Non-additivity of the functional properties of individual P450 species and its manifestation in the effects of alcohol consumption on the metabolism of ketamine and amitriptyline.

To explore functional interconnections between multiple P450 enzymes and their manifestation in alcohol-induced changes in drug metabolism, we implemented a high-throughput study of correlations between the composition of the P450 pool and the substrate saturation profiles (SSP) of amitriptyline and ketamine demethylation in a series of 23 individual human liver microsomes preparations from donors with a known history of alcohol consumption. The SSPs were approximated with linear combinations of three Michaelis-Menten equations with globally optimized KM (substrate affinity) values. This analysis revealed a strong correlation between the rate of ketamine metabolism and alcohol exposure. For both substrates, alcohol consumption caused a significant increase in the role of the low-affinity enzymes. The amplitudes of the kinetic components and the total rate were further analyzed for correlations with the abundance of 11 major P450 enzymes assessed by global proteomics. The maximal rate of metabolism of both substrates correlated with the abundance of CYP3A4, their predicted principal metabolizer. However, except for CYP2D6 and CYP2E1, responsible for the low-affinity metabolism of ketamine and amitriptyline, respectively, none of the other potent metabolizers of the drugs revealed a positive correlation. Instead, in the case of ketamine, we observed negative correlations with the abundances of CYP1A2, CYP2C9, and CYP3A5. For amitriptyline, the data suggest inhibitory effects of CYP1A2 and CYP2A6. Our results demonstrate the importance of functional interactions between multiple P450 species and their decisive role in the effects of alcohol exposure on drug metabolism.

Comparison of Relative Activity versus Relative Expression Factors (RAF versus REF) in Predicting Glucuronidation Mediated Drug Clearance Using Recombinant UGTs.

PURPOSE: Predicting the quantitative fraction of glucuronidation (fgluc) by individual UDP-glucuronosyltransferase enzymes (UGTs) is challenging due to the lack of selective inhibitors and inconsistent activity of recombinant UGT systems (rUGTs). Our study compares the relative expression versus activity factors (REF versus RAF) to predict fgluc based on rUGT data to human liver and intestinal microsomes (HLM and HIM). METHODS: REF scalars were derived from a previous in-house proteomics study for eleven UGT enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17), whereas RAF was calculated by measuring activities in rUGTs to microsomes of selective UGT probe substrates. Protein-normalized activity factor (pnAF) values were generated after correcting activity of individual UGTs to their corresponding protein abundance. The utility of REF and RAF in predicting fgluc was assessed for three UGT substrates-diclofenac, vorinostat, and raltegravir. RESULTS: The REF values ranged from 0.02 to 1.75, RAF based on activity obtained in rUGTs to HLM/HIM were from 0.1 to 274. pnAF values were ~ 5 to 80-fold, except for UGT2B4 and UGT2B15, where pnAF was ~ 180 and > 1000, respectively. The results revealed confounding effect of differential specific activities (per pmol) of rUGTs in fgluc prediction. CONCLUSION: The data suggest that the activity of UGT enzymes was significantly lower when compared to their activity in microsomes at the same absolute protein amount (pmol). Collectively, results of this study demonstrate poor and variable specific activity of different rUGTs (per pmol protein), as determined by pnAF values, which should be considered in fgluc scaling.

Identification of the Biotransformation Pathways of a Potential Oral Male Contraceptive, 11ß-Methyl-19-Nortestosterone (11ß-MNT) and Its Prodrugs: An In Vitro Study Highlights the Contribution of Polymorphic Intestinal UGT2B17.

11ß-Methyl-19-nortestosterone dodecylcarbonate (11ß-MNTDC) is a prodrug of 11ß-MNT and is being considered as a promising male oral contraceptive candidate in clinical development. However, the oral administration of 11ß-MNTDC exhibits an ~200-fold lower serum concentration of 11ß-MNT compared to 11ß-MNTDC, resulting in the poor bioavailability of 11ß-MNT. To elucidate the role of the first-pass metabolism of 11ß-MNT in its poor bioavailability, we determined the biotransformation products of 11ß-MNT and its prodrugs in human in vitro models. 11ß-MNT and its two prodrugs 11ß-MNTDC and 11ß-MNT undecanoate (11ß-MNTU) were incubated in cryopreserved human hepatocytes (HHs) and subjected to liquid chromatography-high resolution tandem mass spectrometry analysis, which identified ten 11ß-MNT biotransformation products with dehydrogenated and glucuronidation (11ß-MNTG) metabolites being the major metabolites. However, 11ß-MNTG formation is highly variable and prevalent in human intestinal S9 fractions. A reaction phenotyping study of 11ß-MNT using thirteen recombinant UDP-glucuronosyltransferase (UGT) enzymes confirmed the major role of UGT2B17 in 11ß-MNTG formation. This was further supported by a strong correlation (R2 > 0.78) between 11ß-MNTG and UGT2B17 abundance in human intestinal microsomes, human liver microsomes, and HH systems. These results suggest that 11ß-MNT and its prodrugs are rapidly metabolized to 11ß-MNTG by the highly polymorphic intestinal UGT2B17, which may explain the poor and variable bioavailability of the drug.

