Stability and Analytical Characterization of Voriconazole as Measured by Immunoassay.

BACKGROUND: Voriconazole is a broad-spectrum triazole antifungal agent recommended for invasive fungal diseases, including invasive aspergillosis. Therapeutic drug monitoring via voriconazole target trough concentration is important to ensure efficacy while preventing toxicity. Our aim was to determine the stability of voriconazole as adapted and measured by an immunoassay. METHODS: Plasma from patient samples (n = 45) evaluated by a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was compared against an ARK immunoassay method, adapted and optimized on the Abbott Alinity c analyzer. Stability of voriconazole and analytical performance of ARK immunoassay was assessed, including functional sensitivity, limit of blank (LoB), limit of detection (LoD), and limit of quantification (LoQ), linearity, and precision. RESULTS: ARK voriconazole immunoassay was highly correlated (Pearson R = 0.988) to the LC-MS/MS method, with an average bias of 0.09 mg/L (2%). CV at LoQ of 0.5 mg/L was 3.7% while the functional sensitivity was established at 0.05 mg/L. Overall imprecision with liquid quality control material obtained from ARK was 5.0%, 6.3%, and 5.9% at 1 mg/L, 5 mg/L, and 10 mg/L, respectively. Limit of blank and LoD were 0.02 mg/L and 0.05 mg/L, respectively. Voriconazole in lithium heparin plasma separator tube declines over time, with a decrease that is more evident near or above toxic concentrations. CONCLUSION: Voriconazole collected in gel separation tubes declines over time, possibly due to absorptive properties. Voriconazole measurements by immunoassay and LC-MS/MS demonstrated acceptable comparability with sufficient level of sensitivity and precision.

Arsenic Speciation and Metallomics Profiling of Human Toenails as a Biomarker to Assess Prostate Cancer Cases: Atlantic PATH Cohort Study.

Chronic exposure to inorganic arsenic and trace metals has been linked to prostate cancer, and altered arsenic methylation capacity may have an important role in arsenic carcinogenesis. Biomarkers may be able to elucidate this role. Our objectives were to characterize profiles of arsenic species and metallome in toenails and urine samples, compare profiles between prostate cancer cases and controls, and determine the discriminant ability of toenail and urine biomarkers. Toenail samples (n = 576), urine samples (n = 152), and questionnaire data were sourced from the Atlantic Partnership for Tomorrow's Health (PATH) cohort study. Healthy controls were matched to prostate cancer cases (3:1 ratio) on sex, age, smoking status, and the province of residence. Metallome profiles and proportions of arsenic species were measured in toenail and urine samples. Analysis of covariance (ANCOVA) was used to compare the mean percent monomethylarsonic acid (%MMA), dimethylarsonic acid (%DMA), inorganic arsenic (%iAs), primary methylation index (PMI, MMA/iAs), and secondary methylation index (SMI, DMA/MMA). Multivariate analysis of covariance (MANCOVA) was used to compare selected metal concentrations. Mean %MMA was significantly lower and SMI was significantly higher in toenails from prostate cancer cases compared to controls in unadjusted and adjusted models. Proportions of arsenic species were correlated with total arsenic in toenails. Arsenic speciation in urine was not different between cases and controls, nor were metallome profiles in toenails and urine. Our results indicate that toenails are a viable biomarker for altered arsenic speciation in prostate cancer cases and may have greater utility than urine in this context.

A Systematic Review and Meta-Analysis of Lipid Signatures in Post-traumatic Stress Disorder.

