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Background: Awake prone positioning (APP) may be beneficial in patients with respiratory failure who are not receiving mechanical ventilation. Randomized controlled trials of APP have been performed during peak COVID-19 periods in unvaccinated populations, with limited data on compliance or patient acceptability. We aimed to evaluate the efficacy and acceptability of APP in a lower-middle income country in an open-label randomized controlled trial using a dedicated APP implementation team and wearable continuous-monitoring devices., Methods: The trial was performed at a tertiary level hospital in Ho Chi Minh City, Vietnam, recruiting adults (≥18 years) hospitalized with moderate or severe COVID-19 and receiving supplemental oxygen therapy via nasal/facemask systems or high-flow nasal cannula (HFNC). Patients were allocated by a computer-generated random number sequence in a 1:1 ratio to standard care or APP, where a dedicated team provided bedside support. Wearable devices continuously recorded pulse oximetry and body position continuously. Our primary outcome was escalation of respiratory support within 28 days of randomization., Results: Ninety-three patients were enrolled in this study between March 2022 and March 2023. Eighty (86%) patients had received ≥2 doses of SARS-CoV2 vaccine. The study was terminated early because of a reduction in the number of eligible patients. Data from 46 patients allocated to APP and 47 to standard care were available for analysis. At baseline, 19/47 (40%) patients allocated to the standard care group and 14/46 (30%) patients allocated to the APP group received HFNC. Continuous monitoring data were available for all patients monitored with wearable devices. Significantly greater mean daily APP times were achieved in those allocated to APP, however, most achieved less than the target 8 h/day. We did not detect clear differences in the primary outcome (relative risk,RR, 0.85, 95% CI 0.40-1.78, p=0.67) or secondary outcomes, including intubation rate and 28-day mortality. Patients reported prone positioning was comfortable, although almost all patients preferred supine positioning. No adverse events associated with the intervention were observed., Conclusions: APP was not associated with benefit, but there was no sign of harm. Continuous monitoring with wearable devices is both feasible and acceptable for patients. In our population, achieving prolonged APP time was challenging despite a dedicated support team, and patients preferred supine positioning., Clinical Trials Registration: NCT05083130., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Phong NT et al.)

Talaromycosis, a severe and invasive fungal infection caused by Talaromyces marneffei, is difficult to treat and impacts those living in endemic regions of Southeast Asia, India, and China. While 30% of infections result in mortality, our understanding of the genetic basis of pathogenesis for this fungus is limited. To address this, we apply population genomics and genome-wide association study approaches to a cohort of 336 T. marneffei isolates collected from patients who enrolled in the Itraconazole vs Amphotericin B for Talaromycosis trial in Vietnam. We find that isolates from northern and southern Vietnam form two distinct geographical clades, with isolates from southern Vietnam associated with increased disease severity. Leveraging longitudinal isolates, we identify multiple instances of disease relapse linked to unrelated strains, highlighting the potential for multistrain infections. In more frequent cases of persistent talaromycosis caused by the same strain, we identify variants arising over the course of patient infections that impact genes predicted to function in the regulation of gene expression and secondary metabolite production. By combining genetic variant data with patient metadata for all 336 isolates, we identify pathogen variants significantly associated with multiple clinical phenotypes. In addition, we identify genes and genomic regions under selection across both clades, highlighting loci undergoing rapid evolution, potentially in response to external pressures. With this combination of approaches, we identify links between pathogen genetics and patient outcomes and identify genomic regions that are altered during T. marneffei infection, providing an initial view of how pathogen genetics affects disease outcomes., Competing Interests: Conflicts of interest statement The author(s) declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Vanadium is available as a dietary supplement and also is known to be toxic if inhaled, yet little information is available concerning the effects of vanadium on mammalian metabolism when concentrations found in food and water. Vanadium pentoxide (V +5 ) is representative of the most common dietary and environmental exposures, and prior research shows that low-dose V +5 exposure causes oxidative stress measured by glutathione oxidation and protein S-glutathionylation. We examined the metabolic impact of V +5 at relevant dietary and environmental doses (0.01, 0.1, and 1 ppm for 24 h) in human lung fibroblasts (HLFs) and male C57BL/6J mice (0.02, 0.2, and 2 ppm in drinking water for 7 mo). Untargeted metabolomics using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) showed that V +5 induced significant metabolic perturbations in both HLF cells and mouse lungs. We noted 30% of the significantly altered pathways in HLF cells, including pyrimidines and aminosugars, fatty acids, mitochondrial and redox pathways, showed similar dose-dependent patterns in mouse lung tissues. Alterations in lipid metabolism included leukotrienes and prostaglandins involved in inflammatory signaling, which have been associated with the pathogenesis of idiopathic pulmonary fibrosis (IPF) and other disease processes. Elevated hydroxyproline levels and excessive collagen deposition were also present in lungs from V +5 -treated mice. Taken together, these results show that oxidative stress from environmental V +5 , ingested at low levels, could alter metabolism to contribute to common human lung diseases. NEW & NOTEWORTHY We used relevant dietary and environmental doses of Vanadium pentoxide (V +5 ) to examine its metabolic impact in vitro and in vivo. Using liquid chromatography-high-resolution mass spectrometry (LC-HRMS), we found significant metabolic perturbations, with similar dose-dependent patterns observed in human lung fibroblasts and male mouse lungs. Alterations in lipid metabolism included inflammatory signaling, elevated hydroxyproline levels, and excessive collagen deposition were present in V +5 -treated lungs. Our findings suggest that low levels of V +5 could trigger pulmonary fibrotic signaling.

