rAAV-compatible human mini promoters enhance transgene expression in rat retinal ganglion cells.

Recombinant adeno-associated viral vectors (rAAV) are the safest and most effective gene delivery platform to drive the treatment of many inherited eye disorders in well-characterized animal models. The use in rAAV of ubiquitous promoters derived from viral sequences such as CMV/CBA (chicken ß-actin promoter with cytomegalovirus enhancer) can lead to unwanted side effects such as pro-inflammatory immune responses and retinal cytotoxicity, thus reducing therapy efficacy. Thus, an advance in gene therapy is the availability of small promoters, that potentiate and direct gene expression to the cell type of interest, with higher safety and efficacy. In this study, we used six human mini-promoters packaged in rAAV2 quadruple mutant (Y-F) to test for transduction of the rat retina after intravitreal injection. After four weeks, immunohistochemical analysis detected GFP-labeled cells in the ganglion cell layer (GCL) for all constructs tested. Among them, Ple25sh1, Ple25sh2 and Ple53 promoted a widespread reporter-transgene expression in the GCL, with an increased number of GFP-expressing retinal ganglion cells when compared with the CMV/CBA vector. Moreover, Ple53 provided the strongest levels of GFP fluorescence in both cell soma and axons of retinal ganglion cells (RGCs) without any detectable adverse effects in retina function. Remarkably, a nearly 50-fold reduction in the number of intravitreally injected vector particles containing Ple53 promoter, still attained levels of transgene expression similar to CMV/CBA. Thus, the tested MiniPs show great potential for protocols of retinal gene therapy in therapeutic applications for retinal degenerations, especially those involving RGC-related disorders such as glaucoma.

Global DNA methylation changes in adults with attention deficit-hyperactivity disorder and its comorbidity with bipolar disorder: links with polygenic scores.

Genetic and environmental factors contribute to the etiology of Attention Deficit-Hyperactivity Disorder (ADHD). In this sense, the study of epigenetic mechanisms could contribute to the understanding of the disorder's neurobiology. Global DNA methylation (GMe) evaluated through 5-methylcytosine levels could be a promising epigenetic biomarker to capture long-lasting biological effects in response to environmental and hormonal changes. We conducted the first assessment of GMe levels in subjects with ADHD (n = 394) and its main comorbidities in comparison to populational controls (n = 390). Furthermore, given the high genetic contribution to ADHD (heritability of 80%), polygenic risk scores (PRS) were calculated to verify the genetic contribution to GMe levels in ADHD and the comorbidities associated with GMe levels. The GMe levels observed in patients were lower than controls (P = 1.1e-8), with women being significantly less globally methylated than men (P = 0.002). Regarding comorbidities, the presence of bipolar disorder (BD) among patients with ADHD was associated with higher methylation levels compared to patients with ADHD without BD (P = 0.031). The results did not change when pharmacological treatment was accounted for in the analyses. The ADHD and BD most predictive PRSs were negatively (P = 0.0064) and positively (P = 0.0042) correlated with GMe, respectively. This study is the first to report an association between GMe, ADHD, and its comorbidity with BD and associations between PRSs for specific psychiatric disorders and GMe. Our findings add to previous evidence that GMe may be a relevant piece in the psychiatric disorders' etiological landscape.

Residual contamination in antineoplastic drug packaging.

INTRODUCTION: The handling of antineoplastic drugs should follow strict supervision and safety rules to minimize the occupational exposure risks to professionals involved. The external surface contamination of drug vials is recognized as a health risk. So, our goal was to determine if there is residual contamination on the vials and containers surface of the antineoplastic drugs doxorubicin (DOX) and cyclophosphamide (CP). METHODS: A cross-sectional study was conducted. Samples were collected using a uniform sampling procedure on the inner surfaces of the packages/boxes and the outer surfaces of the vials. The analyzes were executed by high-performance liquid chromatography/mass spectrometry (UHPLC-MS/MS). RESULTS: A total of 209 samples were analyzed, 66 of CP and 143 of DOX. CP levels were detected in nine samples (13.63%), three were below the lower limit of quantification (LLQ) and the other six had contamination levels ranging from 1.24 to 28.04 ng/filter. DOX levels were detected in 36 samples (25.17%), two were below the LLQ and the others had levels between 1.32 and 664.84 ng/filter. The majority of samples with residual contamination were in vials (80.0%), however, boxes also showed contamination. CONCLUSIONS: The results revealed the presence of residual contamination in the vials and packages of CP and DOX drugs. Although the residues found in each sample are small, special care should be taken in the handling and disposal of the antineoplastic drugs. The use of personal protective equipment is fundamental while handling the vials and packaging of cytotoxic drugs.