Background: Research assessing lipid levels in individuals diagnosed with post-traumatic stress disorder (PTSD) has yielded mixed results. This study aimed to employ meta-analytic techniques to characterize the relationship between the levels of lipid profiles and PTSD. Methods: We performed meta-analyses of studies comparing profiles and levels of lipids between PTSD patients and healthy individuals by searching Embase, Ovid Medline, Scopus, PsycINFO, and Cochrane databases for the studies until March 2021. Meta-analyses were performed using random-effects models with the restricted maximum-likelihood estimator to synthesize the effect size assessed by standardized mean difference (SMD) across studies. Findings: A total of 8,657 abstracts were identified, and 17 studies were included. Levels of total cholesterol (TC) (SMD = 0.57 95% CI, 0.27-0.87, p = 0.003), low-density lipoprotein (LDL) (SMD = 0.48, 95% CI, 0.19-0.76, p = 0.004), and triglyceride (TG) (SMD = 0.46, 95% CI, 0.22-0.70, p = 0.001) were found to be higher, while levels of high-density lipoprotein (HDL) (SMD = -0.47, -0.88 to -0.07, p = 0.026) were found to be lower in PTSD patients compared to healthy controls. Subgroup analysis showed that TG levels were higher in PTSD patients who were on or off of psychotropic medications, both < 40 and ≥ 40 years of age, and having body mass index of < 30 and ≥ 30 compared to healthy controls. Interpretation: This work suggested dysregulation of lipids in PTSD that may serve as biomarker to predict the risk. The study will be useful for physicians considering lipid profiles in PTSD patients to reduce cardiovascular morbidity and mortality.

A systematic review and meta-analysis of lipid metabolomic signatures of Major Depressive Disorder.

The aim of this meta-analysis was to provide a comprehensive synthesis of the evidence examining biomarker signatures in MDD patients including lipids, lipid regulatory proteins (LRP), and polyunsaturated fatty acid (PUFA) as compared to healthy individuals. We performed meta-analyses and meta-regression of the studies comparing lipid, LRP, and PUFA levels between MDD patients and healthy individuals by searching Embase, Ovid Medline, Scopus, PsycINFO, PubMed, and Cochrane databases. Search was performed in these databases up to September 2019 and 29 studies were included. Levels of lipid parameter triglyceride (TG) (SMD 0.55, 95% CI 0.30-0.80, p < 0.0001) were higher while total cholesterol (TC) (SMD = -0.46, 95%CI -0.93 to -0.001, p = 0.04) and very low-density lipoprotein (VLDL) (SMD = -0.46, 95%CI -0.71 to -0.20, p = 0.02) were lower in MDD patients than controls. Subgroup analysis for age showed that the levels of high-density lipoprotein (HDL) were lower in ≥40-year age group (SMD = -0.38, 95%CI -0.70 to -0.06, p = 0.01) and levels of TC was lower in MDD patients in studies from Asian countries (SMD = -0.74, 95%CI -1.37 to -0.10, p = 0.02). TG levels were found to be high all subgroups in MDD patients than controls. A negative association between TC levels and use of lipid lowering medications and a positive association between smoking and LDL levels was found using meta-regression analysis. This study will be useful for physicians when considering the assessment of lipidand LRP profiles in MDD patients to reduce the cardiovascular morbidity and mortality.

Upregulation of antioxidant thioredoxin by antidepressants fluoxetine and venlafaxine.

RATIONALE: Selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) are the most commonly used drugs for the treatment of depression. Studies have shown that chronic treatment with SSRIs and SNRIs produces a protective effect against oxidative stress. Thioredoxin (Trx) is an antioxidant protein that reverses protein cysteine oxidation and facilitates scavenging reactive oxygen species. OBJECTIVES: The current study is to determine whether the SSRI fluoxetine and the SNRI venlafaxine regulate Trx and protect neuronal cells against protein cysteine oxidation. METHODS: HT22 mouse hippocampal cells were incubated with fluoxetine or venlafaxine for 5 days. Protein levels of Trx, Trx reductase (TrxR), and Trx-interacting protein (Txnip) were measured by immunoblotting analysis. Trx and TrxR activities were analyzed by spectrophotometric method. Protein cysteine sulfenylation was measured by dimedone-conjugation assay, while nitrosylation was measured by biotin-switch assay. RESULTS: We found that treatment with fluoxetine or venlafaxine for 5 days increased Trx and TrxR protein levels but produced no effect on Txnip protein levels. These treatments also increased Trx and TrxR activities. Although treatment with fluoxetine or venlafaxine alone had no effect on sulfenylated and nitrosylated protein levels, both drugs inhibited H2O2-increased sulfenylated protein levels and nitric oxide donor nitrosoglutathione-increased nitrosylated protein levels. Stress increases risk of depression. We also found that treatment with fluoxetine or venlafaxine for 5 days inhibited stress hormone corticosterone-increased total sulfenylated and nitrosylated protein levels. CONCLUSIONS: Our findings suggest that chronic treatment with antidepressants may upregulate Trx, subsequently inhibiting protein sulfenylation and nitrosylation, which may contribute to the protective effect of antidepressants against oxidative stress. Our findings also indicate that thioredoxin is a potential therapeutic target for the treatment of depression.