Vanadium is a toxic metal listed by the IARC as possibly carcinogenic to humans. Manufactured nanosize vanadium pentoxide (V 2 O 5 ) materials are used in a wide range of industrial sectors and recently have been developed as nanomedicine for cancer therapeutics, yet limited information is available to evaluate relevant nanotoxicity. In this study we used high-resolution metabolomics to assess effects of two V 2 O 5 nanomaterials, nanoparticles and nanofibers, at exposure levels (0.01, 0.1, and 1 ppm) that did not cause cell death (i.e., non-cytotoxic) in a human airway epithelial cell line, BEAS-2B. As prepared, V 2 O 5 nanofiber exhibited a fibrous morphology, with a width approximately 63 ± 12 nm and length in average 420 ± 70 nm; whereas, V 2 O 5 nanoparticles showed a typical particle morphology with a size 36 ± 2 nm. Both V 2 O 5 nanoparticles and nanofibers had dose-response effects on aminosugar, amino acid, fatty acid, carnitine, niacin and nucleotide metabolism. Differential effects of the particles and fibers included dibasic acid, glycosphingolipid and glycerophospholipid pathway associations with V 2 O 5 nanoparticles, and cholesterol and sialic acid metabolism associations with V 2 O 5 nanofibers. Examination by transmission electron microscopy provided evidence for mitochondrial stress and increased lysosome fusion by both nanomaterials, and these data were supported by effects on mitochondrial membrane potential and lysosomal activity. The results showed that non-cytotoxic exposures to V 2 O 5 nanomaterials impact major metabolic pathways previously associated with human lung diseases and suggest that toxico-metabolomics may be useful to evaluate health risks from V 2 O 5 nanomaterials., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Organ-on-a-chip systems combine microfluidics, cell biology, and tissue engineering to culture 3D organ-specific in vitro models that recapitulate the biology and physiology of their in vivo counterparts. Here, we have developed a multiplex platform that automates the culture of individual organoids in isolated microenvironments at user-defined media flow rates. Programmable workflows allow the use of multiple reagent reservoirs that may be applied to direct differentiation, study temporal variables, and grow cultures long term. Novel techniques in polydimethylsiloxane (PDMS) chip fabrication are described here that enable features on the upper and lower planes of a single PDMS substrate. RNA sequencing (RNA-seq) analysis of automated cerebral cortex organoid cultures shows benefits in reducing glycolytic and endoplasmic reticulum stress compared to conventional in vitro cell cultures., (© 2022. The Author(s).)

Project-based learning (PBL) has long been recognized as an effective way to teach complex biology concepts. However, not all institutions have the resources to facilitate effective project-based coursework for students. We have developed a framework for facilitating PBL using remote-controlled internet-connected microscopes. Through this approach, one lab facility can host an experiment for many students around the world simultaneously. Experiments on this platform can be run on long timescales and with materials that are typically unavailable to high school classrooms. This allows students to perform novel research projects rather than just repeating standard classroom experiments. To investigate the impact of this program, we designed and ran six user studies with students worldwide. All experiments were hosted in Santa Cruz and San Francisco, California, with observations and decisions made remotely by the students using their personal computers and cellphones. In surveys gathered after the experiments, students reported increased excitement for science and a greater desire to pursue a career in STEM. This framework represents a novel, scalable, and effective PBL approach that has the potential to democratize biology and STEM education around the world., Competing Interests: The authors declare the following conflict of interests: P.V.B., K.V., V.T.L., Y.M.R., D.H. and M.T. have submitted patent applications related to Internet-of-Things-enabled microscopy. M.A.M.-R. is a cofounder of Paika, a company for remote people-to-people interactions. The authors declare no other conflicts of interests., (© 2022 The Author(s).)

The Internet of Things (IoT) provides a simple framework to control online devices easily. IoT is now a commonplace tool used by technology companies but is rarely used in biology experiments. IoT can benefit cloud biology research through alarm notifications, automation, and the real-time monitoring of experiments. We developed an IoT architecture to control biological devices and implemented it in lab experiments. Lab devices for electrophysiology, microscopy, and microfluidics were created from the ground up to be part of a unified IoT architecture. The system allows each device to be monitored and controlled from an online web tool. We present our IoT architecture so other labs can replicate it for their own experiments., 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.

Simultaneous longitudinal imaging across multiple conditions and replicates has been crucial for scientific studies aiming to understand biological processes and disease. Yet, imaging systems capable of accomplishing these tasks are economically unattainable for most academic and teaching laboratories around the world. Here, we propose the Picroscope, which is the first low-cost system for simultaneous longitudinal biological imaging made primarily using off-the-shelf and 3D-printed materials. The Picroscope is compatible with standard 24-well cell culture plates and captures 3D z-stack image data. The Picroscope can be controlled remotely, allowing for automatic imaging with minimal intervention from the investigator. Here, we use this system in a range of applications. We gathered longitudinal whole organism image data for frogs, zebrafish, and planaria worms. We also gathered image data inside an incubator to observe 2D monolayers and 3D mammalian tissue culture models. Using this tool, we can measure the behavior of entire organisms or individual cells over long-time periods., (© 2021. The Author(s).)

