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A rapid, selective, and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitation of MO-OH-Nap tropolone (MO-OH-Nap) in mouse plasma. MO-OH-Nap is an α-substituted tropolone with anti-proliferative properties in various cancer cell lines. Detection and separation of analytes was achieved on an ACE Excel C18 (1.7 µm, 100 × 2.1 mm, MAC-MOD Analytical, Chadds Ford, PA, USA) column with mobile phase consisting of 0.05% trifluoroacetic acid in water (mobile phase A) and 0.05% trifluoroacetic acid in acetonitrile (mobile phase B), with an isocratic elution of 15:85% (A:B) at a total flow rate of 0.25 mL/min. The LC-MS/MS system was operated at unit resolution in multiple reaction monitoring (MRM) mode, using precursor ion > product ion combination of 249.10 > 202.15 m/z for MO-OH-Nap and 305.10 > 215.05 m/z for the internal standard (IS), BA-SM-OM. The MS/MS response was linear over a concentration range of 1 to 500 ng/mL with a correlation coefficient (r2) of ≥0.987. The within- and between-batch precision (%RSD) and accuracy (%Bias) were within acceptable limits. The validated method was successfully applied to determine MO-OH-Nap metabolic stability, plasma protein binding, and bio-distribution studies of MO-OH-Nap in CD-1 mice. [ABSTRACT FROM AUTHOR]

Highlights: What are the main findings? The validated method was robust, reliable, and sensitive for quantifying ABZ, ABZ-OX, ABZ-ON, and PR simultaneously from the plasma and dried blood spots. The method is well suited for therapeutic drug monitoring due to its high sensitivity, minimal sample volume requirement, and the ability for remote collection. What is the implication of the main finding? Dried blood spot (DBS) analysis represents an alternative sampling technique over conventional blood-sampling methods, particularly for sample collection, enabling a non-clinical setup without requiring special equipment or trained healthcare personnel. Additionally, it minimizes the solvent volume and analysis time and reduces transportation and storage needs. Millions of individuals throughout the world suffer from lymphatic filariasis (LF), which is a morbid disease caused by Wuchereria bancrofti, Brugia malayi, and Brugia timori. These infections belong to tissue-invading nematodes and are one of the major neglected tropical diseases that often result in permanent and enduring disability among individuals in endemic regions. Due to combination therapy, LF eradication has drastically decreased infections globally. The development of blood micro-sampling techniques allowing precise quantitation of drugs in blood would facilitate pharmacokinetic (PK) studies in remote populations. Therefore, an LC-MS/MS bioanalytical method was utilized to analyze albendazole (ABZ), albendazole sulfone (ABZ-ON), albendazole sulfoxide (ABZ-OX), and probenecid (PR) in plasma and dried blood spots. Solid-phase extraction was utilized to extract the analyte from both plasma and blood-spiked DBS. Analytes of interest were eluted with a gradient mobile system using 0.05% formic acid in water (A) and 0.05% formic acid in methanol (B) and separated using a reversed-phase Acquity ®BEH C18 UPLC column (100 × 2.1 mm, 1.7 µm). Precision and accuracy at each QC level were within the acceptable limit, i.e., ±15% for all analytes in both the matrices. Tests for stability under laboratory and storage conditions indicated that no notable changes were observed for plasma and DBS. The LC-MS/MS method demonstrated its capability to consistently identify all target analytes (ABZ, ABZ-ON, ABZ-OX, and PR) at low concentrations, even at the small specimen volumes obtained from DBS cards. This confirms the efficacy and durability of DBS cards as a micro-sampling technique. Moreover, it enhances collection efforts for therapeutic drug monitoring in remote locations for patients infected with lymphatic filariasis. [ABSTRACT FROM AUTHOR]