ChaQra: a cellular unit of the Indian quantum network.

Major research interests on quantum key distribution (QKD) are primarily focused on increasing 1. Point-to-point transmission distance (1000 km). 2. Secure key rate (Mbps). 3. Security of quantum layer (device-independence). It is great to push the boundaries in these fronts but these isolated approaches are neither scalable nor cost-effective due to requirements of specialised hardware and different infrastructure. Current and future QKD network requires addressing different set of challenges apart from distance, key rate and quantum security. In this regard, we present ChaQra-a sub quantum network with core features as 1. Crypto agility (integration in the already deployed telecommunication fibres). 2. Software defined networking (SDN paradigm for routing different nodes). 3. reliability (addressing denial-of-service with hybrid quantum safe cryptography). 4. upgradability (modules upgradation based on scientific and technological advancements). 5. Beyond QKD (using QKD network for distributed computing, multi-party computation etc). Our results demonstrate a clear path to create and accelerate quantum secure Indian subcontinent under national quantum mission.

Ontogeny of Human Liver Aldehyde Oxidase: Developmental Changes and Implications for Drug Metabolism.

Despite the increasing importance of aldehyde oxidase (AO) in the drug metabolism of clinical candidates, ontogeny data for AO are limited. The objective of our study was to characterize the age-dependent AO content and activity in the human liver cytosolic fraction (HLC) and human hepatocytes (HH). HLC (n = 121 donors) and HH (n = 50 donors) were analyzed for (1) AO protein content by quantitative proteomics and (2) enzyme activity using carbazeran as a probe substrate. AO activity showed high technical variability and poor correlation with the content in HLC samples, whereas hepatocyte samples showed a strong correlation between the content and activity. Similarly, AO content and activity showed no significant age-dependent differences in HLC samples, whereas the average AO content and activity in hepatocytes increased significantly (∼20-40-fold) from the neonatal levels (0-28 days). Based on the hepatocyte data, the age at which 50% of the adult AO content is reached (age50) was 3.15 years (0.32-13.97 years, 95% CI). Metabolite profiling of carbazeran revealed age-dependent metabolic switching and the role of non-AO mechanisms (glucuronidation and desmethylation) in carbazeran elimination. The content-activity correlation in hepatocytes improved significantly (R2 = 0.95; p < 0.0001) in samples showing <10% contribution of glucuronidation toward the overall metabolism, confirming that AO-mediated oxidation and glucuronidation are the key routes of carbazeran metabolism. Considering the confounding effect of glucuronidation on AO activity, AO content-based ontogeny data are a more direct reflection of developmental changes in protein expression. The comprehensive ontogeny data of AO in HH samples are more reliable than HLC data, which are important for developing robust physiologically based pharmacokinetic models for predicting AO-mediated metabolism in children.

Effects of alcohol consumption and tobacco smoking on the composition of the ensemble of drug metabolizing enzymes and transporters in human liver.