Talaromycosis (penicilliosis) is an invasive mycosis that is endemic in tropical and subtropical Asia. Talaromycosis primarily affects individuals with advanced HIV disease and other immunosuppressive conditions, and the disease disproportionally affects people in low-income and middle-income countries, particularly agricultural workers in rural areas during their most economically productive years. Approximately 17 300 talaromycosis cases and 4900 associated deaths occur annually. Talaromycosis is highly associated with the tropical monsoon season, when flooding and cyclones can exacerbate the poverty-inducing potential of the disease. Talaromycosis can present as localised or disseminated disease, the latter causing cutaneous lesions that are disfiguring and stigmatising. Despite up to a third of diagnosed cases resulting in death, talaromycosis has received little attention and investment from regional and global funders, policy makers, researchers, and industry. Diagnostic and treatment modalities remain extremely insufficient, however control of talaromycosis is feasible with known public health strategies. This Viewpoint is a global call for talaromycosis to be recognised as a neglected tropical disease to alleviate its impact on susceptible populations., Competing Interests: Declaration of interests NPG reports grants from US National Institutes of Health, US Centers for Disease Control and Prevention, the CDC Foundation, the Bill & Melinda Gates Foundation, the UK Medical Research Council, and the South African National Health Laboratory Service Research Trust, outside the submitted work. JF-WC and K-YY have an issued patent on antibodies targeting T marneffei Mp1p proteins and their methods of use. JP reports grants from Merck, Astellas, Pfizer, Amplyx, and Minnetronix; and membership on advisory boards or consulting roles for Merck, Amplyx, Minnetronix, F2G, Scynexis, Ampili, and Matinas, outside the submitted work. TL reports research funding from Gilead Sciences, outside the submitted work. All other authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

First-line treatment of talaromycosis with amphotericin B deoxycholate (DAmB) is labor-intensive and toxic. Itraconazole is an appealing alternative antifungal agent. Pharmacokinetic data were obtained from 76 patients who were randomized to itraconazole in the Itraconazole versus Amphotericin B for Talaromycosis (IVAP) trial. Plasma levels of itraconazole and its active metabolite, hydroxyitraconazole, were analyzed alongside longitudinal fungal CFU counts in a population model. Itraconazole and hydroxyitraconazole pharmacokinetic variability was considerable, with areas under the concentration-time curve over 24 h (AUC 24 ) of 3.34 ± 4.31 mg·h/liter and 3.57 ± 4.46 mg·h/liter (mean ± standard deviation), respectively. Levels of both analytes were low; itraconazole minimum concentration ( C min ) was 0.11 ± 0.16 mg/liter, and hydroxyitraconazole C min was 0.13 ± 0.17 mg/liter. The mean maximal rates of drug-induced killing were 0.206 and 0.208 log 10 CFU/ml/h, respectively. There were no associations between itraconazole C min /MIC and time to sterilization of the bloodstream (hazard ratio [HR], 1.01; 95% confidence interval [CI], 0.99 to 1.03; P  = 0.43), time to death (HR, 0.99; 95% CI, 0.96 to 1.02; P  = 0.77), or early fungicidal activity (EFA) (coefficient, -0.004; 95% CI, -0.010 to 0.002; P  = 0.18). Similarly, there was no relationship between AUC/MIC and time to sterilization of the bloodstream (HR, 1.00; 95% CI, 0.99 to 1.00; P  = 0.50), time to death (HR, 1.00; 95% CI, 0.99 to 1.00; P  = 0.91), or EFA (coefficient, -0.0001; 95% CI, -0.0003 to 0.0001; P  = 0.19). This study raises the possibility that the failure of itraconazole to satisfy noninferiority criteria against DAmB for talaromycosis in the IVAP trial was a pharmacokinetic and pharmacodynamic failure.

Objective: Chiropractic trainees require exposure to a diverse patient base, including patients with multiple medical conditions. The Veterans Affairs (VA) Chiropractic Residency Program aims for its doctor of chiropractic (DC) residents to gain experience managing a range of multimorbid cases, yet to our knowledge there are no published data on the comorbidity characteristics of patients seen by VA DC residents. We tested 2 approaches to obtaining Charlson Comorbidity Index (CCI) scores and compared CCI scores of resident patients with those of staff DCs at 1 VA medical center., Methods: Two processes of data collection to calculate CCI scores were developed. Time differences and agreement between methods were assessed. Comparison of CCI distribution between resident DC and staff DCs was done using 100 Monte Carlo simulation iterations of Fisher's exact test., Results: Both methods were able to calculate CCI scores (n = 22). The automated method was faster than the manual (13 vs 78 seconds per patient). CCI scores agreement between methods was good (κ = 0.67). We failed to find a significant difference in the distribution of resident DC and staff DC patients (mean p = .377; 95% CI, .375-.379)., Conclusion: CCI scores of a VA chiropractic resident's patients are measurable with both manual and automated methods, although automated may be preferred for its time efficiency. At the facility studied, the resident and staff DCs did not see patients with significantly different distributions of CCI scores. Applying CCI may give better insight into the characteristics of DC trainee patient populations., (© 2021 Association of Chiropractic Colleges.)