Chemotherapy together with surgery and/or radiotherapy are the most common therapeutic methods for treating cancer. However, the off-target effects of chemotherapy are known to produce side effects and dose-limiting toxicities. Novel delivery platforms based on natural and synthetic polymers with enhanced pharmacokinetic and therapeutic potential for the treatment of cancer have grown tremendously over the past 10 years. Polymers can facilitate selective targeting, enhance and prolong circulation, improve delivery, and provide the controlled release of cargos through various mechanisms, including physical adsorption, chemical conjugation, and/or internal loading. Notably, polymers that are biodegradable, biocompatible, and physicochemically stable are considered to be ideal delivery carriers. This biomimetic and bio-inspired system offers a bright future for effective drug delivery with the potential to overcome the obstacles encountered. This review focuses on the barriers that impact the success of chemotherapy drug delivery as well as the recent developments based on natural and synthetic polymers as platforms for improving drug delivery for treating cancer. [ABSTRACT FROM AUTHOR]

Osteosarcoma (OS) is a primary malignant bone tumor characterized by frequent metastasis, rapid disease progression, and a high rate of mortality. Treatment options for OS have remained largely unchanged for decades, consisting primarily of cytotoxic chemotherapy and surgery, thus necessitating the urgent need for novel therapies. Tropolones are naturally occurring seven‐membered non‐benzenoid aromatic compounds that possess antiproliferative effects in a wide array of cancer cell types. MO‐OH‐Nap is an α‐substituted tropolone that has activity as an iron chelator. Here, we demonstrate that MO‐OH‐Nap activates all three arms of the unfolded protein response (UPR) pathway and induces apoptosis in a panel of human OS cell lines. Co‐incubation with ferric chloride or ammonium ferrous sulfate completely prevents the induction of apoptotic and UPR markers in MO‐OH‐Nap‐treated OS cells. MO‐OH‐Nap upregulates transferrin receptor 1 (TFR1) protein levels, as well as TFR1, divalent metal transporter 1 (DMT1), iron‐regulatory proteins (IRP1, IRP2), ferroportin (FPN), and zinc transporter 14 (ZIP14) transcript levels, demonstrating the impact of MO‐OH‐Nap on iron‐homeostasis pathways in OS cells. Furthermore, MO‐OH‐Nap treatment restricts the migration and invasion of OS cells in vitro. Lastly, metabolomic profiling of MO‐OH‐Nap‐treated OS cells revealed distinct changes in purine and pyrimidine metabolism. Collectively, we demonstrate that MO‐OH‐Nap‐induced cytotoxic effects in OS cells are dependent on the tropolone's ability to alter cellular iron availability and that this agent exploits key metabolic pathways. These studies support further evaluation of MO‐OH‐Nap as a novel treatment for OS. [ABSTRACT FROM AUTHOR]

Moxidectin (MOX) is a milbemycin endectocide recently approved by the U.S. FDA for the treatment of onchocerciasis in persons at least 12 years of age. MOX has been shown to have a good safety profile in recent clinical trials. The efficacy of MOX for the treatment of lymphatic filariasis (LF) and its potential use in mass drug administration protocols for the elimination of LF is currently under evaluation. In the context of a clinical trial, we investigated the pharmacokinetics and drug interactions of a combination of MOX plus albendazole (ALB) with or without diethylcarbamazine (DEC) compared to ivermectin (IVM) plus ALB with or without DEC in the following four different treatment arms: (I) IVM (0.2mg/kg) plus DEC (6 mg/kg) and ALB (400mg); (II) IVM plus ALB; (III) MOX (8 mg) plus DEC and ALB; and (IV) MOX plus ALB. Drug concentrations were determined using validated liquid chromatography-mass spectrometric methods. Pharmacokinetic parameters were determined using standard non-compartmental analysis methods. Statistical analysis was performed using JMP software. Fifty-eight of 164 study participants (53 men and five women) were included with ages ranging from 18 to 63 yrs (mean = 37). MOX apparent oral clearance (Cl/F) ranged from 0.7 to 10.8 L/hr with Cmax values ranging from 20.8 to 314.5 ng/mL. The mean (range) area under the curve (AUC)0-∞ for MOX, 3405 ng*hr/mL (742–11376), and IVM 1906 ng*hr/mL (692–5900), varied over a ~15.3 and ~8.5-fold range, respectively. The geometric mean ratio for Cmax, AUC0–t, and AUC0–∞ were within the no-drug interaction range of 80–125% for all drugs. This indicates that the addition of MOX to ALB alone or ALB plus DEC for LF therapy did not alter the drug exposure of co-administered drugs compared to IVM combinations. Clinical Trial Registration: NCT04410406, https://clinicaltrials.gov/. Author summary: Mass Drug administration (MDA) with multiple drugs is common for the treatment and control of lymphatic filariasis (LF). This study aimed to determine whether the incorporation of moxidectin (MOX) into MDA regimens for LF will alter the pharmacokinetics (PK) of co-administered drugs. In the context of a clinical trial, we investigated the PK and drug interactions of a combination of moxidectin (MOX) plus albendazole (ALB) with or without diethylcarbamazine (DEC) compared to ivermectin (IVM) plus ALB with or without DEC. The study was conducted in adult patients located in Côte d'Ivoire. The addition of MOX did not alter the exposure (AUC0-t) or maximum concentration (Cmax) of co-administered drugs. These results suggest the incorporation of MOX into current MDA LF treatment programs will not result in clinically significant alterations in drug exposure. [ABSTRACT FROM AUTHOR]