We examined the effect of alcohol consumption and smoking on the abundance of drug-metabolizing enzymes and transporters (DMET) in human liver microsomes (HLM) isolated from liver tissues of 94 donors. Global proteomics analysis was performed and DMET protein levels were analyzed in relation to alcohol consumption levels, smoking history, and sex using non-parametric tests (p-value ≤ 0.05; cutoff of 1.25-fold change, FC). The examination of the alcohol-induced changes was further enforced by correlational analysis, where we used arbitrary alcohol consumption grade (ACG) scaling from 0 to 4 to establish a set of protein markers. We elaborated a provisional index of alcohol exposure (PIAE) based on a combination of relative abundances of four proteins (ER chaperone HSPA5, protein disulfide isomerases PDIA3 and P4HB, and cocaine esterase CES2) best correlating with ACG. The PIAE index was then used to find its correlations with the abundances of DMET proteins. Our results demonstrate considerable alcohol-induced changes in composition of the pool of cytochrome P450 enzymes in HLM. We observed significantly increased abundances of CYP2E1, CYP2B6, CYP2J2, and NADPH-cytochrome P450 reductase. In contrast, CYP1A2, CYP2C8, CYP2C9, CYP4A11, and cytochrome b5 protein levels were downregulated. Significant alteration in abundances of UDP-glucuronosyltransferase (UGT) were also detected, comprising of elevated UGT1A6, UGT1A9, and UGT2A1, and reduced UGT1A3, UGT1A4, UGT2B7, UGT2B10, and UGT2B15 levels. Important alcohol-induced changes were also observed in the expression of non-CYP and non-UGT DMET. Additionally, tobacco smoke was associated with elevated CYP1A2, UGT1A6, UGT2A1, and UGT2B4 and decreased FMO3, FMO4, and FMO5 levels.

Differential tissue abundance of membrane-bound drug metabolizing enzymes and transporter proteins by global proteomics.

Protein abundance data of drug-metabolizing enzymes and transporters (DMETs) are critical for scaling in vitro and animal data to humans for accurate prediction and interpretation of drug clearance and toxicity. Targeted DMET proteomics which relies on synthetic stable isotope-labeled surrogate peptides as calibrators, is routinely used for the quantification of selected proteins; however, the technique is limited to the quantification of a small number of proteins. Although the global proteomics-based total protein approach (TPA) is emerging as a better alternative for large-scale protein quantification, the conventional TPA doesn't consider differential sequence coverage by identifying unique peptides across proteins. Here, we optimized the TPA approach by correcting protein abundance data by the sequence coverage (SC-TPA), which was applied to quantify 54 DMETs for characterization of i) differential tissue DMET abundance in the human liver, kidney, and intestine, and ii) interindividual variability of DMET proteins in individual intestinal samples (n=13). UGT2B7, MGST1, MGST2, MGST3, CES2, and MRP2 were expressed in all three tissues, whereas, as expected CYP3A4, CYP3A5, CYP2C9, CYP4F2, UGT1A1, UGT2B17, CES1, FMO5, MRP3, and P-gp were present in the liver and intestine. The top three DMET proteins in individual tissues were: CES1>CYP2E1>UGT2B7 (liver), CES2>UGT2B17>CYP3A4 (intestine), and MGST1>UGT1A6>MGST2 (kidney). CYP3A4, CYP3A5, UGT2B17, CES2, and MGST2 showed high interindividual variability in the intestine. These data are relevant for enhancing in vitro to in vivo extrapolation (IVIVE) of drug absorption and disposition and can be used to enhance the accuracy of physiologically based pharmacokinetic (PBPK) prediction of systemic and tissue concentration of drugs. Significance Statement We quantified the abundance and compositions of drug-metabolizing enzymes and transporters (DMETs) in pooled human liver, intestine, and kidney microsomes using an optimized sequence coverage-informed total protein approach. The quantification of DMETs revealed quantitative differences in their levels in the liver, intestine, and kidney. Further, the analysis of individual intestine samples confirmed high variability in the levels of CYP3A4, CYP3A5, UGT2B17, CES2, and MGST2. These data are applicable for the prediction of first-pass metabolism and tissue-specific drug clearance.

Effect of Cimetidine on Metformin Pharmacokinetics and Endogenous Metabolite Levels in Rats.

Tubular secretion is a primary mechanism along with glomerular filtration for renal elimination of drugs and toxicants into urine. Organic cation transporters (OCTs) and multidrug and toxic extrusion (MATE) transporters facilitate the active secretion of cationic substrates, including drugs such as metformin and endogenous cations. We hypothesized that administration of cimetidine, an Oct/Mate inhibitor, will result in increased plasma levels and decreased renal clearance of metformin and endogenous Oct/Mate substrates in rats. A paired rat pharmacokinetic study was carried out in which metformin (5 mg/kg, intravenous) was administered as an exogenous substrate of Oct/Mate transporters to six Sprague-Dawley rats with and without cimetidine (100 mg/kg, intraperitoneal). When co-administered with cimetidine, metformin area under the curve increased significantly by 3.2-fold, and its renal clearance reduced significantly by 73%. Untargeted metabolomics was performed to investigate the effect of cimetidine on endogenous metabolome in the blood and urine samples. Over 8,000 features (metabolites) were detected in the blood, which were shortlisted using optimized criteria, i.e., a significant increase (P value < 0.05) in metabolite peak intensity in the cimetidine-treated group, reproducible retention time, and quality of chromatogram peak. The metabolite hits were classified into three groups that can potentially distinguish inhibition of i) extra-renal uptake transport or catabolism, ii) renal Octs, and iii) renal efflux transporters or metabolite formation. The metabolomics approach identified novel putative endogenous substrates of cationic transporters that could be tested as potential biomarkers to predict Oct/Mate transporter mediated drug-drug interactions in the preclinical stages. SIGNIFICANCE STATEMENT: Endogenous substrates of renal transporters in animal models could be used as potential biomarkers to predict renal drug-drug interactions in early drug development. Here we demonstrated that cimetidine, an inhibitor of organic cation transporters (Oct/Mate), could alter the pharmacokinetics of metformin and endogenous cationic substrates in rats. Several putative endogenous metabolites of Oct/Mate transporters were identified using metabolomics approach, which could be tested as potential transporter biomarkers to predict renal drug-drug interaction of Oct/Mate substrates.