Background: Talaromycosis is an invasive mycosis endemic in Southeast Asia and causes substantial morbidity and mortality in individuals with advanced human immunodeficiency virus (HIV) disease. Current diagnosis relies on isolating Talaromyces marneffei in cultures, which takes up to 14 days and is detectable only during late-stage infection, leading to high mortality., Methods: In this retrospective case-control study, we assessed the accuracy of a novel Mp1p antigen-detecting enzyme immunoassay (EIA) in stored plasma samples of 372 patients who had culture-proven talaromycosis from blood or sterile body fluids (reference standard) and 517 individuals without talaromycosis (338 healthy volunteers; 179 with other infections). All participants were recruited between 2011 and 2017 in Vietnam., Results: Of cases and controls, 66.1% and 75.4%, respectively, were male; the median age was 33 and 37, respectively. All cases were HIV infected; median CD4 count was 10 cells/μL. At an optical density cutoff of 0.5, the specificity was 98.1% (95% CI, 96.3%-99.0%); the sensitivity was superior to blood culture (86.3% [95% CI, 82.3%-89.5%] vs 72.8% [95% CI, 68.0%-77.2%]) (P < .001, McNemar test). The time to diagnosis was 6 hours vs 6.6 ± 3.0 days for blood culture. Paired plasma and urine testing in the same patients (n = 269) significantly increased sensitivity compared to testing plasma alone or testing urine alone (P < .001 and P = .02, respectively, McNemar test)., Conclusions: The Mp1p EIA is highly specific and is superior in sensitivity and time to diagnosis compared to blood culture for the diagnosis of talaromycosis. Paired plasma and urine testing further increases sensitivity, introducing a new tool for rapid diagnosis, enabling early treatment and potentially reducing mortality., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

Talaromycosis is a leading cause of AIDS-associated opportunistic infections and death in Southeast Asia. We have recently shown in the Itraconazole versus Amphotericin for Talaromycosis (IVAP) trial that induction therapy with amphotericin B reduced mortality over 24 weeks, but not during the first 2 weeks. Antifungal treatment effects in real-world settings have not been rigorously evaluated. Using data obtained from patient records at the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam from 2004 to 2009, we first developed a prognostic model using Bayesian logistic regression to identify predictors of death. Second, we developed a causal model using propensity score matching to assess the treatment effects of amphotericin B and itraconazole. Our prognostic model identified intravenous drug use (odds ratio [OR] = 2.01), higher respiratory rate (OR = 1.12), higher absolute lymphocyte count (OR = 1.62), a concurrent respiratory infection (OR = 1.67) or central nervous system infection (OR = 2.66) as independent predictors of death. Fever (OR = 0.56) was a protective factor. Our prognostic model exhibits good in-sample performance and out-of-sample validation, with a discrimination power of 0.85 and 0.91, respectively. Our causal model showed no significant difference in treatment outcomes between amphotericin B and itraconazole over the first 2 weeks (95% credible interval: 0.62, 2.50). Our prognostic model provides a simple tool based on routinely collected clinical data to predict individual patient outcome. Our causal model shows similar results to the IVAP trial at 2 weeks, demonstrating an agreement between real-world data and clinical trial data., (© The Author(s) 2021. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Background: Long-term neurocognitive outcomes following first-line suppressive anti-retroviral therapy (ART) remain uncertain for individuals with HIV and hepatitis C (HCV) co-infection. The study examined neurocognitive performance before and after 72 weeks of ART using repeated multivariate analyses and latent trajectory models. Methods: One hundred and sixty adults with chronic, untreated HIV infection ( n = 80 with HCV co-infection and n = 80 HIV mono-infected) and 80 demographically similar healthy controls were recruited from the Hospital for Tropical Diseases in Ho Chi Minh City and the surrounding community, respectively. Neurocognitive measures (adapted for use in Vietnam) and liver enzyme tests were compared across groups at baseline. Repeated multivariate and group-based trajectory analyses (GBTA) examined neurocognitive subgroup profiles of the co-infected individuals after 72 weeks of de novo efavirenz- ( n = 41) or raltegravir-based ( n = 39) ART. Results: Baseline analyses revealed worse motor function in HIV-HCV co-infected individuals compared to both comparison groups. Longitudinal analyses revealed improved neurocognitive performance by week 48 for most participants regardless of treatment arm. GBTA identified a subgroup (35% of HIV-HCV sample) with persistent motor impairment despite otherwise successful ART. Higher HIV viral load and lower CD4 + T cell count at baseline predicted persistent motor dysfunction. Liver indices and ART regimen did not predict neurocognitive outcomes in HIV-HCV co-infected individuals. Conclusions: Most HIV-HCV co-infected individuals achieve normative neurocognitive performance after 48 weeks of de novo suppressive ART. However, individuals with more severe HIV disease prior to ART exhibited motor impairment at baseline and 72 weeks after otherwise successful treatment. Interventions aimed at improving motor symptoms at the time of HIV treatment onset may improve long-term clinical outcomes in HIV-HCV co-infected adults., 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 © 2021 Paul, Shikuma, Chau, Ndhlovu, Thanh, Belden, Chow, Chew, Premeaux, Ly, McBride, Bolzenius and Le.)

