) facilities in the Northwest US (VISN 20). We identified male Veterans aged 40-89 years and examined yearly proportions of men tested for T, found to have low T levels (total T < 280 ng/dL, free T < 34 pg/mL, or bioavailable T < 84 ng/dL), and subsequently treated with T from 2002 to 2011. We excluded men who had T treatment in the year prior and men with diagnoses of prostate or breast cancer. Treatment initiation was defined as the first prescription for T within a year following a low T test. From 2002 to 2011, the yearly population of eligible men in VISN 20 increased from 129 247 to 163 572. The proportion of men who had serum T tests increased from 3.2% in 2002 to 5.8% in 2011. Among the tested men, the percentage of men with low T levels increased from 35.0 to 47.3%. However, the proportion of men with low T levels who were given T treatment within a year decreased from 31.0 to 28.0%. Despite large increases in T testing, and detection of men with low T levels, there was a slight decrease in the proportion of men with low T levels who were treated with T. The decrease in T treatment during this time period contrasts with other studies and may be related to higher comorbidity in Veterans and/or VA formulary restrictions on the use of transdermal T formulations., (© 2015 American Society of Andrology and European Academy of Andrology.)

Nagase analbuminemic rats (NAR) are natural mutant Sprague-Dawley rats which do not express albumin due to a single splice mutation in the albumin gene. We accidentally discovered that NAR have a significant bradycardia compared with wild type Sprague-Dawley rats, and the present study was carried out to investigate the mechanism of bradycardia in analbuminemic rats. In vitro studies showed that the basal spontaneous beating rate of isolated atria is similar in NAR compared with wild type animals. However, the chronotropic responsiveness of isolated atria to cholinergic stimulation was markedly increased in NAR, an effect which was prevented by incubation with a nitric oxide synthase (NOS) or guanylyl cyclase inhibitor. NAR had a significant increase in plasma nitrite/nitrate concentrations. Administration of a NOS inhibitor for 5 days normalized heart rate in NAR. The level of NOS isoforms, caveolin-1 and caveolin-3 expression in the atria was assessed by real time PCR. There was no significant difference in the expression of NOS isoforms or caveolin-3 in NAR compared with wild type controls. However, NAR exhibited a significant decrease in caveolin-1 expression in the atria. Since caveolin-1 is known to inhibit endothelial NOS activity in cardiomyocytes, we suggest that decreased caveolin-1 levels may have a role in increased nitric oxide production in NAR. Our data suggest that a NOS/cGMP-dependent mechanism might be involved in increased responsiveness to vagal stimulation and bradycardia in analbuminemic condition., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Relaxation of corpus cavernosum during penile erection is mediated by a non-adrenergic non-cholinergic (NANC) neurotransmission and by the endothelium via the release of nitric oxide. Hydrogen sulfide (H(2)S) is an endogenous gaseous mediator which is a potent vasodilator and a neurotransmitter. This study was initiated to characterize the role of H(2)S in NANC neurogenic transmission in rat corpus cavernosum. The expression of H(2)S producing enzymes was assessed using RT-PCR as well as Western blotting and showed the expression of cystathionine γ-lyase (CSE) in rat corporal tissue. Homogenates from rat corpus cavernosum convert l-cysteine to H(2)S and this was partially inhibited by a CSE inhibitor, propargylglycine. Electrical stimulation of corporal tissue strips caused NANC relaxation. This neurogenic relaxation was significantly enhanced by inhibition of CSE by propargylglycine indicating that endogenously produced H(2)S may have a negative regulatory role in neurogenic relaxation of rat corpus cavernosum. To investigate this further we used physiologically relevant concentrations of exogenous NaHS, and showed that nanomolar concentrations could inhibit corporal relaxation induced by a nitroxyl (HNO) donor (Angeli's salt) but not with nitrosonium (NO(+)) or NO donors. This suggests that an interaction between endogenously produced H(2)S and nitroxyl (HNO) might be involved in erectile function., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cardiac cycle is regulated by a complex interplay between autonomic nervous system and cardiac pacemaker cells. Decreased heart rate variability (HRV) and increased cardiac rhythm regularity are associated with poor prognosis in patients with systemic inflammation (e.g., sepsis). However, the underlying mechanism of decreased HRV in systemic inflammation is not understood. It is known that greater regularity in a complex system could indicate uncoupling of the system's components. The present study aimed to test the hypothesis that impaired responsiveness of cardiac pacemaker to autonomic nervous system may lead to uncoupling of the cardiovascular regulatory mechanisms during systemic inflammation. Systemic inflammation was induced by intraperitoneal injection of endotoxin (lipopolysaccharide, 1 mg/kg) in rats. Cardiovascular signals were recorded in conscious animals using a telemetric system. Heart rate dynamics was analyzed using Poincaré plot, and cardiac cycle regularity was assessed by sample entropy analysis. Spontaneously beating atria were isolated, and chronotropic responsiveness to adrenergic and cholinergic stimulation was assessed using standard organ bath. Sample entropy decreased significantly 4 h after endotoxin injection in conscious rats. Vagal modulation of cardiac cycle (as assessed by Poincaré plot) also exhibited a significant reduction in endotoxemic rats. Acute endotoxin challenge was associated with a significant hyporesponsiveness of isolated spontaneously beating atria to cholinergic stimulation. The chronotropic responsiveness to adrenergic stimulation was identical in controls and endotoxin-treated rats. These data propose that systemic inflammation is linked to reduced cardiac responsiveness to cholinergic stimulation. This may lead to partial uncoupling of cardiac pacemaker cells from autonomic neural control and can explain decreased HRV during systemic inflammation.