Txnip mediates glucocorticoid-activated NLRP3 inflammatory signaling in mouse microglia.

Many studies indicate that chronic stress and excessive stress hormone can cause an inflammatory response. Thioredoxin-interacting protein (Txnip) as an endogenous thioredoxin inhibitor suppresses thioredoxin-produced antioxidant effects. Txnip was also found to interact with nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), which activates NLRP3 inflammasome and promotes inflammatory processes. Recently our laboratory found that chronic stress can increase Txnip protein levels in mouse brain, indicating that Txnip may mediate chronic stress-induced inflammation. Microglia play an important role in neuroinflammation. The purpose of this study is to investigate the effect of chronic stress hormone treatment on Txnip and NLRP3 inflammasome signaling in cultured microglia cells. Our result showed that chronic treatment with stress hormone corticosterone increased Txnip protein levels and Txnip-NLRP3 binding in N9 mouse microglia, in primary cultured mouse microglia and in mouse brain. Our result also showed that chronic corticosterone treatment increased procaspase-1 cleavage, caspase-1 activity and interleukin-1ß release in N9 microglia. Using CRISPR/Cas9 method we found that knocking out Txnip inhibited corticosterone-increased caspase-1 activity and interleukin-1ß release. Our results suggest that chronic corticosterone treatment upregulates Txnip and increases Txnip-NLRP3 binding, which activates NLRP3 inflammasome, resulting in activation of caspase-1 and in further releasing of interleukin-1ß. It is therefore likely that Txnip-activated NLRP3 inflammasome contributes to corticosterone-caused neuroinflammation.

Upregulation of Thioredoxin-Interacting Protein in Brain of Amyloid-ß Protein Precursor/Presenilin 1 Transgenic Mice and Amyloid-ß Treated Neuronal Cells.

Oxidative stress has been hypothesized to play a role in the pathophysiology of Alzheimer's disease (AD). Previously, we found that total nitrosylated protein levels were increased in the brain of amyloid-ß protein precursor (AßPP) and presenilin 1 (PS1) double transgenic mice, an animal model for AD, suggesting that cysteine oxidative protein modification may contribute to this disease. Thioredoxin (Trx) is a major oxidoreductase that can reverse cysteine oxidative modifications such as sulfenylation and nitrosylation, and inhibit oxidative stress. Thioredoxin-interacting protein (Txnip) is an endogenous Trx inhibitor. To understand the involvement of Trx and Txnip in AD development, we investigated Trx and Txnip in the brain of AßPP/PS1 mice. Using immunoblotting analysis, we found that although Trx protein levels were not changed, Txnip protein levels were significantly increased in hippocampus and frontal cortex of 9- and 12-month-old AßPP/PS1 mice when compared to wild-type mice. Txnip protein levels were also increased by amyloid-ß treatment in primary cultured mouse cerebral cortical neurons and HT22 mouse hippocampal cells. Using biotin switch and dimedone conjugation methods, we found that amyloid-ß treatment increased protein nitrosylation and sulfenylation in HT22 cells. We also found that downregulation of Txnip, using CRISPR/Cas9 method in HT22 cells, attenuated amyloid-ß-induced protein nitrosylation and sulfenylation. Our findings suggest that amyloid-ß may increase Txnip levels, subsequently inhibiting Trx reducing capability and enhancing protein cysteine oxidative modification. Our findings also indicate that Txnip may be a potential target for the treatment of AD.