Talaromyces marneffei causes fatal invasive mycosis in Southeast Asia. Diagnosis by culture has limited sensitivity and can result in treatment delay. We describe the use of a novel Mp1p enzyme immunoassay (EIA) to identify blood culture-negative talaromycosis, subsequently confirmed by bone marrow cultures. This EIA has the potential to speed diagnosis, enabling early therapy initiation., (© The Author(s) 2020. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Quantification of plasma cell-free Epstein Barr virus DNA (cf EBV DNA) has been suggested as a promising liquid biopsy assay for screening and early detection of nasopharyngeal carcinoma (NPC). However, the diagnostic value of this assay is currently not known in the population of Vietnam, one of the countries which contributed the most to the NPC cases. Herein, we have reported a highly sensitive quantitative polymerase chain reaction (qPCR)-based assay targeting cf EBV DNA for the detection of NPC. A standard curve with linear regression, R 2 = 0.9961 (range: 25-150 000 copies/mL) and a detection limit of 25 copies/mL were obtained using an EBV standard panel provided by the Chinese University of Hong Kong. The clinical performance of this assay was assessed using plasma samples obtained from 261 Vietnamese individuals. The optimized qPCR assay detected cf EBV DNA in plasma with a sensitivity of 97.4% and a specificity of 98.2%. The absolute quantitative results of pretreatment cf EBV DNA and patient overall clinical stages were statistically correlated ( P < .05). In summary, the remarkably high sensitivity and specificity of our optimized qPCR assay strongly supports the wide use of cf EBV DNA quantification as a routine noninvasive method in early diagnosis and management of patients with NPC.

Accurate prediction of in vivo metabolic pathways in humans can be challenging because in vitro liver matrices may fail to produce certain in vivo metabolites.Rat and human spheroids, generated from cryopreserved hepatocytes in media that contained minimal amount of serum, maintained morphology, viability and cytochrome P450 (CYP) activities for at least a week without media exchange.With spheroid cultures, multiple Phase I and Phase II metabolites were observed in rat and human spheroid cultures that were incubated with loratadine (LOR) for multiple days. Consistent with in vivo observations, 3-hydroxydesloratadine, (3-OH-DL), along with its glucuronide, were observed in human spheroids, but not in rat spheroids. Interestingly, the putative intermediate metabolite leading to 3-OH-DL, DL-N-glucuronide, was observed in incubations with both rat and human spheroids. In conclusion, hepatocyte spheroid were capable of recapitulating the inter-species differences in metabolism between human and rat for LOR, therefore, it may represent a viable model for studying complex metabolic pathways.

Amphotericin B deoxycholate (DAmB) is a first-line agent for the initial treatment of talaromycosis. However, little is known about the population pharmacokinetics and pharmacodynamics of DAmB for talaromycosis. Pharmacokinetic data were obtained from 78 patients; among them, 55 patients had serial fungal CFU counts in blood also available for analysis. A population pharmacokinetic-pharmacodynamic model was fitted to the data. The relationships between the area under the concentration-time curve (AUC)/MIC and the time to blood culture sterilization and the time to death were investigated. There was only modest pharmacokinetic variability in the average AUC, with a mean ± standard deviation of 11.51 ± 3.39 mg·h/liter. The maximal rate of drug-induced kill was 0.133 log 10 CFU/ml/h, and the plasma concentration of the DAmB that induced the half-maximal rate of kill was 0.02 mg/liter. Fifty percent of patients sterilized their bloodstreams by 83.16 h (range, 13 to 264 h). A higher initial fungal burden was associated with a longer time to sterilization (hazard ratio [HR], 0.51; 95% confidence interval [CI], 0.36 to 0.70; P  < 0.001). There was a weak relationship between AUC/MIC and the time to sterilization, although this did not reach statistical significance (HR, 1.03; 95% CI, 1.00 to 1.06, P  = 0.091). Furthermore, there was no relationship between the AUC/MIC and time to death (HR, 0.97; 95% CI, 0.88 to 1.08; P  = 0.607) or early fungicidal activity {slope = log[(0.500 - 0.003·(AUC/MIC)]; P  = 0.319} adjusted for the initial fungal burden. The population pharmacokinetics of DAmB are surprisingly consistent. The time to sterilization of the bloodstream may be a useful pharmacodynamic endpoint for future studies. (This study has been registered at the ISRCTN registry under no. ISRCTN59144167.)., (Copyright © 2019 Le et al.)

Recurrent viral diseases often occur after the viruses evade the hosts' immune system, by inducing exhaustion of antiviral T cells. In the present study, we found that functionally exhausted herpes simplex virus type 1 (HSV-1) -specific CD8 + T cells, with elevated expression of lymphocyte activation gene-3 (LAG-3), an immune checkpoint receptor that promotes T cell exhaustion, were frequent in symptomatic (SYMP) patients with a history of numerous episodes of recurrent corneal herpetic disease. Similarly, following UV-B induced virus reactivation from latency the symptomatic wild-type (WT) B6 mice that developed increase virus shedding and severe recurrent corneal herpetic disease had more exhausted HSV-specific LAG-3 + CD8 + T cells in both trigeminal ganglia (TG) and cornea. Moreover, a therapeutic blockade of LAG-3 immune checkpoint with antagonist antibodies combined with a therapeutic immunization with gB 498-505 peptide immunodominant epitope of latently infected B6 mice significantly restored the quality and quantity of functional HSV-1 gB 498-505 specific CD8 + T cells in both TG and cornea and protected against UV-B induced recurrent corneal herpes infection and disease. In contrast to dysfunctional HSV-specific CD8 + T cells from WT B6 mice, more functional HSV-specific CD8 + T cells were detected in LAG-3 -/- deficient mice and were associated with less UV-B induced recurrent corneal herpetic disease. Thus, the LAG-3 pathway plays a fundamental role in ocular herpes T cell immunopathology and provides an important immune checkpoint target that can synergizes with T cell-based therapeutic vaccines against symptomatic recurrent ocular herpes.