Evaluation of oxidative stress and its association with drug therapy in inpatients treated for cocaine dependence.

The use of cocaine affects several systems and organs of the human body and the consumption of this substance leads to an increase in the production of reactive oxygen species, and to the reduction of antioxidant defenses. The aim of this study was to evaluate the oxidative stress (OS), biochemical and hematological parameters in patients hospitalized for treatment of cocaine addiction, comparing levels at hospital admission and discharge. Forty patients were included in the study. OS was evaluated using catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GPx), total antioxidant power (FRAP), malondialdehyde (MDA), and sulfhydryl group (GS). The medications used during hospitalization were registered and their influence on the parameters of OS was analyzed. After the hospitalization period, there was an increase in GGT levels, a reduction in SOD activity, and an increase in GPx activity and FRAP levels. Carbamazepine users had higher SOD values and lower FRAP values at hospital discharge. The use of chlorpromazine caused differences in creatinine and gamma-glutamyltransferase (GGT) serum leves, and the levels of glutamic oxalacetic transaminase (TGO), MDA, and FRAP were increased at hospital discharge. Haloperidol and thiamine during hospitalization interfered with alkaline phosphatase levels. The use of risperidone caused an increase in the levels of SOD, and folic acid use was associated with lower levels of GPx and higher levels of glutamic-pyruvic transaminase (TGP) and alkaline phosphatase. Drug rehabilitation treatment was effective in decreasing oxidative damage represented by the reduction of biological markers.

De novo genesis of retinal ganglion cells by targeted expression of Klf4 in vivo.

Retinal ganglion cell (RGC) degeneration is a hallmark of glaucoma, the most prevalent cause of irreversible blindness. Thus, therapeutic strategies are needed to protect and replace these projection neurons. One innovative approach is to promote de novo genesis of RGCs via manipulation of endogenous cell sources. Here, we demonstrate that the pluripotency regulator gene Krüppel-like factor 4 (Klf4) is sufficient to change the potency of lineage-restricted retinal progenitor cells to generate RGCs in vivo Transcriptome analysis disclosed that the overexpression of Klf4 induces crucial regulators of RGC competence and specification, including Atoh7 and Eya2 In contrast, loss-of-function studies in mice and zebrafish demonstrated that Klf4 is not essential for generation or differentiation of RGCs during retinogenesis. Nevertheless, induced RGCs (iRGCs) generated upon Klf4 overexpression migrate to the proper layer and project axons aligned with endogenous fascicles that reach the optic nerve head. Notably, iRGCs survive for up to 30 days after in vivo generation. We identified Klf4 as a promising candidate for reprogramming retinal cells and regenerating RGCs in the retina.This article has an associated 'The people behind the papers' interview.

Evaluation of oxidative stress biomarkers and liver and renal functional parameters in patients during treatment a mental health unit to treat alcohol dependence.

Alcohol dependence is one of the main reasons for inpatient admission to psychiatric hospitals. The abuse of this chemical substance can cause modifications in our organism and among them, variations in the oxidative stress parameters. Therefore, the aim of this study is to evaluate patients admitted to a psychiatric hospital unit to treat alcohol dependence, comparing oxidative stress, renal and hepatic function parameters from the moment of admission to those obtained at discharge. Hepatic function was verified through gamma-glutamyl-transferase (GGT), alkaline-phosphatase (ALP), aspartate-aminotransferase (AST) and alanine-aminotransferase (ALT) activity measurements. Urea and creatinine serum levels were measured for kidney function evaluation. Oxidative stress was evaluated by superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), ferric reducing antioxidant power (FRAP) and malondialdehyde (MDA). Medications used during hospital stay were record and their influence over the measured parameters analyzed. Twenty-eight patients (82% male, 44 ± 13 years old) were included in this study. A significant increase in BMI of patients after the period of hospitalization could be observed. There were reductions in creatinine, AST, ALT, GGT and ALP serum levels. SOD levels were lower at discharge, while GPx and FRAP presented higher levels. Chlorpromazine use showed influence over some hepatic function markers (ALT, GGT and ALP) and oxidative stress parameters (CAT and GPx); while carbamazepine use influenced GGT and FRAP. Patients on alcohol dependence treatment had significant improvements of renal and hepatic function parameters and higher GPx and FRAP values after the hospitalization period, which indicates reversion of alcohol effects over oxidative stress parameters.