A sensitive and selective liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantitation of dual PI3K/BRD4 inhibitor SF2523 in mouse plasma. The analysis was performed on a UPLC system connected to a Shimadzu 8060 mass spectrometer by electrospray ionization in positive multiple reaction monitoring mode. Chromatographic separation was carried out on an ACE Excel C18 column with a gradient elution containing 0.1% formic acid and methanol as the mobile phase. The linearity was conducted in the concentration range 0.1–500 ng/ml for SF2523 in 100 μl of plasma. The inter‐ and intra‐batch precision (RSD) were both lower than 13.5%, with the accuracy (percentage bias) ranging from −10.03 to 11.56%. The validated method was successfully applied to plasma protein binding and in vitro metabolism studies. SF2523 was highly bound to mouse plasma proteins (>95% bound). Utilizing mouse S9 fractions, a total of seven phase I and II metabolites were identified with hydroxylation found to be the major metabolic pathway. Metabolite identification included analysis of retention behaviors, molecular weight changes and MS/MS fragment patterns of SF2523 and the metabolites. This newly developed and validated method allows the rapid and easy determination of the SF2523 concentration with high sensitivity in a low sample volume and can be applied to future pre‐clinical studies. [ABSTRACT FROM AUTHOR]

Hydroxychloroquine (HCQ) is Food and Drug Administration (FDA)‐approved for malaria, systemic and chronic discoid lupus erythematosus, and rheumatoid arthritis. Because HCQ has a proposed multimodal mechanism of action and a well‐established safety profile, it is often investigated as a repurposed therapeutic for a range of indications. There is a large degree of uncertainty in HCQ pharmacokinetic (PK) parameters which complicates dose selection when investigating its use in new disease states. Complications with HCQ dose selection emerged as multiple clinical trials investigated HCQ as a potential therapeutic in the early stages of the COVID‐19 pandemic. In addition to uncertainty in baseline HCQ PK parameters, it was not clear if disease‐related consequences of SARS‐CoV‐2 infection/COVID‐19 would be expected to impact the PK of HCQ and its primary metabolite desethylhydroxychloroquine (DHCQ). To address the question whether SARS‐CoV‐2 infection/COVID‐19 impacted HCQ and DHCQ PK, dried blood spot samples were collected from SARS‐CoV‐2(−)/(+) participants administered HCQ. When a previously published physiologically based pharmacokinetic (PBPK) model was used to fit the data, the variability in exposure of HCQ and DHCQ was not adequately captured and DHCQ concentrations were overestimated. Improvements to the previous PBPK model were made by incorporating the known range of blood to plasma concentration ratios (B/P) for each compound, adjusting HCQ and DHCQ distribution settings, and optimizing DHCQ clearance. The final PBPK model adequately captured the HCQ and DHCQ concentrations observed in SARS‐CoV‐2(−)/(+)participants, and incorporating COVID‐19‐associated changes in cytochrome P450 activity did not further improve model performance for the SARS‐CoV‐2(+) population. [ABSTRACT FROM AUTHOR]

