Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells.

Genetic screens in mammalian cells commonly focus on loss-of-function approaches. To evaluate the phenotypic consequences of extra gene copies, we used bulk segregant analysis (BSA) of radiation hybrid (RH) cells. We constructed six pools of RH cells, each consisting of ∼2500 independent clones, and placed the pools under selection in media with or without paclitaxel. Low pass sequencing identified 859 growth loci, 38 paclitaxel loci, 62 interaction loci, and three loci for mitochondrial abundance at genome-wide significance. Resolution was measured as ∼30 kb, close to single-gene. Divergent properties were displayed by the RH-BSA growth genes compared to those from loss-of-function screens, refuting the balance hypothesis. In addition, enhanced retention of human centromeres in the RH pools suggests a new approach to functional dissection of these chromosomal elements. Pooled analysis of RH cells showed high power and resolution and should be a useful addition to the mammalian genetic toolkit.

Development and validation of gas chromatography with electron capture detection method using QuEChERS for pesticide residue determination in cucumber.

A modified quick, easy, cheap, efficient, rugged, and safe (QuEChERS) method coupled to gas chromatography with electron capture detection was developed for the simultaneous determination of selected electronegative pesticides, namely, chlorpyrifos-methyl (1), chlorpyrifos (2), quinolphos (3), profenofos (4), myclobutanil (5), ethion (6), fenpropathrin (7), and cypermethrin (8), in vegetables with high water content. The selected compounds and some of their metabolites have even been found in human body fluids. In addition, some of them are known or suspected carcinogens according to the World Health Organization. Extraction and cleanup parameters were optimized; thus, the original QuEChERS method was modified to minimize solvent usage by making the study eco-friendly. The developed method was validated for selectivity, specificity, linearity, precision, and accuracy using SANTE guidelines. Calibration curves showed good linearity (r > 0.99) within the test range. Precision was evaluated by intra- and inter-day experiments with an acceptable range of less than 20.0% of relative standard deviation. Recovery was evaluated at limit of quantification and was found to be in the range of 70-120%, with relative standard deviations lower than 4.21%. The proposed method is applicable for detection and monitoring of selected pesticides in one run not only in fruits and vegetables with high water content but also in samples containing large quantities of pigments/dyes.

Extreme phenotypic diversity in operant response to intravenous cocaine or saline infusion in the hybrid mouse diversity panel.

Cocaine self-administration is a complexly determined trait, with a substantial proportion of individual differences being determined by genetic variation. However, the relevant genetic variants that drive heritable differences in cocaine use remain undiscovered. Cocaine intravenous self-administration (IVSA) procedures in laboratory animals provide opportunities to prospectively investigate neurogenetic influences on the acquisition of voluntary cocaine use. Here, we provide information on cocaine (or saline-as a control) IVSA in 84 members of the hybrid mouse diversity panel (HMDP), an array of genetically distinct classical or recombinant inbred strains. We found cocaine IVSA to be substantially heritable in this population, with strain-level intake ranging for near 0 to >25 mg/kg/session. Though saline IVSA was also found to be heritable, a modest genetic correlation between cocaine and saline IVSA indicates that operant responding for the cocaine reinforcer was influenced, at least in part, by unique genetic variants. Genome-wide association studies (GWAS) of infusions earned in cocaine and saline groups revealed significant quantitative trait loci (QTL) on Chromosomes 3 and 14 for cocaine, but not saline, IVSA. Positional candidates were further prioritized through use of bulk RNA sequencing data that revealed genes with cis-eQTL and genetic correlation to number of infusions. Additionally, these data identify reference strains with extreme cocaine IVSA phenotypes, revealing them as polygenic models of risk and resilience to cocaine reinforcement. This work is part of an ongoing effort to characterize genetic variation that moderates cocaine IVSA that may, in turn, provide a more comprehensive understanding of cocaine risk genetics and neurobiology.

Development of a bioanalytical method for the quantification of sinococuline in human plasma and its application in phase I clinical pharmacokinetic study.