The EphA4 receptor and its ephrin ligands are involved in astrocytic gliosis following CNS injury. Therefore, a strategy aimed at the blockade of EphA4 signaling could have broad therapeutic interest in brain disorders. We have identified novel small molecule inhibitors of EphA4 kinase in specific enzymatic and cell-based assays. In addition, we have demonstrated in two in vitro models of scratch injury that EphA4 receptor kinase is activated through phosphorylation and is involved in the repopulation of the wound after the scratch. A potent EphA4 kinase inhibitor significantly inhibited wound closure and reduced the accumulation of the reactive astrocytes inside the scratch. We have also shown that after the transient focal cerebral ischemia in rats, a large glial scar is formed by the accumulation of astrocytes and chondroitin sulfate proteoglycan surrounding the infarcted tissue at 7 days and 14 days of reperfusion. EphA4 protein expression is highly up-regulated in the same areas at these time points, supporting its potential role in the glial scar formation and maintenance. Taken together, these results suggest that EphA4 kinase inhibitors might interfere with the astrogliosis reaction and thereby lead to improved neurological outcome after ischemic injury., (© 2011 Merck Sharp & Dohme Corp. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

d-Serine, an endogenous co-agonist for the N-methyl-d-aspartate (NMDA) receptor in mammals, is synthesized from l-serine by serine racemase. Although much attention has been focused on the role of d-serine within the central nervous system, the physiological role of d-serine in peripheral nerves such as corpus cavernosal nerves has not been investigated. The present study was aimed to study the expression, cellular localization and function of serine racemase/d-serine system in isolated rat corpus cavernosum. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis showed the expression of serine racemase in rat corpus cavernosum. Immunogold electron microscopy demonstrated the cellular localization of serine racemase in the cavernosal nerves' membrane of the tissue. The organ bath studies on isolated rat corpus cavernosum showed that d-serine increases the non-adrenergic non-cholinergic neurogenic relaxation of isolated rat corpus cavernosum in vitro. This effect of d-serine was inhibited by a variety of NMDA receptor antagonists (ketamine, MK 801 and ifenprodil), suggesting that NMDA receptors are involved in the effects of d-serine on the neurogenic relaxation of corporal tissue strips. These observations provide the first evidence for the role of d-serine in modulating the neurogenic relaxation of rat corpus cavernosum, and may open new therapeutic avenues for the treatment of impotence., (Copyright 2010 Elsevier Inc. All rights reserved.)

Hemoproteins, hemoglobin and myoglobin, once released from cells can cause severe oxidative damage as a consequence of heme redox cycling between ferric and ferryl states that generates radical species that induce lipid peroxidation. We demonstrate in vitro that acetaminophen inhibits hemoprotein-induced lipid peroxidation by reducing ferryl heme to its ferric state and quenching globin radicals. Severe muscle injury (rhabdomyolysis) is accompanied by the release of myoglobin that becomes deposited in the kidney, causing renal injury. We previously showed in a rat model of rhabdomyolysis that redox cycling between ferric and ferryl myoglobin yields radical species that cause severe oxidative damage to the kidney. In this model, acetaminophen at therapeutic plasma concentrations significantly decreased oxidant injury in the kidney, improved renal function, and reduced renal damage. These findings also provide a hypothesis for potential therapeutic applications for acetaminophen in diseases involving hemoprotein-mediated oxidative injury.