Background: Ivermectin (IVM) is a broad–spectrum anthelmintic drug used to treat diseases caused by filarial worms, such as onchocerciasis and lymphatic filariasis (LF). IVM is part of a triple–drug therapy used by the Mass Drug Administration (MDA) as a preventive strategy to eradicate LF in sub–Saharan Africa. The drug shows high variability in drug exposure in previous pharmacokinetic studies. This study aims to build a population pharmacokinetic (PopPK) model to identify and quantify the possible sources of the variability of IVM exposure after a single–oral dose in LF–infected subjects and healthy individuals. Methodology / Principal findings: In this analysis, 724 samples were collected from treatment–naïve Wuchereria bancrofti–infected (n = 32) and uninfected (n = 24) adults living in Côte d'Ivoire who had received one dose of IVM as a part of triple–drug therapy. PopPK analysis was conducted using Phoenix NLME 8.3 software. The Monte Carlo simulation based on the final model was performed to simulate drug exposure among different dosing groups (200 μg/kg, 18 mg, and 36 mg). A two–compartment model with zero–order dose input into the absorption compartment with a lag time function followed by first–order absorption and linear elimination best described the IVM's pharmacokinetic (PK) parameters. The final model identifies that the PK parameters of IVM are not affected by LF infection. Sex was a significant covariate on the peripheral volume of distribution (Vp/F, 53% lower in men than in women). IVM drug exposure shows linear pharmacokinetic behavior among the simulated dosing groups with similar drug exposure based on sex. Conclusion/Significance: We have developed a PopPk model to describe and identify possible sources of the variability of IVM exposure. To our knowledge, this is the first PopPK study of IVM in patients with LF. Trial registration: NCT02845713; NCT03664063 Author summary: Ivermectin has been shown to be a valuable tool in mass drug administration campaigns against lymphatic filariasis. The drug shows high variability in drug exposure in previous pharmacokinetic studies. Variability in drug exposure can lead to either a higher exposure with the potential for toxic effects or a lower exposure that may result in treatment failure. Identifying and quantifying the source of high variability using non–linear mixed effects modeling is crucial for future dosing recommendations that seek to maximize therapeutic benefit while minimizing toxicity risks. In this study, the variability of IVM exposure after a single oral dose was investigated with a population pharmacokinetic model using data from Lymphatic filariasis infected subjects and healthy individuals. [ABSTRACT FROM AUTHOR]

Background: Prior work supports delayed gastric emptying in anorexia nervosa and bulimia nervosa (BN) but not binge-eating disorder, suggesting that neither low body weight nor binge eating fully accounts for slowed gastric motility. Specifying a link between delayed gastric emptying and self-induced vomiting could offer new insights into the pathophysiology of purging disorder (PD). Methods: Women (N = 95) recruited from the community meeting criteria for DSM-5 BN who purged (n = 26), BN with nonpurging compensatory behaviors (n = 18), PD (n = 25), or healthy control women (n = 26) completed assessments of gastric emptying, gut peptides, and subjective responses over the course of a standardized test meal under two conditions administered in a double-blind, crossover sequence: placebo and 10 mg of metoclopramide. Results: Delayed gastric emptying was associated with purging with no main or moderating effects of binge eating in the placebo condition. Medication eliminated group differences in gastric emptying but did not alter group differences in reported gastrointestinal distress. Exploratory analyses revealed that medication caused increased postprandial PYY release, which predicted elevated gastrointestinal distress. Conclusions: Delayed gastric emptying demonstrates a specific association with purging behaviors. However, correcting disruptions in gastric emptying may exacerbate disruptions in gut peptide responses specifically linked to the presence of purging after normal amounts of food. [ABSTRACT FROM AUTHOR]