Herpes simplex virus 1 (HSV-1) is a prevalent human pathogen that infects the cornea, causing potentially blinding herpetic disease. A clinical herpes vaccine is still lacking. In the present study, a novel prime/pull vaccine was tested in a human leukocyte antigen (HLA) transgenic rabbit model of ocular herpes (HLA Tg rabbits). Three peptide epitopes were selected, from the HSV-1 membrane glycoprotein C (UL44 400-408 ), the DNA replication binding helicase (UL9 196-204 ), and the tegument protein (UL25 572-580 ), all preferentially recognized by CD8 + T cells from "naturally protected" HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who never had recurrent corneal herpetic disease). HLA Tg rabbits were immunized with a mixture of these three ASYMP CD8 + T cell peptide epitopes (UL44 400-408 , UL9 196-204 , and UL25 572-580 ), which were delivered subcutaneously with CpG 2007 adjuvant (prime). Fifteen days later, half of the rabbits received a topical ocular treatment with a recombinant neurotropic adeno-associated virus type 8 (AAV8) vector expressing the T cell-attracting CXCL10 chemokine (pull). The frequency and function of HSV-specific CD8 + T cells induced by the prime/pull vaccine were assessed in the peripheral blood, cornea, and trigeminal ganglion (TG). Compared to the cells generated in response to peptide immunization alone, the peptide/CXCL10 prime/pull vaccine generated frequent polyfunctional gamma interferon-positive (IFN-γ + ) CD107 + CD8 + T cells that infiltrated both the cornea and TG. CD8 + T cell mobilization into the cornea and TG of prime/pull-vaccinated rabbits was associated with a significant reduction in corneal herpesvirus infection and disease following an ocular HSV-1 (strain McKrae) challenge. These findings draw attention to the novel prime/pull vaccine strategy for mobilizing antiviral CD8 + T cells into tissues to protect against herpesvirus infection and disease. IMPORTANCE There is an urgent need for a vaccine against widespread herpes simplex virus infections. The present study demonstrates that immunization of HLA transgenic rabbits with a peptide/CXCL10 prime/pull vaccine triggered mobilization of HSV-specific CD8 + T cells locally into the cornea and TG, the sites of acute and latent herpesvirus infections, respectively. Mobilization of antiviral CD8 + T cells into the cornea and TG of rabbits that received the prime/pull vaccine was associated with protection against ocular herpesvirus infection and disease following an ocular HSV-1 challenge. These results highlight the importance of the prime/pull vaccine strategy to bolster the number and function of protective CD8 + T cells within infected tissues., (Copyright © 2018 American Society for Microbiology.)

Circulating conventional memory CD8 + T cells (i.e., the CD8 + effector memory T [T EM ] cell and CD8 + central memory T [T CM ] cell subsets) and the noncirculating CD8 + tissue-resident memory T (T RM ) cell subset play a critical role in mucosal immunity. Mucosal chemokines, including the recently discovered CXCL17, are also important in mucosal immunity because they are homeostatically expressed in mucosal tissues. However, whether the CXCL17 chemokine contributes to the mobilization of memory CD8 + T cell subsets to access infected mucosal tissues remains to be elucidated. In this study, we report that after intravaginal HSV type 1 infection of B6 mice, we detected high expression levels of CXCL17 and increased numbers of CD44 high CD62L low CD8 + T EM and CD103 high CD8 + T RM cells expressing CXCR8, the cognate receptor of CXCL17, in the vaginal mucosa (VM) of mice with reduced genital herpes infection and disease. In contrast to wild-type B6 mice, the CXCL17 -/- mice developed 1) fewer CXCR8 + CD8 + T EM and T RM cells associated with more virus replication in the VM and more latency established in dorsal root ganglia, and 2) reduced numbers and frequencies of functional CD8 + T cells in the VM. These findings suggest that the CXCL17/CXCR8 chemokine pathway plays a crucial role in mucosal vaginal immunity by promoting the mobilization of functional protective CD8 + T EM and CD8 + T RM cells, within this site of acute and recurrent herpes infection., (Copyright © 2018 by The American Association of Immunologists, Inc.)

vic -Dibromides containing the α-bromocarbonyl or α-bromoaromatic moieties were reductively debrominated to furnish alkenes in high yield. o - and m -Anisidines but not p -anisidine were found to be effective debrominating agents. The reductive debrominations were found to be trans -stereospecific.