Roles of glutamate receptors in a novel in vitro model of early, comorbid cerebrovascular, and Alzheimer's diseases.

Systemic multimorbidity is highly prevalent in the elderly and, remarkably, coexisting neuropathological markers of Alzheimer's (AD) and cerebrovascular (CVD) diseases are found at autopsy in most brains of patients clinically diagnosed as AD. Little is known on neurodegeneration peculiar to comorbidities, especially at early stages when pathogenesis may propagate at subclinical levels. We developed a novel in vitro model of comorbid CVD/AD in organotypic hippocampal cultures, by combining oxygen-glucose deprivation (OGD) and exposure to amyloid-Aß oligomers (AßOs), both applied at levels subtoxic to neurons when used in isolation. We focused on synaptic proteins and the roles of glutamate receptors, which have been implicated in many basic and clinical approaches to either CVD or AD. Subtoxic insults by OGD and AßOs synergized to reduce levels of synaptophysin (SYP) and PSD-95 without cell death, while effects of antagonists of either metabotropic or ionotropic glutamate receptors were distinct from reports in models of isolated CVD or AD. In particular, modulation of glutamate receptors differentially impacted SYP and PSD-95, and antagonists of a single receptor subtype had distinct effects when either isolated or combined. Our findings highlight the complexity of CVD/AD comorbidity, help understand variable responses to glutamate receptor antagonists in patients diagnosed with AD and may contribute to future development of therapeutics based on investigation of the pattern of progressive comorbidity.

Cell proliferation in the central nervous system of an adult semiterrestrial crab.

Neurogenesis occurs in adults of most organisms, both vertebrates and invertebrates. In semiterrestrial crabs of the infraorder Brachyura, the deutocerebrum, where neurogenesis occurs, processes the olfactory sensory information from the antennae. The deutocerebrum is composed of a pair of olfactory lobes associated with cell clusters 9 and 10 (Cl 9 and Cl 10), containing proliferating cells. Because the location of the neurogenic niche in brachyuran semiterrestrial crabs has not been defined, here we describe a neurogenic niche in the central olfactory system of the crab Ucides cordatus and report two types of glial cells in the deutocerebrum, based on different markers. Serotonin (5-hydroxytryptamine) labeling was used to reveal neuroanatomical aspects of the central olfactory system and the neurogenic niche. The results showed a zone of proliferating neural cells within Cl 10, which also contains III beta-tubulin (Tuj1)+ immature neurons, associated with a structure that has characteristics of the neurogenic niche. For the first time, using two glial markers, glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS), we identified two types of astrocyte-like cells in different regions of the deutocerebrum. This study adds to the understanding of neurogenesis in a brachyuran semiterrestrial crustacean and encourages comparative studies between crustaceans and vertebrates, including mammals, based on shared aspects of both mechanisms of neurogenesis and regenerative potentials.

Alternative Sampling Devices to Collect Dried Blood Microsamples: State-of-the-Art.

ABSTRACT: Dried blood spots (DBS) have been used in newborn screening programs for several years. More recently, there has been growing interest in using DBS as a home sampling tool for the quantitative determination of analytes. However, this presents challenges, mainly because of the well-known hematocrit effect and other DBS-specific parameters, including spotted volume and punch site, which could add to the method uncertainty. Therefore, new microsampling devices that quantitatively collect capillary dried blood are continuously being developed. In this review, we provided an overview of devices that are commercially available or under development that allow the quantitative (volumetric) collection of dried blood (-based) microsamples and are meant to be used for home or remote sampling. Considering the field of therapeutic drug monitoring (TDM), we examined different aspects that are important for a device to be implemented in clinical practice, including ease of patient use, technical performance, and ease of integration in the workflow of a clinical laboratory. Costs related to microsampling devices are briefly discussed, because this additionally plays an important role in the decision-making process. Although the added value of home sampling for TDM and the willingness of patients to perform home sampling have been demonstrated in some studies, real clinical implementation is progressing at a slower pace. More extensive evaluation of these newly developed devices, not only analytically but also clinically, is needed to demonstrate their real-life applicability, which is a prerequisite for their use in the field of TDM.

