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.

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.

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.

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.

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.

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.

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.

Dimethandrolone, a Potential Male Contraceptive Pill, is Primarily Metabolized by the Highly Polymorphic UDP-Glucuronosyltransferase 2B17 Enzyme in Human Intestine and Liver.

Dimethandrolone undecanoate (DMAU), an oral investigational male hormonal contraceptive, is a prodrug that is rapidly converted to its active metabolite, dimethandrolone (DMA). Poor and variable oral bioavailability of DMA after DMAU dosing is a critical challenge to develop it as an oral drug. The objective of our study was to elucidate the mechanisms of variable pharmacokinetics of DMA. We first identified DMA metabolites formed in vitro and in vivo in human hepatocyte incubation and serum samples following oral DMAU administration in men, respectively. The metabolite identification study revealed two metabolites, DMA-glucuronide (DMA-G; major) and the androstenedione analog of DMA (minor), in the hepatocyte incubations. After oral DMAU administration, only DMA-G was detected in serum, which was >100-fold compared with DMA levels, supporting glucuronidation as the major elimination mechanism for DMA. Next, 13 clinically relevant UDP-glucuronosyltransferase (UGT) enzymes were tested for their involvement in DMA-G formation, which revealed a major role of UDP-glucuronosyltransferase 2B17 (UGT2B17) isoform with a smaller contribution of UGT1A9 in DMA-G formation. These data were confirmed by dramatically higher DMA glucuronidation rates (>200- and sevenfold) in the high versus the null UGT2B17-expressing human intestinal and liver microsomes, respectively. Since human UGT2B17 is a highly variable enzyme with a 20%-80% gene deletion frequency, the in vitro data suggest a major role of UGT2B17 polymorphism on the first-pass metabolism of DMA. Further, considering DMA is a selective and sensitive UGT2B17 substrate, it could be used as a clinical probe of UGT2B17 activity. SIGNIFICANCE STATEMENT: Dimethandrolone (DMA) is an active metabolite of dimethandrolone undecanoate (DMAU), an investigational male hormonal contraceptive. Previous studies have indicated poor and inconsistent bioavailability of DMAU following oral administration. This study found that UDP-glucuronosyltransferase 2B17-mediated high intestinal first-pass metabolism is the key mechanism of variable DMA bioavailability.

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.

Synthesis and characterization of a series of N,N'-substituted urea derivatives by using electrospray ionization tandem mass spectrometry: Differentiation of positional isomers.

RATIONALE: Characterization of N,N'-substituted ureas was found to be challenging by nuclear magnetic resonance (NMR) spectroscopy, particularly N-di- and tri-alkylated ureas because of the absence of adjacent protons. In the present study, electrospray ionization tandem mass spectrometry has been used to differentiate positional isomeric pairs and to characterize a series of N,N'-substituted ureas, as these compounds have significant importance for drug discovery. Additionally, urea is an essential functionality in several bioactive compounds as well as a variety of clinically approved therapies. METHODS: High-resolution electrospray ionization tandem mass spectrometry (ESI-HR-MS/MS) has been used to characterize a series of N,N'-substituted urea derivatives and differentiate two pairs of positional isomers. The data was acquired by Xcaliber application in positive ionization mode. RESULTS: ESI-HR-MS/MS spectra of [M + H]+ ions of the positional isomeric urea derivatives 8a and 8b show distinct fragmentation patterns. For example, the MS/MS spectrum of the [M + H]+ ion of isomer 8a displays the abundant fragment ion at m/z 285.1595, which was totally absent in isomer 8b. This would be plausibly formed by the cleavage of the C-N bond of the urea group with the elimination of the isocyanate moiety. In contrast, the MS/MS spectrum of the [M + H]+ ion of isomer 8b shows an intense ion at m/z 311.1389 which is completely absent in isomer 8a which would be formed by the cleavage of the C-N bond attached to the ring nitrogen. Similarly, another pair of positional isomers, 8c and 8d, have been clearly distinguished by their fragmentation behaviour. In addition, a series of N,N'-substituted urea derivatives were studied to investigate the impact of different substitution on the fragmentation behaviour. CONCLUSIONS: The present study demonstrates that ESI-HR-MS/MS can be used to differentiate pairs of N,N'-substituted urea positional isomers and characterize a series of derivatives. It was observed that a characteristic fragment ion was formed by the C-N bond cleavage with the elimination of an isocyanate moiety. The proposed mechanism of fragmentation was supported by the change in the fragmentation pathway upon alkylation of the NH. In order to generalize this fragmentation pattern, a series of N-alkylated ureas was synthesized and studied by MS/MS.