Extraction, separation and purification of fatty acid ethyl esters for biodiesel and DHA from Thraustochytrid biomass.

A novel approach for in situ transesterification, extraction, separation, and purification of fatty acid ethyl esters (FAEE) for biodiesel and docosahexaenoic acid (DHA) from Thraustochytrid biomass has been developed. The downstream processing of Thraustochytrids oil necessitates optimization, considering the higher content of polyunsaturated fatty acids (PUFA). While two-step methods are commonly employed for extracting and transesterifying oil from oleaginous microbes, this may result in oxidation/epoxidation of omega-3 oil due to prolonged exposure to heat and oxygen. To address this issue, a rapid single-step method was devised for in situ transesterification of Thraustochytrid oil. Through further process optimization, a 50% reduction in solvent requirement was achieved without significantly impacting fatty acid recovery or composition. Scale-up studies in a 4 L reactor demonstrated complete FAEE recovery (99.98% of total oil) from biomass, concurrently enhancing DHA yield from 16% to nearly 22%. The decolorization of FAEE oil with fuller's earth effectively removed impurities such as pigments, secondary metabolites, and waxes, resulting in a clear, shiny appearance. High-performance liquid chromatography (HPLC) analysis indicated that the eluted DHA was over 94.5% pure, as corroborated by GC-FID analysis.

Characterization of Gla proteoforms and non-Gla peptides of gamma carboxylated proteins: Application to quantification of prothrombin proteoforms in human plasma.

Gamma (γ) carboxylation is an essential post-translational modification in vitamin K-dependent proteins (VKDPs), involved in maintaining critical biological homeostasis. Alterations in the abundance or activity of these proteins have pharmacological and pathological consequences. Importantly, low levels of fully γ-carboxylated clotting factors increase plasma des-γ-carboxy precursors resulting in little or no biological activity. Therefore, it is important to characterize the levels of γ-carboxylation that reflect the active state of these proteins. The conventional enzyme-linked immunosorbent assay for protein induced by vitamin K absence or antagonist II (PIVKA-II) quantification uses an antibody that is not applicable to distinguish different γ-carboxylation states. Liquid chromatography-mass spectrometry (LC-MS) approaches have been utilized to distinguish different γ-carboxylated proteoforms, however, these attempts were impeded by poor sensitivity due to spontaneous neutral loss of CO2 and simultaneous cleavage of the backbone bond in the collision cell. In this study, we utilized an alkaline mobile phase in combination with polarity switching (positive and negative ionization modes) to simultaneously identify and quantify γ-carboxylated VKDPs. The method was applied to compare Gla proteomics of prothrombin (FII) in 10 µL plasma samples of healthy control and warfarin-treated adults. We also identified surrogate non-Gla peptides for seven other VKDPs to quantify total (active plus inactive) protein levels. The total protein approach (TPA) was used to quantify absolute levels of the VKDPs in human plasma.

Effect of probenecid on blood levels and renal elimination of furosemide and endogenous compounds in rats: Discovery of putative organic anion transporter biomarkers.