Dried Plasma Spots and Oral Fluid as Alternative Matrices for Therapeutic Drug Monitoring of Busulfan: Analytical Method Development and Clinical Evaluation.

BACKGROUND: Busulfan (BU) is an alkylating agent with a narrow therapeutic index and high intraindividual pharmacokinetic variability used in conditioning therapy for hematopoietic stem cell transplantation. Monitoring BU exposure during high-dose conditioning regimens is recommended and positively impacts outcomes. We aimed to develop, validate, and apply a ultra-high-performance liquid chromatography-mass spectrometry (MS)/MS assay to measure BU concentrations in oral fluid and dried plasma spots (DPS) as alternative matrices to plasma. METHODS: We prepared plasma and oral fluid samples by protein precipitation and DPS after liquid extraction. We analyzed extracts using an LC-MS/MS system with an Acquity HSS T3 column in the positive electrospray ionization mode. The method was validated and applied to 79 paired plasma and oral fluid samples from 7 patients on BU conditioning treatment. DPS were prepared by pipetting plasma onto Whatman 903 paper. The correlation between BU in plasma, oral fluid, and DPS samples was evaluated. RESULTS: Run time was 4.0 minutes. The assay was linear at 50-5000 ng mL-1 (r > 0.99), precise (1.9%-5.3% oral fluid and 1.8%-5.9% DPS), and accurate (98.1%-108.9% oral fluid and 93%-103.1% DPS). BU was stable in DPS at 23°C for 24 hours. BU levels in oral fluid (r = 0.927) and DPS (r = 0.982) were significantly correlated with plasma. Despite the good correlation, we found a wide variation between oral fluid and plasma levels. The area under curves (AUCs) calculated with oral fluid concentrations were 79.1%-167.1% of plasma AUCs. Bland-Altman plots found a better agreement for DPS, with AUCs estimated from corrected DPS levels at 83.1%-114.1% of plasma values. CONCLUSIONS: We developed and validated a simple and fast ultra-high-performance liquid chromatography-MS/MS assay to measure BU in oral fluid and DPS. The results do not support the use of oral fluid as a matrix for routine therapeutic drug monitoring of BU. The AUC estimated from BU measurements in DPS was comparable to that in plasma, supporting the use of DPS in BU therapeutic drug monitoring as an alternative matrix, with adequate short-term stability and logistic advantages.

Simple extraction of toxicologically relevant psychotropic compounds and metabolites from whole blood using mini-QuEChERS followed by UPLC-MS/MS analysis.

The determination of psychotropic drugs and metabolites in blood is relevant in the context of both therapeutic drug monitoring and clinical and forensic toxicology. LC-MS/MS is the preferred method for these assays. However, LC-MS/MS is particularly susceptible to matrix ionization effects and appropriate sample preparation is required to minimize these effects. In this study, a simple, single-step, mini-QuEchERS extraction procedure, coupled to UPLC-MS/MS, was developed and validated for the determination of 15 toxicologically relevant compounds in whole blood, including psychoactive drugs and some metabolites. The assay was linear in the range of 25-1,000 ng ml-1 , fulfilling criteria for accuracy and precision. Extraction yields (71.9-87.7%) and matrix effects (-3.3 to +4.4%, with the exception of codeine, which had matrix effects of -35.36 to -28.14%) were acceptable for the majority of the evaluated compounds, using a single internal standard. The assay was applied to 238 clinical specimens from patients admitted to an emergency service, with 22 samples presenting quantifiable concentrations of 11 different compounds. The developed assay is a simple and efficient strategy for determination of target psychotropic drugs and metabolites in forensic and clinical toxicology.

A quick UPLC-MS/MS method for therapeutic drug monitoring of abiraterone and delta(4)-abiraterone in human plasma.