Vitamin D plays a critical role in bone development and maintenance, and in other physiological functions. The quantitation of endogenous levels of individual vitamin D and its metabolites is crucial for assessing several disease state conditions. With cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leading to the coronavirus disease 2019 (COVID-19) pandemic, there are several studies that have associated lower levels of serum vitamin D with severity of infection in COVID-19 patients. In this context, we have developed and validated a robust LC–MS/MS method for simultaneous quantitation of vitamin D and its metabolites in human dried blood spot (DBS) obtained from participants tested for COVID-19. The chromatographic separation for vitamin D and metabolites was performed using an ACE Excel C18 PFP column protected with a C18 guard column (Phenomenex, Torrance, CA, USA). The mobile phase consisted of formic acid in water (0.1% v/v) as mobile phase A and formic acid in methanol (0.1% v/v) as mobile phase B, operated at a flow rate of 0.5 mL/min. Analysis was performed utilizing the LC–MS/MS technique. The method was sensitive with a limit of quantification of 0.78 ng/mL for all analytes, and had a large dynamic range (200 ng/mL) with a total run time of 11 min. The inter- and intraday accuracy and precision values met the acceptance criteria per the US Food and Drug Administration guidelines. Blood concentrations of 25(OH)D3, vitamin D3, 25(OH)D2, and vitamin D2 over a range of 2–195.6, 0.5–121.5, 0.6–54.9, and 0.5–23.9 ng/mL, respectively, were quantified in 909 DBS samples. In summary, our developed LC−MS/MS method may be used for quantification of vitamin D and its metabolites in DBS, and may be applied to investigations of the emerging role of these compounds in various physiological processes. [ABSTRACT FROM AUTHOR]

Extracorporeal membrane oxygenation (ECMO) is becoming increasingly utilized to support critically ill patients who experience life-threatening cardiac or pulmonary compromise. The provision of this intervention poses challenges related to its complications and the optimization of medication therapy. ECMO's mechanical circulatory support is facilitated via various devices and equipment that have been shown to sequester lipophilic- and protein-bound medications, including anti-infectives. Since infectious outcomes are dependent on achieving specific anti-infectives' pharmacodynamic targets, the understanding of these medications' pharmacokinetic parameters in the setting of ECMO is important to clinicians. This narrative, non-systematic review evaluated the findings of the most recent and robust pharmacokinetic analyses for commonly utilized anti-infectives in the setting of ECMO. The data from available literature indicates that anti-infective pharmacokinetic parameters are similar to those observed in other non-ECMO critically ill populations, but considerable variability in the findings was observed between patients, thus prompting further evaluation of therapeutic drug monitoring in this complex population. [ABSTRACT FROM AUTHOR]

Rab GTPases are critical regulators of protein trafficking in the cell. To ensure proper cellular localization and function, Rab proteins must undergo a posttranslational modification, termed geranylgeranylation. In the isoprenoid biosynthesis pathway, the enzyme geranylgeranyl diphosphate synthase (GGDPS) generates the 20‐carbon isoprenoid donor (geranylgeranyl pyrophosphate [GGPP]), which is utilized in the prenylation of Rab proteins. We have pursued the development of GGDPS inhibitors (GGSI) as a novel means to target Rab activity in cancer cells. Osteosarcoma (OS) and Ewing sarcoma (ES) are aggressive childhood bone cancers with stagnant survival statistics and limited treatment options. Here we show that GGSI treatment induces markers of the unfolded protein response (UPR) and triggers apoptotic cell death in a variety of OS and ES cell lines. Confirmation that these effects were secondary to cellular depletion of GGPP and disruption of Rab geranylgeranylation was confirmed via experiments using exogenous GGPP or specific geranylgeranyl transferase inhibitors. Furthermore, GGSI treatment disrupts cellular migration and invasion in vitro. Metabolomic profiles of OS and ES cell lines identify distinct changes in purine metabolism in GGSI‐treated cells. Lastly, we demonstrate that GGSI treatment slows tumor growth in a mouse model of ES. Collectively, these studies support further development of GGSIs as a novel treatment for OS and ES. [ABSTRACT FROM AUTHOR]

A selective, sensitive, and rapid liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitation of ICG in rat plasma. The chromatographic separation was achieved using an ACE excel C18 (3 µm, 50 × 3.0 mm) column, with a mobile phase composition of 0.1% formic acid and 0.1% formic acid in acetonitrile, using a gradient flow at a rate of 0.3 mL/min. The MS was operated at a unit resolution in the multiple reaction monitoring mode, using the precursor ion → product ion combinations of 753.3 → 330.2 m/z (ICG) and 747.45 → 717.50 (Cy7.5 amine) with a run time of 5 min. The assay was linear over a concentration range of 1–1000 ng/mL with a regression coefficient (r2) of 0.998 or better. The inter and intra-batch precision (% relative standard deviation, %RSD) was lower than 13.5%, with accuracy (%Bias) between −10.03% and 11.56%. The ICG was stable under laboratory storage and handling conditions. The validated method was successfully applied to preclinical pharmacokinetic (PK) studies of ICG at a dose of 0.39 mg/kg in rats. PK parameters suggested the highest plasma concentration within 2 min of intravenous dosing with restricted systemic distribution and rapid clearance. [ABSTRACT FROM AUTHOR]