Identification of novel glutathione conjugates of terbinafine in liver microsomes and hepatocytes across species.

1. Terbinafine (TBF), a common antifungal agent, has been associated with rare incidences of hepatotoxicity. It is hypothesized that bioactivation of TBF to reactive intermediates and subsequent binding to critical cellular proteins may contribute to this toxicity. In the present study, we have characterized the bioactivation pathways of TBF extensively in human, mouse, monkey, dog and rat liver microsomes and hepatocytes. 2. A total of twenty glutathione conjugates of TBF were identified in hepatocytes; thirteen of these conjugates were also detected in liver microsomes. To the best of our knowledge, only two of these conjugates have been reported previously. The conjugates were categorized into three groups based on their mechanism of formation: (a) alkene/alkyne oxidation followed by glutathione conjugation, with or without N-demethylation, (b) arene oxidation followed by glutathione conjugation, with or without N-demethylation, and (c) N-dealkylation followed by glutathione conjugation of the allylic aldehyde, alcohol and acid intermediates. 3. Differences were observed across species in the contributions of these pathways toward overall metabolic turnover. We conclude that, in addition to the glutathione conjugates known to form by Michael addition to the allylic aldehyde, there are other pathways involving the formation of arene oxides and alkene/alkyne epoxides that may be relevant to the discussion of TBF-mediated idiosyncratic drug reactions.

Immuno-protective role of biologically synthesized dietary selenium nanoparticles against multiple stressors in Pangasinodon hypophthalmus.

An environment friendly and sustainable approach is being emerged in the area of nanotechnology for accelerated growth and development of culturable aquatic animals hence green chemistry is gaining momentum in recent years. The present study has been carried out to delineate the effects of selenium nanoparticles (Se-NPs) on growth performance, antioxidative status and immunity of fish reared under lead (Pb) and high temperature (34 °C). Three hundred and fifteen fish were equally distributed in seven treatments in triplicates. Three isocaloric and isonitrogenous experimental diets viz. control (Se-NPs-0 mg/kg), Se-NPs at 1 mg/kg and Se-NPs at 2 mg/kg were formulated. The fish were reared under lead (Pb, 1/21st of LC50 (4 ppm)) and high temperature (34 °C) stress and fed with or without dietary Se-NPs. The effects of dietary Se-NPs were studied in terms of growth performance (Weight gain %, feed conversion ratio, protein efficiency ratio and specific growth rate), antioxidative status (catalase, superoxide dismutase, glutathione-S-transferase and glutathione peroxidase), neurotransmitter enzymes (AChE), stress biomarkers (heat shock protein 70, serum cortisol, blood glucose, vitamin C), immunological status (total protein, A/G ratio and respiratory burst activity) in Pangasinodon hypophthalmus post challenge with Aeromonas veronii biovar sobria. Results of the investigation demonstrated significant improvement of growth performance, antioxidative status, neurotransmitter enzyme activity, stress markers and more importantly enhanced immunity of the fish with dietary incorporation of Se-NPs at 1 mg/kg. In addition, post bacterial infection, the relative % survival increased and cumulative mortality % decreased in the group fed with Se-NPs at 1 mg/kg diet. Pb and high temperature treated and fed with control diet group showed devastating impact on the growth performance, antioxidative status, stress markers and immunity of the fish. Similarly, application of Se-NPs at 2 mg/kg showed poor growth performance and elevated level of oxidative stress and other stress biomarkers including other biochemical attributes. Inclusive results indicated that, Se-NPs at 1 mg/kg has capability to enhance overall performance and alleviate multiple stresses in P. hypophthalmus. Hence, Se-NPs at optimum level have ability to develop green chemistry in feed industry for better growth performance of the fish.