Hydrogen sulfide is rapidly emerging as an important vasoactive mediator formed in health and disease. Its biological action is centered on its reactivity with heme-proteins and its ability to activate K(ATP) channels. Hydrogen sulfide is a signalling molecule of the inflammatory and nervous systems, and in particular the cardiovascular system where it regulates vascular tone, cardiac work, and exerts cardioprotection. This has led to an explosion of papers in which the role of hydrogen sulfide generated in vitro has been used to stimulate biological responses, and where a variety of methods have been used to measure the concentration of this compound in biological fluids. Understanding the chemistry and the inherent problems in the analytical techniques used to measure hydrogen sulfide concentrations is critical to our expanding knowledge on the biology of hydrogen sulfide. In this brief review we will cover the chemistry of hydrogen sulfide, including sources of hydrogen sulfide, its speciation at physiological pH, the susceptibility of sulfide to aerobic oxidation, and the methods used to measure hydrogen sulfide concentrations in solution, including biological fluids. We also give a brief overview of knockout animals and inhibition of the enzymes involved in the formation of hydrogen sulfide in vivo.

Heart rate variability (HRV) is reduced in several clinical settings associated with either systemic inflammation or neuropsychiatric impairment. The possibility that the changes in HRV observed in patients with neuropsychiatric impairment might relate to the overproduction of inflammatory cytokines does not seem to have been considered in the studies undertaken to date. HRV is decreased in patients with liver cirrhosis but its relationship to the impairment of neuropsychiatric performance, commonly observed in these patients, is unknown. The aim of this study was to investigate the relationship between HRV, hepatic encephalopathy, and production of inflammatory cytokines in patients with cirrhosis. Eighty patients with cirrhosis [53 men, 27 women; mean (+/-1SD) age 54 +/- 10 yr], classified as neuropsychiatrically unimpaired or as having minimal or overt hepatic encephalopathy, and 11 healthy subjects were studied. HRV was assessed by applying Poincaré plot analysis to the R-R interval series on a 5-min ECG. Inflammatory cytokines (TNF-alpha, IL-6, IL-10, and IL-12) were measured in a subgroup of patients. Long-term R-R variability was significantly decreased in the patients with cirrhosis, in parallel with the degree of neuropsychiatric impairment (P < 0.01) and independently of the degree of hepatic dysfunction (P = 0.011). The relative risk of death increased by 7.7% for every 1-ms drop in this variable. Plasma levels of IL-6 significantly correlated with indexes of both HRV and neuropsychiatric performance. The changes observed in HRV and in neuropsychiatric status in patients with cirrhosis are significantly correlated, most likely reflecting a common pathogenic mechanism mediated by inflammatory cytokines.

beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic A beta 42 peptides in Alzheimer's disease. Although previous mouse studies have shown brain A beta lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of A beta in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC(50) approximately 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain A beta levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma A beta levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPP beta, A beta 40, A beta 42, and plasma A beta 40 levels. CSF A beta 42 lowering showed an EC(50) of approximately 20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF A beta lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

Background: Newborn infants are exposed to various sources of oxidative and/or nitrative stress, which refers to either oxidation and/or nitration of endogenous proteins including loss of their original function. Nitrative stress is predominantly caused following synthesis of peroxynitrite. Particularly preterm infants with immature defense mechanisms against free radical injury appear at risk., Objective: To test the feasibility of quantifying the degradation products of the peroxynitrite marker nitrotyrosine [3-nitro-4-hydroxyphenylacetic acid (NHPA) and para-hydroxyphenylacetic acid (PHPA)] in neonatal urine samples., Methods: NHPA and PHPA were determined by gas chromatography/mass spectroscopy in urinary samples of preterm and term infants (mean gestational age = 28.4 and 39.6 weeks, respectively)., Results: The urinary NHPA levels were lower in preterm infants in comparison with term infants. When the NHPA levels were adjusted to the urinary PHPA levels, no differences were found between the two groups., Conclusions: Nitrotyrosine can be quantified in urinary samples of even the most immature infants. Nitration of endogenous PHPA in the gastrointestinal tract of term infants may have masked potentially higher levels of NHPA in preterm infants., ((c) 2007 S. Karger AG, Basel)

This letter describes replacements for the P3 amide moiety present in previously reported tertiary carbinamine macrolactones. Although P-gp efflux issues associated with these amide-macrolactones were solved and full brain penetration was measured in one case, potency was compromised in the process.