Nucleic acid (NA) therapy has gained importance over the past decade due to its high degree of selectivity and minimal toxic effects over conventional drugs. Currently, intravenous (IV) or intramuscular (IM) formulations constitute majority of the marketed formulations containing nucleic acids. However, oral administration is traditionally preferred due to ease of administration as well as higher patient compliance. To leverage the benefits of oral delivery for NA therapy, the NA of interest must be delivered to the target site avoiding all degrading and inhibiting factors during its transition through the gastrointestinal tract. The oral route presents myriad of challenges to NA delivery, making formulation development challenging. Researchers in the last few decades have formulated various delivery systems to overcome such challenges and several reviews summarize and discuss these strategies in detail. However, there is a need to differentiate between the approaches based on target so that in future, delivery strategies can be developed according to the goal of the study and for efficient delivery to the desired site. The goal of this review is to summarize the mechanisms for target specific delivery, list and discuss the formulation strategies used for oral delivery of NA therapies and delineate the similarities and differences between local and systemic targeting oral delivery systems and current challenges. [ABSTRACT FROM AUTHOR]

Fatty acids are widespread naturally occurring compounds, and essential constituents for living organisms. Short chain fatty acids (SCFAs) appeared as physiologically relevant metabolites for their involvement with gut microbiota, immunology, obesity, and other pathophysiological functions. This has raised the demand for reliable analytical detection methods in a variety of biological matrices. Here, we describe an updated overview of sample pretreatment techniques and liquid chromatography-mass spectrometry (LC-MS)-based methods for quantitative analysis of SCFAs in blood, plasma, serum, urine, feces and bacterial cultures. The present review incorporates various procedures and their applications to help researchers in choosing crucial parameters, such as pretreatment for complex biological matrices, and variables for chromatographic separation and detection, to establish a simple, sensitive, and robust quantitative method to advance our understanding of the role of SCFAs in human health and disease as potential biomarkers. [ABSTRACT FROM AUTHOR]

A sensitive, specific and rapid liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method was developed and validated to quantify azithromycin concentrations in human plasma. Azithromycin (AZI) is the most common outpatient prescribed antibiotic in the US and clinical studies have demonstrated the efficacy and safety of AZI in many bacterial infections. To support a clinical study, we developed a high‐throughput LC–MS/MS method to process up to 250 samples per day to quantify AZI in human plasma. Samples were prepared by solid‐phase extraction. Separation was achieved with an ACE C18 column (2.1 × 100 mm, 1.7 μm) equipped with a C18 guard column. The mobile phase consisted of 0.1% formic acid and methanol–acetonitrile (1:1, v/v) at a flow rate of 0.25 ml/min. The ionization was optimized with positive electrospray source using multiple reaction monitoring transition, m/z 749.50 > 591.45 for AZI and m/z 754.50 > 596.45 for AZI‐d5. Extraction recoveries were approximately 90% for AZI. The assay was linear from 0.5 to 2,000 ng/ml and required only 100 μl of plasma with a total analysis time of 4.5 min. The method was successfully applied to pharmacokinetic studies of a weight‐based dosing protocol for AZI. [ABSTRACT FROM AUTHOR]