A selective, highly sensitive, precise, and novel bioanalytical method has been developed and validated to quantify sinococuline, an active constituent present in the phytopharmaceutical drug product containing Cocculus hirsutus plant extract, in vivo. Chromatographic separation was achieved on a Luna Omega Polar-C18 bonded analytical column maintained at 45°C. The isocratic mobile phase consisted of methanol and ammonium formate buffer (60:40, v/v) at acidic pH with a low flow rate of 0.250 mL/min. Detection was performed on an API 4000 mass spectrometer using electrospray ionization in positive polarity and multiple reaction monitoring mode to achieve a lower limit of quantification of 1.50 ng/mL. Excellent accuracy and precision were obtained after extracting the analyte from plasma samples using a chemical analogue as an internal standard in the absence of an isotope-labeled compound. The extraction efficacy was evidenced from recovery study, and the analyte was found to be stable in plasma. Validation study demonstrated linearity with coefficient of correlation, r ≥ 0.99, and minimal matrix effect. This bioanalytical method was successfully applied to evaluate pharmacokinetic parameters of sinococuline from a phase I clinical trial of an aqueous extract of C. hirsutus in healthy human volunteers.

Genotoxic effects of drospirenone and ethinylestradiol in human breast cells (in vitro) and bone marrow cells of female mice (in vivo).

Estrogen and progesterone congeners as found in various oral contraceptive formulations have been implicated as the cause of cancer in sex and tissue-specific targets. The mechanism of carcinogenesis by sex steroids is still debatable. In this study, we evaluated the genotoxicity induced by two components of one of the commonly used oral contraceptive formulation; drospirenone and ethinylestradiol in human breast cells (MCF-7) in vitro and in bone marrow cells of female mice in vivo. DNA damage was assessed by alkaline comet assay. Both of the drugs produced DNA damage in human breast cells at exposure concentrations which are about 100-fold and above than normally found in human blood after their lowest recommended doses. The DNA damage was produced only after metabolic activation by mice liver S-9 fraction in both cases. The co-exposure with both the compounds at median exposure levels resulted in potentiation of DNA damage. In bone marrow cells of adult female mice, both the compounds produced DNA damage at human equivalent doses after exposure was carried out repeatedly for approximately one estrus cycle (5 days). The co-administration with the compounds resulted in potentiation of DNA damage as indicated by percent tail DNA in comet assay. Thus it is concluded that drospirenone and ethinylestradiol cause DNA damage in certain target specific tissue (mammary epithelial cells) and in female bone marrow cells. The co-exposure with drospirenone and ethinylestradiol results in potentiation of genotoxicity which may pose a threat of cancer development in women taking these drugs for long periods.

Reduced Folate Carrier: an Entry Receptor for a Novel Feline Leukemia Virus Variant.

Feline leukemia virus (FeLV) is horizontally transmitted among cats and causes a variety of hematopoietic disorders. Five subgroups of FeLV, A to D and T, each with distinct receptor usages, have been described. Recently, we identified a new FeLV Env (TG35-2) gene from a pseudotyped virus that does not belong to any known subgroup. FeLV-A is the primary virus from which other subgroups have emerged via mutation or recombination of the subgroup A env gene. Retrovirus entry into cells is mediated by the interaction of envelope protein (Env) with specific cell surface receptors. Here, phenotypic screening of a human/hamster radiation hybrid panel identified SLC19A1, a feline reduced folate carrier (RFC) and potential receptor for TG35-2-phenotypic virus. RFC is a multipass transmembrane protein. Feline and human RFC cDNAs conferred susceptibility to TG35-2-pseudotyped virus when introduced into nonpermissive cells but did not render these cells permissive to other FeLV subgroups or feline endogenous retrovirus. Moreover, human cells with genomic deletion of RFC were nonpermissive for TG35-2-pseudotyped virus infection, but the introduction of feline and human cDNAs rendered them permissive. Mutation analysis of FeLV Env demonstrated that amino acid substitutions within variable region A altered the specificity of the Env-receptor interaction. We isolated and reconstructed the full-length infectious TG35-2-phenotypic provirus from a naturally FeLV-infected cat, from which the FeLV Env (TG35-2) gene was previously isolated, and compared the replication of the virus in hematopoietic cell lines with that of FeLV-A 61E by measuring the viral RNA copy numbers. These results provide a tool for further investigation of FeLV infectious disease.IMPORTANCE Feline leukemia virus (FeLV) is a member of the genus Gammaretrovirus, which causes malignant diseases in cats. The most prevalent FeLV among cats is FeLV subgroup A (FeLV-A), and specific binding of FeLV-A Env to its viral receptor, thiamine transporter feTHTR1, is the first step of infection. In infected cats, novel variants of FeLV with altered receptor specificity for viral entry have emerged by mutation or recombination of the env gene. A novel FeLV variant arose from a subtle mutation of FeLV-A Env, which altered the specific interaction of the virus with its receptor. RFC, a folate transporter, is a potential receptor for the novel FeLV variant. The perturbation of specific retrovirus-receptor interactions under selective pressure by the host results in the emergence of novel viruses.