Transporter-mediated drug-drug interactions (DDIs) are assessed using probe drugs and in vitro and in vivo models during drug development. The utility of endogenous metabolites as transporter biomarkers is emerging for prediction of DDIs during early phases of clinical trials. Endogenous metabolites such as pyridoxic acid and kynurenic acid have shown potential to predict DDIs mediated by organic anion transporters (OAT1 and OAT3). However, these metabolites have not been assessed in rats as potential transporter biomarkers. We carried out a rat pharmacokinetic DDI study using probenecid and furosemide as OAT inhibitor and substrate, respectively. Probenecid administration led to a 3.8-fold increase in the blood concentrations and a 3-fold decrease in renal clearance of furosemide. High inter-individual and intra-day variability in pyridoxic acid and kynurenic acid, and no or moderate effect of probenecid administration on these metabolites suggest their limited utility for prediction of Oat-mediated DDI in rats. Therefore, rat blood and urine samples were further analysed using untargeted metabolomics. Twenty-one m/z features (out of >8000 detected features) were identified as putative biomarkers of rat Oat1 and Oat3 using a robust biomarker qualification approach. These m/z features belong to metabolic pathways such as fatty acid analogues, peptides, prostaglandin analogues, bile acid derivatives, flavonoids, phytoconstituents, and steroids, and can be used as a panel to decrease variability caused by processes other than Oats. When validated, these putative biomarkers will be useful in predicting DDIs caused by Oats in rats.

The Plasma Membrane Monoamine Transporter is Highly Expressed in Neuroblastoma and Functions as an mIBG Transporter.

Neuroblastoma (NB) is a pediatric cancer with low survival rates in high-risk patients. 131I-mIBG has emerged as a promising therapy for high-risk NB and kills tumor cells by radiation. Consequently, 131I-mIBG tumor uptake and retention are major determinants for its therapeutic efficacy. mIBG enters NB cells through the norepinephrine transporter (NET), and accumulates in mitochondria through unknown mechanisms. Here we evaluated the expression of monoamine and organic cation transporters in high-risk NB tumors and explored their relationship with MYCN amplification and patient survival. We found that NB mainly expresses NET, the plasma membrane monoamine transporter (PMAT), and the vesicular membrane monoamine transporter 1/2 (VMAT1/2), and that the expression of these transporters is significantly reduced in MYCN-amplified tumor samples. PMAT expression is the highest and correlates with overall survival in high-risk NB patients without MYCN amplification. Immunostaining showed that PMAT resides intracellularly in NB cells and co-localizes with mitochondria. Using cells expressing PMAT, mIBG was identified as a PMAT substrate. In mitochondria isolated from NB cell lines, mIBG uptake was reduced by ∼50% by a PMAT inhibitor. Together, our data suggest that PMAT is a previously unrecognized transporter highly expressed in NB and could impact intracellular transport and therapeutic response to 131I-mIBG. SIGNIFICANCE STATEMENT: This study identified that plasma membrane monoamine transporter (PMAT) is a novel transporter highly expressed in neuroblastoma and its expression level is associated with overall survival rate in high-risk patients without MYCN amplification. PMAT is expressed intracellularly in neuroblastoma cells, transports meta-iodobenzylguanidine (mIBG) and thus could impact tumor retention and response to 131I-mIBG therapy. These findings have important clinical implications as PMAT could represent a novel molecular marker to help inform disease prognosis and predict response to 131I-mIBG therapy.

High-Throughput Assay of Cytochrome P450-Dependent Drug Demethylation Reactions and Its Use to Re-Evaluate the Pathways of Ketamine Metabolism.

In a search for a reliable, inexpensive, and versatile technique for high-throughput kinetic assays of drug metabolism, we elected to rehire an old-school approach based on the determination of formaldehyde (FA) formed in cytochrome P450-dependent demethylation reactions. After evaluating several fluorometric techniques for FA detection, we chose the method based on the Hantzsch reaction with acetoacetanilide as the most sensitive, robust, and adaptable to high-throughput implementation. Here we provide a detailed protocol for using our new technique for automatized assays of cytochrome P450-dependent drug demethylations and discuss its applicability for high-throughput scanning of drug metabolism pathways in the human liver. To probe our method further, we applied it to re-evaluating the pathways of metabolism of ketamine, a dissociative anesthetic and potent antidepressant increasingly used in the treatment of alcohol withdrawal syndrome. Probing the kinetic parameters of ketamine demethylation by ten major cytochrome P450 (CYP) enzymes, we demonstrate that in addition to CYP2B6 and CYP3A enzymes, which were initially recognized as the primary metabolizers of ketamine, an important role is also played by CYP2C19 and CYP2D6. At the same time, the involvement of CYP2C9 suggested in the previous reports was deemed insignificant.

Dissecting Parameters Contributing to the Underprediction of Aldehyde Oxidase-Mediated Metabolic Clearance of Drugs.