We present a comprehensive in vitro approach to assessing metabolism-mediated hepatotoxicity using male Sprague-Dawley rat liver slices incubated with the well characterized hepatotoxicant, precocene I, and inhibitors of cytochrome P450 (CYP) enzymes. This approach combines liquid chromatography mass spectrometry (LC MS) detection methods with multiple toxicity endpoints to enable identification of critical metabolic pathways for hepatotoxicity. The incubations were performed in the absence and presence of the non-specific CYP inhibitor, 1-aminobenzotriazole (ABT) and isoform-specific inhibitors. The metabolite profile of precocene I in rat liver slices shares some features of the in vivo profile, but also had a major difference in that epoxide dihydrodiol hydrolysis products were not observed to a measurable extent. As examples of our liver slice metabolite identification procedure, a minor glutathione adduct and previously unreported 7-O-desmethyl and glucuronidated metabolites of precocene I are reported. Precocene I induced hepatocellular necrosis in a dose- and time-dependent manner. ABT decreased the toxicity of precocene I, increased exposure to parent compound, and decreased metabolite levels in a dose-dependent manner. Of the isoform-specific CYP inhibitors tested for an effect on the precocene I metabolite profile, only tranylcypromine was noticeably effective, indicating a role of CYPs 2A6, 2C9, 2Cl9, and 2E1. With respect to toxicity, the order of CYP inhibitor effectiveness was ABT>diethyldithiocarbamate∼tranylcypromine>ketoconazole. Furafylline and sulfaphenazole had no effect, while quinidine appeared to augment precocene I toxicity. These results suggest that rat liver slices do not reproduce the reported in vivo biotransformation of precocene I and therefore may not be an appropriate model for precocene I metabolism. However, these results provide an example of how small molecule manipulation of CYP activity in an in vitro model can be used to confirm metabolism-mediated toxicity., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Microplate scintillation counters are utilized routinely in drug metabolism laboratories for the off-line radioanalysis of fractions collected during HPLC radioprofiling. In this process, the current fraction collection technology is limited by the number of plates that can be used per injection as well as the potential for sample loss due to dripping or spraying as the fraction collector head moves from well to well or between plates. More importantly, sample throughput is limited in the conventional process, since the collection plates must be manually exchanged after each injection. The Collect PAL, an innovative multiple-plate fraction collector, was developed to address these deficiencies and improve overall sample throughput. It employs a zero-loss design and has sub-ambient temperature control. Operation of the system is completely controlled with software and up to 24 (96- or 384-well) fraction collection plates can be loaded in a completely automated run. The system may also be configured for collection into various-sized tubes or vials. At flow rates of 0.5 or 1.0 mL/min and at collection times of 10 or 15s, the system precisely delivered 83-μL fractions (within 4.1% CV) and 250-μL fractions (within 1.4% CV), respectively, of three different mobile phases into 12 mm × 32 mm vials. Similarly, at a flow rate of 1 mL/min and 10s collection times, the system precisely dispensed mobile phase containing a [(14)C]-radiolabeled compound across an entire 96-well plate (% CV was within 5.3%). Triplicate analyses of metabolism test samples containing [(14)C]buspirone and its metabolites, derived from three different matrices (plasma, urine and bile), indicated that the Collect PAL produced radioprofiles that were reproducible and comparable to the current technology; the % CV for 9 selected peaks in the radioprofiles generated with the Collect PAL were within 9.3%. Radioprofiles generated by collecting into 96- and 384-well plates were qualitatively comparable; however, the peak resolution was greater in the profiles that were collected in 384-well plates due to the collection of a larger number of fractions per minute. In conclusion, this new and innovative fraction collector generated radioprofile results that were comparable to current technology and should provide a major improvement in capacity and throughput for radioprofiling studies., (Copyright © 2010 Elsevier B.V. All rights reserved.)

3'-tert-Butyl-3'-N-tert-butyloxycarbonyl-4-deacetyl-3'-dephenyl-3'-N-debenzoyl-4-O-methoxy-paclitaxel (BMS-275183) is a taxane analog that has the potential for oral use in the treatment of various types of cancer. In this study, the metabolism and excretion of [(14)C]BMS-275183 were evaluated after a single oral administration of [(14)C]BMS-275183 to rats and dogs (15 and 1 mg/kg, respectively). To aid metabolite identification by mass spectrometry (MS), a stable labeled (phenyl-(13)C(6)) BMS-275183 was included in 1:1 ratio of (13)C(6)/(12)C in the dose administration. Fecal excretion was the major route of elimination for [(14)C]BMS-275183 in both species (85-86 and <9% of the dose in feces and urine, respectively). The highest radioactivity in plasma was observed at 1 h postdose, suggesting rapid absorption of the drug in both species. The total radioactivity in plasma was measurable up to 24 h postdose. Metabolites were identified by liquid chromatography-MS and/or NMR spectroscopy. [(14)C]BMS-275183 was the prominent component in rat and dog plasma and was detected up to 24 h along with various oxidative and hydrolytic metabolites. [(14)C]BMS-275183 was extensively metabolized in both species, forming mainly oxidative metabolites, and unchanged parent drug accounted for <3.5% of the administered dose in urine and feces. The prominent metabolites resulted from oxidation of the tert-butyl groups on the side chain and further oxidation and cyclization of the tert-butylhydroxylated metabolites. A total of 30 oxidative metabolites including M13, a prominent ester cleavage metabolite, were identified in rat and dog samples.

3'-tert-Butyl-3'-N-tert-butyloxycarbonyl-4-deacetyl-3'-dephenyl-3'-N-debenzoyl-4-O-methoxycarbonyl-paclitaxel (BMS-275183) is an orally available taxane analog that has the potential to be used as an oral agent to treat cancers. The compound is similar to the two clinically intravenously administered taxanes, paclitaxel and docetaxel, in that it contains a baccatin ring linked to a side chain through an ester bond. Unlike the other taxanes, the hydrolysis of this ester bond leads to formation of a free baccatin core (M13) that was the major metabolism pathway in incubations of [(14)C]BMS-275183 in human liver microsomes (HLMs) in the presence of NADPH, but it was not formed in incubations with human liver cytosol or HLM in the absence of NADPH. The other prominent metabolites formed in HLM incubations resulted from oxidation of t-butyl groups on the side chain (M20, M20B, M21, M22, and M23). All these metabolites were formed by cDNA-expressed CYP3A and not by other cytochrome P450 (P450) enzymes tested. Formation of these metabolites was selectively inhibited by ketoconazole and troleandomycin. The formation of M13 followed Michaelis-Menten kinetics with the K(m) values of 1.3 to 2.4 muM in HLM or CYP3A4; the V(max) value for the formation of M13 and M23 in the cDNA-expressed CYP3A4 matched well (within 2-fold difference) with that determined in HLM when expressed in units of per picomole of P450. These results showed that BMS-275183 is metabolized by CYP3A4 to yield baccatin through oxidation of side-chain t-butyl groups. An intramolecular cyclization of a side-chain hydroxylation metabolite is proposed to be responsible for the formation of M13, the side-chain hydrolysis metabolite.