Acquisition, remission, and persistence of eczema, asthma, and rhinitis in children.

BACKGROUND: Allergic sensitization is associated with eczema, asthma, and rhinitis. However, it is unknown whether and how allergic sensitization is associated over time with acquisition, remission, and persistence of these diseases and their comorbidity. OBJECTIVE: To gain a better understanding of factors including allergic sensitization transitions that influence the temporal pattern of asthma, eczema, and rhinitis and their comorbidity during childhood. METHODS: In the Isle of Wight birth cohort, information on allergic sensitization to common allergens was collected at ages 4, 10, and 18 years along with asthma, rhinitis, and eczema status determined by clinical diagnosis. Logistic regressions were used to estimate subsequent and concurrent odds ratios of diseases transition with allergic sensitization transition status as the main independent variable. Two transition periods were considered, 4 to 10 years of age and 10 to 18 years of age. RESULTS: The odds of new diagnosis of allergic disease (no-yes) was increased among subjects with acquired or persistent allergic sensitization to common allergens compared to subjects with no sensitization (acquisition of sensitization odds ratio [OR]=3.22, P < .0001; persistence of sensitization, OR=6.33, P < .0001). The odds of remission of allergic diseases (yes-no) was lower among subjects with acquired or sustained allergic sensitization (acquisition, OR=0.18, P = .0001; persistence, OR=0.085, P < .0001), compared to subjects not sensitized. Subjects with acquired or persistent allergic sensitization were also had higher odds for persistence of disease (yes-yes) than subjects not sensitized (acquisition, OR=5.49, P = .0001; persistence, OR=11.79, P < .0001). CONCLUSION: Transition of allergic sensitizations to common allergens is a prognostic factor for subsequent or concurrent transition of eczema, asthma, and rhinitis. Prevention or reduction in allergic sensitization has a potential to lead to remission of these conditions.

Treatment options for partial auricle reconstruction: a prospective study of outcomes and patient satisfaction.

OBJECTIVE: To evaluate the various methods and outcomes of post-traumatic partial auricle wound reconstruction, and to review the benefit to the patient's quality of life and their psychological improvement after the operation. METHODS: The prospective study included patients who suffered from post-traumatic partial auricular wounds. The defects were repaired using various techniques including simple local cutaneous advancement flaps, tube flaps, cartilage framework using conchal and costal cartilage with local skin flap cover. RESULTS: A total of 18 patients were included, with a male predominance (sex ratio: 7:2), mean age with standard deviation of 31.66 ±9.27 years. Causes included road traffic accident (RTA), assault, human and insect bite and avulsion injuries. Injuries were sustained in the upper third of the auricle (n=8); middle third (n=5), lower third (n=3), and upper two-thirds (n=2). Out of the 18 patients, wound were repaired using post auricular mastoid skin flap (n=7); local superior and inferior chondrocutaneous flap (n=3); costal cartilage as a cartilage framework (n=4), temporoparietal fascia used to cover the costal cartilage graft (n=1), and conchal cartilage as a cartilage framework (n=3). In our study 13/18 patients were 'highly satisfied' with the aesthetic outcome, 3/18 were 'moderately satisfied', and 2/18 were 'slightly satisfied'. None were dissatisfied by the postoperative result. In terms of objective assessment, patient outcome in two patients was graded 'good' while the outcome of remaining patients (n=16) was graded as 'excellent'. CONCLUSIONS: The use of skin flaps in the post-auricular region and the mastoid region associated with or without cartilage framework yields good cosmetic and functional result. The various techniques used for ear reconstruction yielded 100% satisfactory results in terms of functional outcome as well as boosting the confidence of patients.