We investigated the effect of variability and instability in aldehyde oxidase (AO) content and activity on the scaling of in vitro metabolism data. AO content and activity in human liver cytosol (HLC) and five recombinant human AO preparations (rAO) were determined using targeted proteomics and carbazeran oxidation assay, respectively. AO content was highly variable as indicated by the relative expression factor (REF; i.e., HLC to rAO content) ranging from 0.001 to 1.7 across different in vitro systems. The activity of AO in HLC degrades at a 10-fold higher rate in the presence of the substrate as compared with the activity performed after preincubation without substrate. To scale the metabolic activity from rAO to HLC, a protein-normalized activity factor (pnAF) was proposed wherein the activity was corrected by AO content, which revealed up to sixfold higher AO activity in HLC versus rAO systems. A similar value of pnAF was observed for another substrate, ripasudil. Physiologically based pharmacokinetic (PBPK) modeling revealed a significant additional clearance (CL; 66%), which allowed for the successful prediction of in vivo CL of four other substrates, i.e., O-benzyl guanine, BIBX1382, zaleplon, and zoniporide. For carbazeran, the metabolite identification study showed that the direct glucuronidation may be contributing to around 12% elimination. Taken together, this study identified differential protein content, instability of in vitro activity, role of additional AO clearance, and unaccounted metabolic pathways as plausible reasons for the underprediction of AO-mediated drug metabolism. Consideration of these factors and integration of REF and pnAF in PBPK models will allow better prediction of AO metabolism. SIGNIFICANCE STATEMENT: This study elucidated the plausible reasons for the underprediction of aldehyde oxidase (AO)-mediated drug metabolism and provided recommendations to address them. It demonstrated that integrating protein content and activity differences and accounting for the loss of AO activity, as well as consideration of extrahepatic clearance and additional pathways, would improve the in vitro to in vivo extrapolation of AO-mediated drug metabolism using physiologically based pharmacokinetic modeling.

Characterization of Xenobiotic and Steroid Disposition Potential of Human Placental Tissue and Cell Lines (BeWo, JEG-3, JAR, and HTR-8/SVneo) by Quantitative Proteomics.

The placenta is a fetal organ that performs critical functions to maintain pregnancy and support fetal development, including metabolism and transport of xenobiotics and steroids between the maternal-fetal unit. In vitro placenta models are used to study xenobiotic and steroid disposition, but how well these models recapitulate the human placenta is not well understood. We first characterized the abundance of proteins involved in xenobiotic and steroid disposition in human placental tissue. In pooled human placenta, the following xenobiotic and steroid disposition proteins were detected (highest to lowest), 1) enzymes: glutathione S-transferase P, carbonyl reductase 1, aldo-keto reductase 1B1, hydroxysteroid dehydrogenases (HSD3B1 and HSD11B1), aromatase, epoxide hydrolase 1 (EPHX1) and steryl-sulfatase, and 2) transporters: monocarboxylate transporters (MCT1 and 4), organic anion transporting polypeptide 2B1, organic anion transporter 4, and breast cancer resistance protein (BCRP). Then, the tissue proteomics data were compared with four placental cell lines (BeWo, JEG-3, JAR, and HTR-8/SVneo). The differential global proteomics analysis revealed that the tissue and cell lines shared 1420 cytosolic and 1186 membrane proteins. Although extravillous trophoblast and cytotrophoblast marker proteins were detected in all cell lines, only BeWo and JEG-3 cells expressed the syncytiotrophoblast marker, chorionic somatomammotropin hormone 1. BeWo and JEG-3 cells expressed most target proteins including aromatase, HSDs, EPHX1, MCT1, and BCRP. JEG-3 cells treated with commonly detected phthalates in human biofluids showed dysregulation of steroid pathways. The data presented here show that BeWo and JEG-3 cells are closer to the placental tissue for studying xenobiotic and steroid disposition. SIGNIFICANCE STATEMENT: This is the first study to compare proteomics data of human placental tissue and cell lines (BeWo, JAR, JEG-3, and HTR-8/SVneo). The placental cell line and tissue proteomes are vastly different, but BeWo and JEG-3 cells showed greater resemblance to the tissue in the expression of xenobiotic and steroid disposition proteins. These data will assist researchers to select an optimum cell model for mechanistic investigations on xenobiotic and steroid disposition in the placenta.

Continuous Large Area Monolayered Molybdenum Disulfide Growth Using Atmospheric Pressure Chemical Vapor Deposition.