Enterococcus faecium has become an important drug-resistant nosocomial pathogen because of widespread antibiotic abuse. We developed short and chemically simple antimicrobial peptides (AMPs) with a selective amino acid composition, fixed charge, and hydrophobicity ratio based on the core antimicrobial motif of bovine lactoferrin (LfcinB6). Among these peptides, 5L and 6L (both 12 residues long) demonstrated a narrow spectrum and high antibacterial activity against drug-resistant E. faecium isolates with a minimal inhibitory concentration (MIC) that ranged from 4–16 µg/mL. At 32 µg/mL, peptides 5L and 6L inhibited E. faecium strain C68 biofilm formation by 90% and disrupted established biofilms by 75%. At 40 µg/mL, 5L reduced 1 × 107E. faecium persister cells by 3 logs within 120 min of exposure, whereas 6L eliminated all persister cells within 60 min. At 0.5× MIC, 5L and 6L significantly downregulated the expression of a crucial biofilm gene ace by 8 folds (p = 0.02) and 4 folds (p = 0.01), respectively. At 32 µg/mL, peptides 5L and 6L both depolarized the E. faecium membrane, increased fluidity, and eventually ruptured the membrane. Physiologically, 5L (at 8 µg/mL) altered the tricarboxylic acid cycle, glutathione, and purine metabolism. Interestingly, in an ex vivo model of porcine skin infection, compared to no treatment, 5L (at 10× MIC) effectively eliminated all 1 × 106 exponential (p = 0.0045) and persister E. faecium cells (p = 0.0002). In conclusion, the study outlines a roadmap for developing narrow-spectrum selective AMPs and presents peptide 5L as a potential therapeutic candidate to be explored against E. faecium. [ABSTRACT FROM AUTHOR]

Indole is an endogenous substance currently being evaluated as a biomarker for ulcerative colitis, irritable bowel syndrome, Crohn's disease and non-alcoholic fatty liver disease. A novel, selective, and sensitive method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed for quantitation of indole concentrations in mouse plasma and tissues. Samples were prepared by protein precipitation using ice-cold acetonitrile (ACN) followed by injecting the extracted analyte to LC-MS/MS system. Indole was separated using Synergi Fusion C18 (4 µm, 250 × 2.0 mm) column with mobile phase 0.1% aqueous formic acid (A) and methanol (B) using gradient flow with run time 12 min. The mass spectrometer was operated in atmospheric pressure chemical ionization (APCI) positive mode at unit resolution in multiple reaction monitoring (MRM) mode, using precursor ion > product ion combinations of 118.1 > 91.1 m/z for indole and 124.15 > 96.1 m/z for internal standard (IS) indole d7. The MS/MS response was linear over the range of indole concentrations (1–500 ng/mL). The validated method was applied for quantitation of indole concentrations range in mouse lungs (4.3–69.4 ng/g), serum (0.8–38.7 ng/mL) and cecum (1043.8–12,124.4 ng/g). This method would help investigate the role of indole as a biomarker and understand its implications in different disease states. [ABSTRACT FROM AUTHOR]

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, has been proposed to be a mediator of vascular dysfunction during hyperhomocysteinemia. Levels of ADMA are regulated by dimethylarginine dimethylaminohydrolase (DDAH). Using both in vitro and in vivo approaches, we tested the hypothesis that hyperhomocysteinemia causes downregulation of the two genes encoding DDAH (Ddah1 and Ddah2). In the MS-1 murine endothelial cell line, the addition of homocysteine decreased NO production but did not elevate ADMA or alter levels of Ddah1 or Ddah2 mRNA. Mice heterozygous for cystathionine [beta]-synthase (Cbs) and their wild-type littermates were fed either a control diet or a high-methionine/low-folate (HM/LF) diet to produce varying degrees of hyperhomocysteinemia. Maximal relaxation of the carotid artery to the endothelium-dependent dilator acetylcholine was decreased by -50% in [Cbs.sup.+/-] mice fed the HM/LF diet compared with [Cbs.sup.+/+] mice fed the control diet (P < 0.001). Compared with control mice, hyperhomocysteinemic mice had lower levels of Ddahl mRNA in the liver (P < 0.001) and lower levels of Ddah2 mRNA in the liver, lung, and kidney (P < 0.05). Downregulation of DDAH expression in hyperhomocysteinemic mice did not result in an increase in plasma ADMA, possibly due to a large decrease in hepatic methylation capacity (S-adenosyl-methionine-to-Sadenosylhomocysteine ratio). Our findings demonstrate that hyperhomocysteinemia causes tissue-specific decreases in DDAH expression without altering plasma ADMA levels in mice with endothelial dysfunction. asymmetric dimethylarginine; endothelium; homocysteine; vascular function

Genetic variation in drug metanolizing enzymes is an important contributor to interindividual variation in drug disposition and response and is associated with significant clinical consequences. Many commonly used drugs are [...]