Expression of the filaggrin gene in umbilical cord blood predicts eczema risk in infancy: A birth cohort study.

BACKGROUND: Filaggrin gene (FLG) expression, particularly in the skin, has been linked to the development of the skin barrier and is associated with eczema risk. However, knowledge as to whether FLG expression in umbilical cord blood (UCB) is associated with eczema development and prediction is lacking. OBJECTIVE: This study sought to assess whether FLG expression in UCB associates with and predicts the development of eczema in infancy. METHODS: Infants enrolled in a birth cohort study (n=94) were assessed for eczema at ages 3, 6, and 12 months. Five probes measuring FLG transcripts expression in UCB were available from genomewide gene expression profiling. FLG genetic variants R501X, 2282del4, and S3247X were genotyped. Associations were assessed using Poisson regression with robust variance estimation. Area under the curve (AUC), describing the discriminatory/predictive performance of fitted models, was estimated from logistic regression. RESULTS: Increased level of FLG expression measured by probe A_24_P51322 was associated with reduced risk of eczema during the first year of life (RR=0.60, 95% CI: 0.38-0.95). In contrast, increased level of FLG antisense transcripts measured by probe A_21_P0014075 was associated with increased risk of eczema (RR=2.02, 95% CI: 1.10-3.72). In prediction models including FLG expression, FLG genetic variants, and sex, discrimination between children who will and will not develop eczema at 3 months of age was high (AUC: 0.91, 95% CI: 0.84-0.98). CONCLUSIONS AND CLINICAL RELEVANCE: This study demonstrated, for the first time, that FLG expression in UCB is associated with eczema development in infancy. Moreover, our analysis provided prediction models that were capable of discriminating, to a great extent, between those who will and will not develop eczema in infancy. Therefore, early identification of infants at increased risk of developing eczema is possible and such high-risk newborns may benefit from early stratification and intervention.

Molecular diversity of Pakistani mango (Mangifera indica L.) varieties based on microsatellite markers.

Understanding the genetic diversity of different Pakistani mango varieties is important for germplasm management and varietal characterization. Microsatellites are efficient and highly polymorphic markers for comparative genome mapping, and were used in the present study to determine the genetic relatedness and variability among 15 indigenous mango cultivars (Mangifera indica L.). Overall, 181 bands were produced using 12 simple sequence repeat (SSR) primers. Out of the 12 primers used, 10 were polymorphic and two were monomorphic. Genetic relatedness among cultivars was assessed by constructing a dendrogram using the unweighted pair group method of arithmetic means. The accessions exhibited coefficients of similarity ranging from 75 to 100%, indicating the frequent use of only a few parent cultivars and the presence of inbreeding. The primers used in the present study were found to be valuable for identifying genetic relationships among mango cultivars.

Surgical treatment of acute acromioclavicular joint dislocations: hook plate versus minimally invasive reconstruction.

PURPOSE: This study was performed to compare the clinical results of a minimally invasive technique for acute acromioclavicular (AC) joint dislocation repair with the traditional hook plate fixation. METHODS: Forty-four patients with an acute (within 2 weeks after trauma) complete AC joint separation (35 male, nine female; median age 36.2 years, range 18-56) underwent surgical repair with either a minimally invasive AC joint repair or a conventional hook plate. Functional outcome was evaluated using the Constant-Murley Score (CMS), the TAFT score and the AC joint instability score (ACJI). Radiographic evaluation was performed with bilateral anterior-posterior (a.p.) stress and Alexander views. RESULTS: All patients were available after a median follow-up of 32 months (range 24-51). There were no significant differences in the mean CMS, Taft score and the ACJI between the two groups. The radiological assessment revealed no significant difference in the coracoclavicular distance. In both groups, a slight loss of reduction was observed. Periarticular ossification was seen in 11 patients of the minimally invasive AC joint repair and eight patients of the hook plate group but this did not affect the final outcome. Hook plates were removed after a median interval of 11.9 weeks (range 10-13). CONCLUSION: Good clinical results can be achieved with both minimally invasive AC joint repair and hook plate fixation. However, in the hook plate group a second operation is mandatory for plate removal. LEVEL OF EVIDENCE: III.