Glucocorticoid Upregulates Thioredoxin-interacting Protein in Cultured Neuronal Cells.

Previous studies have shown that chronic stress and chronic stress hormone treatment induce oxidative damage in rodents. Thioredoxin (Trx) is a small redox protein that plays an important role in regulation of oxidative protein cysteine modification. A Trx reduced state is maintained by thioredoxin reductase (TrxR), and the thioredoxin-interacting protein (Txnip) is an endogenous inhibitor of Trx. The purpose of this study was to investigate the effects of chronic treatment with stress hormone corticosterone on Trx, TrxR and Txnip in cultured neuronal cells. Using immunoblotting analysis we found that although chronic corticosterone treatment had no effect on Trx and TrxR protein levels, this treatment significantly increased Txnip protein levels. Using immunocytochemistry we also found that chronic corticosterone treatment increased Txnip in both nucleus and cytosol, while glucocorticoid receptor inhibitor RU486 can block corticosterone-increased Txnip protein levels. Using biotin switch, dimedone conjugation and CRISPR/Cas9 methods we found that chronic corticosterone treatment increased protein nitrosylation and sulfenylation, while knocking out Txnip blocked corticosterone-induced protein nitrosylation and sulfenylation. Since Trx can reduce cysteine oxidative protein modification such as nitrosylation and sulfenylation, our findings suggest that chronic corticosterone treatment may upregulate Txnip by targeting glucocorticoid receptor, subsequently inhibiting Trx activity and enhancing oxidative protein cysteine modification, which contributes to corticosterone-caused oxidative damage.

Chronic treatment with mood stabilizer lithium inhibits amphetamine-induced risk-taking manic-like behaviors.

A lack of behavioral tests and animal models for manic-depressive bipolar disorder is recognized as an important factor limiting development of novel pharmaceutical treatments for the disorder. Repeated amphetamine-induced hyperactivity is a commonly used animal model for mania. However, hyperactivity represents only one facet of mania and is also seen in other disorders. Increased engagement in risk taking behavior is frequently observed in the manic phase of bipolar disorder. In the present study, we analyzed the effect of the most commonly used mood stabilizer lithium on repeated amphetamine treatment-induced risk-taking behaviors in rats using elevated plus maze and wire-beam bridge tests. We found that repeated amphetamine treatment not only increased locomotor activity, but also increased risk taking behaviors in rats, and further that chronic lithium treatment inhibited the amphetamine-increased risk taking behavior. Our studies suggest that these tests may be useful tools to analyze the pharmacological validity of new and improved anti-manic drugs in animals.

Anti-oxidant studies and anti-microbial effect of Origanum vulgare Linn in combination with standard antibiotics.

BACKGROUND: Origanum is one of the over 200 genera in the Lamiaceae (mint family), and this genus includes culinary, fragrant, and medicinal properties. The plant is reported to contain anti-microbial properties, but it lacks combination studies with that of synthetic antibiotics. AIM: To investigate the anti-oxidant and anti-microbial interaction studies of Origanum vulgare with standard drugs against Bacillus species of bacteria and Aspergillus niger. MATERIALS AND METHODS: The anti-oxidant properties of phenolic, non-phenolic fractions of chloroform extract and volatile oil were evaluated by free radical-scavenging, hydrogen peroxide radical-scavenging assay, reducing power, and metal chelating assays. RESULTS: The minimum inhibitory concentration and fractional inhibitory concentration index were determined which demonstrates the behavior of volatile oil, phenolic, and non-phenolic fractions of volatile oil with that of ciprofloxacin and fluconazole. The IC50 value for volatile oil was found to be 15, 30, and 30 µg/ml and that of phenolic fraction was 60, 120, and 120 µg/ml for free radical-scavenging, hydrogen peroxide-scavenging, and metal chelating assays respectively. Non-phenolic fraction was found to act antagonistically along with ciprofloxacin against B. cereus and B. subtilis, while the phenolic fraction exhibited indifferent activity along with ciprofloxacin against both the bacterial strains. CONCLUSION: This combination of drug therapy will not only prove effective in antibiotic resistance, but these natural constituents will also help in preventing body from harmful radicals which lead to fatal diseases.