Pulmonary Embolism in Ischemic Stroke.

Silent pulmonary embolism (PE) may be associated with acute ischemic stroke (AIS). We identified 10 patients from 3,132 unique patients (3,431 CT scans). We retrospectively examined CT angiogram of patients with AIS to determine the frequency of concurrent PE in AIS. The period prevalence of PE was 0.32. Seven patients had concurrent PE, whereas three had PE diagnosed 2 days after their AIS presentation. We suspected paradoxical embolism via patent foramen ovale as the cause of stroke in three patients and thrombophilia in four patients. Seven patients had poor outcome including four deaths. CT angiogram stroke protocol images from aortic arch to vertex allows visualization of upper pulmonary arteries and PE detection in AIS.

Dietary nano-silver: Does support or discourage thermal tolerance and biochemical status in air-breathing fish reared under multiple stressors?

Unexpected fluctuations in weather parameters due to global climate change have been observed in all ecosystems worldwide. The aquatic ecosystem shelters a great diversity of fishes in the upper region of the ecosystem which adversely get affected due to their poikilothermic nature. The present study was designed to elucidate the impact of critical temperature minima (CTMin), lethal temperature minima (LTMin), critical temperature maxima (CTMax), and lethal temperature maxima (LTMax) on Channa striatus. Biologically synthesized silver nanoparticles (Ag-NPs) were evaluated for their potential to enhance thermal tolerance and improve the activities of biochemical enzymes of C. striatus reared under lead (Pb) and high temperature (34 °C) for 50 days. Three iso-caloric and iso-nitrogenous diets which included a basal diet and two supplemented diets with Ag-NPs @ 0.5 mg/kg, and 1 mg/kg were used in the study. Results suggested that CTMin and LTMin were significantly (p < 0.01) reduced and CTMax and LTMax were enhanced in the group fed with 0.5 mg/kg Ag-NPs supplemented feed. Pre-exposure to high temperature led to enhanced CTMax and LTMax in C. striatus. The biochemical enzymes involved in protein metabolism, carbohydrate metabolism, acetylcholine esterase and antioxidant activities were found to be normal in fish fed with 0.5 mg/kg Ag-NPs supplemented diet. Bioaccumulation of silver and Pb was determined in different fish tissues and experimental water. Overall, the incorporation of Ag-NPs at 0.5 mg/kg in diet can confer protection to fish against Pb and thermal stress and enhance thermal tolerance of C. striatus.

Role of limited hydrogen and flow interval on the growth of single crystal to continuous graphene by low-pressure chemical vapor deposition.

A method for defect-free large crystallite graphene growth remains unknown despite much research effort. In this work, we discuss the role of flow duration of H2 gas for the production of graphene as per requirement and production at a minimum flow rate considering the safety issue of hydrogen utilization. The copper substrate used for growth was treated for different time intervals (0 to 35 min) in H2 flow prior to growth. Structural and chemical changes occurring in the copper substrate surface were probed by grazing incidence x-ray diffraction and x-ray photoelectron spectroscopy. The results were correlated with the Raman spectroscopy data, which can quantify the quality of graphene. With increasing H2 flow interval, secondary nucleation sites were observed and growth favored few-layer graphene structures. The surface-adsorbed oxygen molecules and its conversion to an OH terminated surface with increasing hydrogen flow interval was found to be a key factor in enhancing nucleation density. The Stranski-Krastanov type of nucleation was observed for samples grown with different time intervals of H2 treatment, except 5 min of H2 flow prior to growth for which the Volmer-Weber type of growth favored monolayer graphene crystallite growth.