MACVIA-ARIA Sentinel NetworK for allergic rhinitis (MASK-rhinitis): the new generation guideline implementation.

Several unmet needs have been identified in allergic rhinitis: identification of the time of onset of the pollen season, optimal control of rhinitis and comorbidities, patient stratification, multidisciplinary team for integrated care pathways, innovation in clinical trials and, above all, patient empowerment. MASK-rhinitis (MACVIA-ARIA Sentinel NetworK for allergic rhinitis) is a simple system centred around the patient which was devised to fill many of these gaps using Information and Communications Technology (ICT) tools and a clinical decision support system (CDSS) based on the most widely used guideline in allergic rhinitis and its asthma comorbidity (ARIA 2015 revision). It is one of the implementation systems of Action Plan B3 of the European Innovation Partnership on Active and Healthy Ageing (EIP on AHA). Three tools are used for the electronic monitoring of allergic diseases: a cell phone-based daily visual analogue scale (VAS) assessment of disease control, CARAT (Control of Allergic Rhinitis and Asthma Test) and e-Allergy screening (premedical system of early diagnosis of allergy and asthma based on online tools). These tools are combined with a clinical decision support system (CDSS) and are available in many languages. An e-CRF and an e-learning tool complete MASK. MASK is flexible and other tools can be added. It appears to be an advanced, global and integrated ICT answer for many unmet needs in allergic diseases which will improve policies and standards.

Low-cost dielectric substrate for designing low profile multiband monopole microstrip antenna.

This paper proposes a small sized, low-cost multiband monopole antenna which can cover the WiMAX bands and C-band. The proposed antenna of 20 × 20 mm(2) radiating patch is printed on cost effective 1.6 mm thick fiberglass polymer resin dielectric material substrate and fed by 4 mm long microstrip line. The finite element method based, full wave electromagnetic simulator HFSS is efficiently utilized for designing and analyzing the proposed antenna and the antenna parameters are measured in a standard far-field anechoic chamber. The experimental results show that the prototype of the antenna has achieved operating bandwidths (voltage stand wave ratio (VSWR) less than 2) 360 MHz (2.53-2.89 GHz) and 440 MHz (3.47-3.91 GHz) for WiMAX and 1550 MHz (6.28-7.83 GHz) for C-band. The simulated and measured results for VSWR, radiation patterns, and gain are well matched. Nearly omnidirectional radiation patterns are achieved and the peak gains are of 3.62 dBi, 3.67 dBi, and 5.7 dBi at 2.66 GHz, 3.65 GHz, and 6.58 GHz, respectively.

Management of recurrent inguinal hernia at a tertiary care hospital of southern Sindh, Pakistan.

BACKGROUND: The goal of the present study was to determine the frequency, mode of presentation, and need for reoperation in the treatment of recurrent inguinal hernia at Liaquat University Hospital, Jamshoro, Sindh, Pakistan. METHODS: This descriptive, cross-sectional study was conducted over a period of four years, from January 2007 through December 2010. A total of 916 patients with inguinal hernia underwent operation in the Department of Surgery at Liaquat University Hospital Jamshoro, Sindh, Pakistan, during the study period. Of them, 62 patients were diagnosed to have recurrent inguinal hernia and were included in the study. Female patients and patients with other types of hernia like femoral, epigastric, and paraumbilical defects were excluded. RESULTS: Among the 62 patients studied, the commonest age group with recurrence was 41-50 years (43.5%). Fifty-one (82.2%) of the patients were ambulatory workers. In 47 cases (75.8%) recurrence was on the right side, and in 15 (24.1%) recurrence was on the left side. First time recurrence was seen in 54 patients (87%) and second time recurrence was seen in 7 patients (11.2 %); third time recurrence was observed in only one patient. All of these patients had an initial open surgery without mesh, except one. Bassini's repair was done as a primary repair in 47 (75.8%) cases. A total of 11 patients (17.7%) had no previous medical records. Darning repair was done in 3 patients (4.8%), and open surgery with mesh was performed in only one patient. The highest recurrence rates were seen in patients whose hernia repairs had been done by postgraduate trainees. That is, 45 of the patients requiring reoperation (72.5%), compared to 11 (17.7%), and 6 (9.6%) operations performed by registrars and consultants, respectively. All patients in our study underwent tension-free Lichtenstein mesh repair. Postoperative complications included retention of urine (40.3%), scrotal hematoma (6.4%), and wound infection (3.2%). CONCLUSIONS: Recurrent inguinal hernia is still frequently observed today, and the Lichtenstein tension-free repair has gained great acceptance worldwide and is currently considered the procedure of choice for primary and recurrent inguinal hernias.