The growth of large crystallite continuous monolayer materials like molybdenum disulfide (MoS2) with the desired morphology via chemical vapor deposition (CVD) remains a challenge. In CVD, the complex interplay of various factors like growth temperatures, precursors, and nature of the substrate decides the crystallinity, crystallite size, and coverage area of the grown MoS2 monolayer. In the present work, we report about the role of weight fraction of molybdenum trioxide (MoO3), sulfur, and carrier gas flow rate toward nucleation and monolayer growth. The concentration of MoO3 weight fraction has been found to govern the self-seeding process and decides the density of nucleation sites affecting the morphology and coverage area. A carrier gas flow of 100 sccm argon results in large crystallite continuous films with a lower coverage area (70%), while a flow rate of 150 sccm results in 92% coverage area with a reduced crystallite size. Through a systematic variation of experimental parameters, we have established the recipe for the growth of large crystallite atomically thin MoS2 suitable for optoelectronic devices.

Nano­zinc enhances gene regulation of non­specific immunity and antioxidative status to mitigate multiple stresses in fish.

The toxicity of ammonia surged with arsenic pollution and high temperature (34 °C). As climate change enhances the pollution in water bodies, however, the aquatic animals are drastically affected and extinct from nature. The present investigation aims to mitigate arsenic and ammonia toxicity and high-temperature stress (As + NH3 + T) using zinc nanoparticles (Zn-NPs) in Pangasianodon hypophthalmus. Zn-NPs were synthesized using fisheries waste to developing Zn-NPs diets. The four isonitrogenous and isocaloric diets were formulated and prepared. The diets containing Zn-NPs at 0 (control), 2, 4 and 6 mg kg-1 diets were included. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-s-transferase (GST) were noticeably improved using Zn-NPs diets in fish reared under with or without stressors. Interestingly, lipid peroxidation was significantly reduced, whereas vitamin C and acetylcholine esterase were enhanced with supplementation of Zn-NPs diets. Immune-related attributes such as total protein, globulin, albumin, myeloperoxidase (MPO), A:G ratio, and NBT were also improved with Zn-NPs at 4 mg kg-1 diet. The immune-related genes such as immunoglobulin (Ig), tumor necrosis factor (TNFα), and interleukin (IL1b) were strengthening in the fish using Zn-NPs diets. Indeed, the gene regulations of growth hormone (GH), growth hormone regulator (GHR1), myostatin (MYST) and somatostatin (SMT) were significantly improved with Zn-NPs diets. Blood glucose, cortisol and HSP 70 gene expressions were significantly upregulated by stressors, whereas the dietary Zn-NPs downregulated the gene expression. Blood profiling (RBC, WBC and Hb) was reduced considerably with stressors (As + NH3 + T), whereas Zn-NPs enhanced the RBC, WBC, and Hb count in fish reread in control or stress conditions. DNA damage-inducible protein gene and DNA damage were significantly reduced using Zn-NPs at 4 mg kg-1 diet. Moreover, the Zn-NPs also enhanced the arsenic detoxification in different fish tissues. The present investigation revealed that Zn-NPs diets mitigate ammonia and arsenic toxicity, and high-temperature stress in P. hypophthalmus.

Quantitative Characterization of Clinically Relevant Drug-Metabolizing Enzymes and Transporters in Rat Liver and Intestinal Segments for Applications in PBPK Modeling.

Rats are extensively used as a preclinical model for assessing drug pharmacokinetics (PK) and tissue distribution; however, successful translation of the rat data requires information on the differences in drug metabolism and transport mechanisms between rats and humans. To partly fill this knowledge gap, we quantified clinically relevant drug-metabolizing enzymes and transporters (DMETs) in the liver and different intestinal segments of Sprague-Dawley rats. The levels of DMET proteins in rats were quantified using the global proteomics-based total protein approach (TPA) and targeted proteomics. The abundance of the major DMET proteins was largely comparable using quantitative global and targeted proteomics. However, global proteomics-based TPA was able to detect and quantify a comprehensive list of 66 DMET proteins in the liver and 37 DMET proteins in the intestinal segments of SD rats without the need for peptide standards. Cytochrome P450 (Cyp) and UDP-glycosyltransferase (Ugt) enzymes were mainly detected in the liver with the abundance ranging from 8 to 6502 and 74 to 2558 pmol/g tissue. P-gp abundance was higher in the intestine (124.1 pmol/g) as compared to that in the liver (26.6 pmol/g) using the targeted analysis. Breast cancer resistance protein (Bcrp) was most abundant in the intestinal segments, whereas organic anion transporting polypeptides (Oatp) 1a1, 1a4, 1b2, and 2a1 and multidrug resistance proteins (Mrp) 2 and 6 were predominantly detected in the liver. To demonstrate the utility of these data, we modeled digoxin PK by integrating protein abundance of P-gp and Cyp3a2 into a physiologically based PK (PBPK) model constructed using PK-Sim software. The model was able to reliably predict the systemic as well as tissue concentrations of digoxin in rats. These findings suggest that proteomics-informed PBPK models in preclinical species can allow mechanistic PK predictions in animal models including tissue drug concentrations.