Combination Studies of Oreganum Vulgare Extract Fractions and Volatile Oil along with Ciprofloxacin and Fluconazole against Common Fish Pathogens.

PURPOSE: The study is aimed at finding new antibiotic therapy for aquaculture due to potential of bacteria to develop resistance to the existing therapies. Use of large quantities of synthetic antibiotics in aquaculture thus has the potential to be detrimental to fish health, to the environment and wildlife and to human health. METHODS: Antimicrobial potential of volatile oil and fractions of chloroform extract of Oreganum vulgare was evaluated alone and in the presence of standard antimicrobials against common fish pathogens by disc-diffusion, agar well assay and two fold microdilution method by nanodrop spectrophotometric method. RESULTS: The best results were represented by volatile oil followed by phenolic fraction by disc-diffusion, agar well and microdilution assays (Minimum inhibitory concentration). By the interaction studies, it was observed that the volatile oil and phenolic fraction were able to inhibit the pathogens at very low concentration compared to standard drugs. The fractional inhibitory concentration index (FICI) was calculated and volatile oil and phenolic fractions were found to be synergistic against Pseudomonas fluorescens and Candida albicans. CONCLUSION: The experimental data suggests the use of volatile oil and phenolic fraction in combination with standard antimicrobials to maintain healthy aquaculture with lesser adverse effects as compared to synthetic antibiotic therapy.

Oreganum vulgare Linn. leaf: An Extensive Pharmacognostical and Phytochemical Quality Assessment.

PURPOSE: Standardization and detailed pharmacognostical studies of Oreganum vulgare Linn. leaf for authentication and commercial utilization. METHODS: Oreganum vulgare Linn. leaf was with standardization according to standard procedures described in WHO, 2011 and I.P. 1996. RESULTS: The physicochemical parameters total ash, acid insoluble ash, water soluble ash and sulphated ash were found to be 11.5%, 11%, 5, 10.5% w/w respectively. Foaming index was found be <100. The trace elements were found to be copper, lead, cadmium, zinc, cobalt, manganese, nickel and copper in ethanol extract and phytochemical screening of aqueous and ethanol extract showed the presence of carbohydrates, flavonoids, anthocyanins, phenolic compounds etc. CONCLUSION: The standardization parameters viz. physico-chemical parameters, macroscopy, microscopy, taxonomy, anatomy and preliminary phytochemical screening, microbial and aflatoxin count, HPTLC profile is being reported to help in authentication and development of monograph of this plant.

Antibacterial activities of Origanum vulgare alone and in combination with different antimicrobials against clinical isolates of Salmonella typhi.

BACKGROUND: Typhoid fever continues to remain a major public health problem especially in the areas where there is problem of sanitation and hygiene. The emergence of multidrug resistance of Salmonella typhi, the bacteria responsible for Typhoid to ampicillin, chloramphenicol, and cotrimoxazole has further complicated the treatment and management of enteric fever. One strategy for the treatment of the multidrug resistant bacteria is to use herbs in combination with conventional drugs. The present study was done to find out the interaction effect of phenolic, nonphenolic fractions, and volatile oil of Origanum vulgare with ciprofloxacin. MATERIALS AND METHODS: The minimum inhibitory concentration (MIC) by microdilution method for individual phytoconstituents and in combination with ciprofloxacin was compared for clinically isolated bacteria from patients infected with S. typhi. Fractional inhibitory concentration (FIC) and Fractional inhibitory concentration index (FICI) were also calculated. RESULTS: The MIC declined to a significant level indicating synergistic relationship between ciprofloxacin and phenolic, nonphenolic fractions and volatile oil in vitro. The FICI exhibits synergistic effect for all the three samples while indifferent and antagonistic for samples and for phenolic and nonphenolic fractions. CONCLUSIONS: Present study shows that not only the formulation using O. vulgare and ciprofloxacin can overcome multidrug resistance but also will reduce the toxic effects of ciprofloxacin.