Combining benefits of an adrenergic and a muscarinic blocker in a single formulation - a pharmacokinetic evaluation.

A pharmacokinetic bioequivalence study was conducted in Asian subjects, to compare a fixed dose combination capsule single oral dose of alpha adrenoceptor blocker-Alfuzosin hydrochloride 10mg extended release and muscarinic antagonists-Solifenacin succinate 5mg against individually administered Xatral XL 10mg tablets (Alfuzosin) of Sanofi Synthelabo Limited, United Kingdom (UK) and Vesicare 5mg tablets (Solifenacin) of Astellas Pharma Limited, UK under fed conditions. Blood samples were collected pre-dose up to 72 h post dose for determination of plasma Alfuzosin and Solifenacin concentrations and calculation of the pharmacokinetic parameters. ANOVA was performed on the log (natural)-transformed pharmacokinetic parameters. A 90% confidence interval for the ratios of the test and reference product averages (least square means) were calculated for alfuzosin and solifenacin. The 90% confidence intervals obtained for alfuzosin for Cmax, AUC0-t and AUC0-∞ were 102.74-122.75%, 95.84-116.96% and 95.82-116.76%, respectively. The 90% confidence intervals obtained for Solifenacin for Cmax, and AUC0-72 were 89.55-97.91% and 90.47-99.38%, respectively. Based on the results, the fixed dose combination was concluded to be bioequivalent to individually administered products.

Routine histopathology of gallbladder after elective cholecystectomy for gallstones: waste of resources or a justified act?

BACKGROUND: Selective approach for sending cholecystectomy specimens for histopathology results in missing discrete pathologies such as premalignant benign lesions such as porcelain gallbladder, carcinoma-in-situ, and early carcinomas. To avoid such blunders therefore, every cholecystectomy specimen should be routinely examined histologically. Unfortunately, the practice of discarding gallbladder specimen is standard in most tertiary care hospitals of Pakistan including the primary investigators' own institution. This study was conducted to assess the feasibility or otherwise of performing histopathology in every specimen of gallbladder. METHODS: This cohort study included 220 patients with gallstones for cholecystectomy. All cases with known secondaries from gallbladder, local invasion from other viscera, traumatic rupture of gallbladder, gross malignancy of gallbladder found during surgery was excluded from the study. Laparoscopic cholecystectomy was performed in majority of cases except in those cases where anatomical distortion and dense adhesions prevented laparoscopy. All gallbladder specimens were sent for histopathology, irrespective of their gross appearance. RESULTS: Over a period of two years, 220 patients with symptomatic gallstones were admitted for cholecystectomy. Most of the patients were females (88%). Ninety two per cent patients presented with upper abdominal pain of varying duration. All specimens were sent for histopathology. Two hundred and three of the specimens showed evidence chronic cholecystitis, 7 acute cholecystitis with mucocele, 3 acute cholecystitis with empyema and one chronic cholecystitis associated with poly. Six gallbladders (2.8%) showed adenocarcinoma of varying differentiation along with cholelithiasis. CONCLUSION: The histopathological spectrum of gallbladder is extremely variable. Incidental diagnosis of carcinoma gall bladder is not rare; if the protocol of routine histopathology of all gallbladder specimens is not followed, subclinical malignancies would fail to be identified with disastrous results. We strongly recommend routine histopathology of all cholecystectomy specimens.

Genetic pathways regulating the longitudinal acquisition of cocaine self-administration in a panel of inbred and recombinant inbred mice.