Health risk assessment and metal contamination in fish, water and soil sediments in the East Kolkata Wetlands, India, Ramsar site.

East Kolkata Wetlands (EKW) is an important site for fish culture in sewage-fed areas, which are major receivers of pollutants and wastages from Kolkata. EKW is internationally important as the Ramsar site was declared on Aug 2002 with an area of 125 km2. EKW is a natural water body where wastewater-fed natural aquaculture has been practiced for more than 70 years. It is ecologically vulnerable due to the discharge of toxic waste through sewage canals from cities. Assessing the EKW to understand the inflow and load of the toxic metal (s) in fish, water, and sediments samples is essential. The field (samples collection from 13 sites) and lab (determination of toxic level of metals) based research were carried out to assess metal toxicity and health risk assessment in EKW. The levels of eighteen metals (18), namely Chromium, Vanadium, Cobalt, Manganese, Copper, Nickel, Zinc, Silver, Molybdenum, Arsenic, Selenium, Tin, Gallium, Germanium, Strontium, Cadmium, Mercury, and Lead, were determined using Inductively coupled plasma mass spectrometry (ICP-MS) in five fish tissues viz. muscle, liver, kidney, gill and brain, along with the water samples and soil sediments in 13 sampling sites. The bioaccumulation and concentration of metals in fish tissues, soil sediments, and water samples were well within the safe level concerning the recommendation of different national and international agencies except for a few metals in a few sampling sites like Cd, As, and Pb. The geoaccumulation index (Igeo) was also determined in the soil sediments, indicating moderate arsenic, selenium, and mercury contamination in a few sites. The contamination index in water was also determined in 13 sampling sites. The estimated daily intake (EDI), reference dose (RfD), target hazard quotient (THQ), slope factor and cancer risk of Cr, Mn, Co, Ni, Cu, Zn, As, Se, Cd, Pb and Hg from fish muscle were determined. Based on the results of the present investigation, it is concluded that fish consumption in the East Kolkata Wetland (EKW) is safe. The effects of bioaccumulation of metals in muscle tissue were well within the safe level for consumption as recommended by WHO/FAO.

Morbidity and Mortality of Necrotizing Fasciitis and Their Prognostic Factors in Children.

Background: This is a prospective study of the clinico-etiologic profile and factors affecting outcomes in 40 children managed for necrotizing fasciitis (NF). Materials and Methods: Demographic details, clinical characteristics, and laboratory parameters were recorded, and the Laboratory Risk Indicator for Necrotizing Fasciitis (LRINEC) score was calculated. Primary outcome (survival vs. nonsurvival) was noted, and prognostic factors were identified. Results: Initiating factors included boils (45%), i.v. cannula extravasations (22.5%), and blunt trauma (17.5%). Lesion (s) were predominantly on the lower limbs (35%) and trunk (25%). Twenty-two patients (55%) had <5% body surface area (BSA) involved. Severely deranged clinical and laboratory parameters were common. Ultrasound localized fluid collections. Pus cultures showed methicillin-resistant Staphylococcus aureus (52.5%), methicillin-sensitive S. aureus [27.5%], and polymicrobial growth (20%). Blood culture was positive in 24 patients (60%). Most isolates were sensitive to clindamycin and amoxy-clavulanate. Prognostic factors for mortality (n = 6; 15%) included categorization as "Sick," BSA involvement >10%, thrombocytopenia, raised serum creatinine, late debridement, and polymicrobial blood culture isolates. All six nonsurvivors had a LRINEC score of ≥8 and positive blood cultures. Six patients (20.7%) developed unsightly scars and 5 (17.24%) contractures across joints. Conclusions: Pediatric NF has significant morbidity and mortality. Patients with adverse prognostic factors can benefit from early referral to a facility with a critical care unit. Adequate wound management is essential to minimize residual deformity.