MR imaging of carotid webs.

PURPOSE: We propose a magnetic resonance (MR) imaging protocol for the characterization of carotid web morphology, composition, and vessel wall dynamics. The purpose of this case series was to determine the feasibility of imaging carotid webs with MR imaging. METHODS: Five patients diagnosed with carotid web on CT angiography were recruited to undergo a 30-min MR imaging session. MR angiography (MRA) images of the carotid artery bifurcation were acquired. Multi-contrast fast spin echo (FSE) images were acquired axially about the level of the carotid web. Two types of cardiac phase resolved sequences (cineFSE and cine phase contrast) were acquired to visualize the elasticity of the vessel wall affected by the web. RESULTS: Carotid webs were identified on MRA in 5/5 (100%) patients. Multi-contrast FSE revealed vessel wall thickening and cineFSE demonstrated regional changes in distensibility surrounding the webs in these patients. CONCLUSION: Our MR imaging protocol enables an in-depth evaluation of patients with carotid webs: morphology (by MRA), composition (by multi-contrast FSE), and wall dynamics (by cineFSE).

Characterization of stress degradation products of amodiaquine dihydrochloride by liquid chromatography with high-resolution mass spectrometry and prediction of their properties by using ADMET Predictor™.

The degradation behavior of amodiaquine dihydrochloride, an antimalarial drug, was investigated in solution as well as solid states. The drug was subjected to hydrolytic, photolytic, oxidative, and thermal stress conditions, according to International Conference on Harmonization guideline Q1A(R2). It showed extensive hydrolysis in acidic, alkaline, and neutral solutions both with and without light, while it proved to be stable to thermal and oxidative conditions. In total, six degradation products were formed, which were separated on a C8 column, employing a gradient reversed-phase high-performance liquid chromatography method in which acetonitrile and 10 mM ammonium formate (pH 3.0) were used in the mobile phase. To characterize the degradation products, mass fragmentation behavior of the drug was established by direct infusion of solution to quadrupole time-of-flight and multiple-stage mass spectrometry systems. Liquid chromatography with high-resolution mass spectrometry studies were subsequently carried out on the stressed samples using the same gradient high-performance liquid chromatography method employed for the separation of the degradation products. Hydrogen/deuterium exchange studies were additionally conducted to determine the number of labile hydrogen atoms. The degradation pathway of the drug was delineated, justified by mechanistic explanation. Lastly, ADMET Predictor™ software was employed to predict relevant physicochemical and toxicity data for the degradation products.

Selective electric and magnetic sensitivity of aperture probes.

We report the effect of geometrical factors governing the polarization profiles of near-field scanning optical microscope (NSOM) probes. The most important physical parameter controlling the selective electric or magnetic field sensitivity is found to be the width of the metal rim surrounding aperture. Probes with metal rim width w < λ/2 selectively senses the optical electric field, while those with w > λ/2 selectively senses the optical magnetic field. Intensity variation of optical Hertz standing wave formed upon reflection at oblique incidence shows a phase difference of π/2 between electric and magnetic probes: an analogue of the classical Wiener's experiment. Our work paves way towards electromagnetic engineering of nanostructures.

Optical field enhancement of nanometer-sized gaps at near-infrared frequencies.

We report near-field and far-field measurements of transmission through nanometer-sized gaps at near-infrared frequencies with varying the gap size from 1 nm to 10 nm. In the far-field measurements, we excluded direct transmission on the metal film surface via interferometric method. Kirchhoff integral formalism was used to relate the far-field intensity to the electric field at the nanogaps. In near-field measurements, field enhancement factors of the nanogaps were quantified by measuring transmission of the nanogaps using near-field scanning optical microscopy. All the measurements produce similar field enhancements of about ten, which we put in the context of comparing with the giant field enhancements in the terahertz regime.