To identify addiction genes, we evaluate intravenous self-administration of cocaine or saline in 84 inbred and recombinant inbred mouse strains over 10 days. We integrate the behavior data with brain RNA-seq data from 41 strains. The self-administration of cocaine and that of saline are genetically distinct. We maximize power to map loci for cocaine intake by using a linear mixed model to account for this longitudinal phenotype while correcting for population structure. A total of 15 unique significant loci are identified in the genome-wide association study. A transcriptome-wide association study highlights the Trpv2 ion channel as a key locus for cocaine self-administration as well as identifying 17 additional genes, including Arhgef26, Slc18b1, and Slco5a1. We find numerous instances where alternate splice site selection or RNA editing altered transcript abundance. Our work emphasizes the importance of Trpv2, an ionotropic cannabinoid receptor, for the response to cocaine.

ESI-MS/MS stability-indicating bioanalytical method development and validation for simultaneous estimation of donepezil, 5-desmethyl donepezil and 6-desmethyl donepezil in human plasma.

A bioanalytical method was developed and validated to estimate donepezil, 6-desmethyl donepezil and 5-desmethyl donepezil simultaneously in human plasma using galantamine as an internal standard (IS). The chromatographic separation was achieved on a reverse-phase XTerra RP (150 × 4.6 mm, 5 µm) column without affecting recovery (mean recovery > 60% with CV < 10%) for all analytes. ESI-MS/MS multiple reaction monitoring in positive polarity was used to detect mass pairs for donepezil (m/z 380.3 → 91.3), 6-desmethyl donepezil (m/z 366.4 → 91.3), 5-desmethyl donepezil (m/z 366.4 → 91.3) and galantamine m/z (288.1 → 213.0). The linearity was established over a dynamic range of 0.339-51.870, 0.100-15.380 and 0.103-15.763 ng/mL for donepezil, 6-desmethyl donepezil and 5-desmethyl donepezil, respectively. The current method shows that minimal conversion of labile metabolites to parent donepezil in plasma as stability was successfully achieved for 211 days at -15 °C storage temperature. The method was successfully applied to a clinical study after administration of 10 mg donepezil tablets to healthy male Indian volunteers.

Single-cell analysis of gene expression in the substantia nigra pars compacta of a pesticide-induced mouse model of Parkinson's disease.

Exposure to pesticides in humans increases the risk of Parkinson's disease (PD), but the mechanisms remain poorly understood. To elucidate these pathways, we dosed C57BL/6J mice with a combination of the pesticides maneb and paraquat. Behavioral analysis revealed motor deficits consistent with PD. Single-cell RNA sequencing of substantia nigra pars compacta revealed both cell-type-specific genes and genes expressed differentially between pesticide and control, including Fam241b, Emx2os, Bivm, Gm1439, Prdm15, and Rai2. Neurons had the largest number of significant differentially expressed genes, but comparable numbers were found in astrocytes and less so in oligodendrocytes. In addition, network analysis revealed enrichment in functions related to the extracellular matrix. These findings emphasize the importance of support cells in pesticide-induced PD and refocus our attention away from neurons as the sole agent of this disorder.

Protective effect of rosuvastatin pretreatment against acute myocardial injury by regulating Nrf2, Bcl-2/Bax, iNOS, and TNF-α expressions affecting oxidative/nitrosative stress and inflammation.

Cardiovascular disorders are the leading cause of death globally. Rosuvastatin is a member of statins (inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase) with many pleiotropic properties. This study investigated cardioprotective effects of rosuvastatin in isoprenaline-induced myocardial injury. Male rats were given rosuvastatin (1, 5, or 10 mg/kg, oral) daily for 1 week and on seventh and eighth day isoprenaline (150 mg/kg, subcutaneous) was given to induce cardiac injury. On ninth day, rats were euthanized and different samples were harvested for analysis. Isoprenaline administration resulted in increased cardiac mass, increased cardiac injury marker levels (cTnI, CK-MB, ALT, and AST), increased lipid/protein oxidation, and increased cardiac nitrite levels. It also decreased superoxide dismutase, CAT, GST, and glutathione reductase activities, and total antioxidant activity. Isoprenaline also increased TNF-α and IL-6 levels. Decreased mRNA expression of Nrf2 and Bcl-2 along with increased mRNA expression of Bax, eNOS and iNOS genes was observed in isoprenaline treated animals. Histopathological evaluations of rosuvastatin pre-treated groups showed reduction of myocardial necrosis. Pretreatment with rosuvastatin (5 and 10 mg/kg) reduced many of these pathological changes. The current study showed that rosuvastatin significantly reduces myocardial injury induced by isoprenaline.