Ixabepilone (BMS-247550, Ixempra is a semi-synthetic analog of the natural product epothilone B and marketed for its use in the treatment of cancer. A conventional human ADME study using decay counting methods for (14)C detection could not be conducted due to the radiolytic instability of [(14)C]ixabepilone at a typical specific activity (generally 1-10 microCi/mg). However, [(14)C]ixabepilone was sufficiently stable at low specific activity (1-2 nCi/mg). These low levels required the use of accelerator mass spectrometry (AMS) for radioactivity detection. The metabolic fate of this compound was investigated in eight patients following single intravenous doses of [(14)C]ixabepilone (70 mg, 80 nCi; specific activity: 1.14 nCi/mg). Metabolite profiles in pooled urine, feces and plasma samples were determined by HPLC-AMS analysis. The major radioactive component in urine and plasma was [(14)C]ixabepilone. Feces had a small amount of ixabepilone. There were numerous other drug-related components in both urine and fecal extracts (each <6% of the administered dose). LC/MS analysis of plasma, urine and feces samples showed the presence of three degradants of ixabepilone and several oxidative metabolites (M+16, M+14 and M-2 metabolites). This study demonstrates the utility of AMS in investigating the metabolite and excretion profiles of [(14)C]ixabepilone following administration to humans.

Purpose: To determine if ixabepilone is a substrate for cytochrome P450 3A4 (CYP3A4) and if its metabolism by this cytochrome is clinically important, we did a clinical drug interaction study in humans using ketoconazole as an inhibitor of CYP3A4., Experimental Design: Human microsomes were used to determine the cytochrome P450 enzyme(s) involved in the metabolism of ixabepilone. Computational docking (CYP3A4) studies were done for epothilone B and ixabepilone. A follow-up clinical study was done in patients with cancer to determine if 400 mg/d ketoconazole (inhibitor of CYP3A4) altered the pharmacokinetics, drug-target interactions, and pharmacodynamics of ixabepilone., Results: Molecular modeling and human microsomal studies predicted ixabepilone to be a good substrate for CYP3A4. In patients, ketoconazole coadministration resulted in a maximum ixabepilone dose administration to 25 mg/m(2) when compared with single-agent therapy of 40 mg/m(2). Coadministration of ketoconazole with ixabepilone resulted in a 79% increase in AUC(0-infinity). The relationship of microtubule bundle formation in peripheral blood mononuclear cells to plasma ixabepilone concentration was well described by the Hill equation. Microtubule bundle formation in peripheral blood mononuclear cells correlated with neutropenia., Conclusions: Ixabepilone is a good CYP3A4 substrate in vitro; however, in humans, it is likely to be cleared by multiple mechanisms. Furthermore, our results provide evidence that there is a direct relationship between ixabepilone pharmacokinetics, neutrophil counts, and microtubule bundle formation in PBMCs. Strong inhibitors of CYP3A4 should be used cautiously in the context of ixabepilone dosing.

The pharmacokinetics and hepatic metabolism of [3H] ivermectin (IVM) and [3H]cyclosporin A (CSA) were investigated in a subpopulation of the CF-1 mouse stock naturally deficient in mdr1a p-glycoprotein (PGP). A survey of key drug-metabolizing activities in liver fractions from PGP-deficient (-/-) or wild-type (+/+) animals indicated the two subpopulations are not different in hepatic metabolic activity and capacity. Intravenous pharmacokinetics of CSA were identical between the two groups, and results from microsomal incubations indicated similar biotransformation of IVM and CSA in liver. Intestinal excretion of [3H]IVM and [3H]CSA was enhanced in PGP (+/+) animals. Absence of PGP resulted in higher blood concentrations of IVM after oral dosing, suggesting enhanced absorption of IVM in (-/-) mice. Concentrations of [3H]IVM and [3H]CSA were always greater in the brains of (-/-) mice compared with (+/+) mice after either i.v. or oral administration. In contrast, liver concentrations of either compound were not different between (+/+) and (-/-) animals after an i.v. dose. These results show the PGP (-/-) and (+/+) subpopulations of CF-1 mice are useful for studying the role of mdr1a PGP in systemic exposure and tissue disposition of PGP substrates in the absence of metabolism differences.

The effect of various hand washing regimens on transfer of bacterial contaminants from the hands to a hydrogel contact lenses was evaluated. Each of 47 subjects performed 5 different hand washing procedures, and then handled a new, sterile hydrogel contact lens. The lenses were cultured to determine colony-forming units (CFUs) and microbial identity. Median CFUs on lenses handled after washing with water, soap and water, or soap and water followed by towel drying were higher than the median CFU for lenses handled after no hand washing. The median CFU for lenses handled after soap and water washing followed by an alcohol wipe was not different from the no washing group. The majority of the contaminants were identified as Staphylococcus epidermidis. These results show that ordinary hand washing alone does not decrease, and may even increase, the amount of contaminants transferred from the hands to a hydrogel lens. Use of an alcoholic wipe after hand washing reverses this effect. Hand washing is still recommended in contact lens hygiene for removal of more pathogenic contaminants.