Abiraterone acetate efficacy against prostate cancer is dependent on the circulating levels of abiraterone and its active metabolites, which present significant pharmacokinetic variability among patients. Thus, therapeutic drug monitoring can be performed to improve treatment outcomes. To support such studies, there are only a limited number of bioanalytical methods in current literature. This work presents a fast method to quantify abiraterone and D4A in plasma in 4 min by UPLC-MS/MS. Bioanalytical method validation was performed according to the recommendations of the US Food and Drug Administration. The method was linear within the range of 1-400 ng/ml for abiraterone and 0.2-20 ng/ml for D4A (r2 > 0.99). Based on the analysis of quality control samples at the lower limit of quantification, low, medium and high concentrations, the method was precise (CVabiraterone ≤ 9.72%; CVD4A ≤ 14.64%) and accurate (CVabiraterone 95.51-107.59%; CVD4A 98.04-99.89%). Application of the method to the quantification of abiraterone and D4A in 10 clinical samples revealed important variability in the conversion ratio of abiraterone to D4A (CV 90.85%). Considering the current literature, this is the fastest method to quantify abiraterone and D4A in plasma, allowing for optimization of the analytical routine.

Determination of cannabinoids in plasma using salting-out-assisted liquid-liquid extraction followed by LC-MS/MS analysis.

The detection of the markers of Cannabis consumption in biological specimens is an important task for drug testing laboratories in varous contexts. A simple assay combining salting-out assisted liquid-liquid extraction sample preparation and LC-MS/MS analysis was applied to the measurement of Δ9 -tetrahydrocannabinol, 11-nor-9-carboxy-Δ9 -tetrahydrocannabinol (THC-COOH), 11-hydroxy-Δ9 -tetrahydrocannabinol, cannabinol and cannabidiol concentrations in 100 µl plasma specimens. The assay had linearity of 1-100 ng ml-1 for THC-COOH and 0.5-50 ng ml-1 for the other tested cannabinoids. Assay validation criteria were fulfilled. Extraction yields (88.7-97.3%) and internal-standard correct matrix effects (-9.6 to +5.4%) were acceptable. The assay was applied to 238 clinical specimens from trauma patients, with 19 samples presenting quantifiable concentrations of at least one of the target compounds. The developed assay is a simple and efficient strategy for simultaneous measurement of Δ9 -tetrahydrocannabinol, THC-COOH, 11-hydroxy-Δ9 -tetrahydrocannabinol, cannabinol and cannabidiol concentrations in plasma specimens.

Investigation of SIRT1 gene variants in HIV-associated lipodystrophy and metabolic syndrome.

HIV-infected individuals on chronic use of highly active antiretroviral therapy (HAART) are more likely to develop adipose tissue and metabolic disorders, such as lipodystrophy (LD) and metabolic syndrome (MetS). The development of these phenotypes is known to be multifactorial. Thus, variants in genes implicated in adipogenesis and lipid metabolism may increase susceptibility to LD and MetS. Sirtuin 1 (SIRT1) may influence the outcome of these disturbances due to its role in the regulation of transcription factors involved in energy regulation. Therefore, we genotyped four polymorphisms located in SIRT1 (rs2273773 T>C, rs12413112 G>A, rs7895833 A>G, rs12049646 T>C) in 832 HIV-infected patients receiving HAART by real-time polymerase chain reaction. The prevalence of LD was 55.8% and MetS was 35.3%. Lipoatrophy was the most prevalent subtype in all samples (38.0%) and showed significant difference between white and non-white individuals (P = 0.002). None of the genetic variants investigated in SIRT1 was associated with LD and MetS. White individuals and those in longer time of HAART use were more likely to develop LD. We concluded that these SIRT1 polymorphisms are not predictive factors to the development of lipodystrophy and metabolic syndrome in HIV-infected individuals from Brazil.

Retina transduction by rAAV2 after intravitreal injection: comparison between mouse and rat.

Adeno-associated virus vectors (rAAV) are currently the most common vehicle used in clinical trials of retinal gene therapy, usually delivered through subretinal injections to target cells of the outer retina. However, targeting the inner retina requires intravitreal injections, a simple and safe procedure, which is effective for transducing the rodent retina, but still of low efficiency in the eyes of primates. We investigated whether adjuvant pharmacological agents may enhance rAAV transduction of the retinas of mouse and rat after intravitreal delivery. Tyrosine kinase inhibitors were highly efficient in mice, especially imatinib and genistein, and promoted transduction even of the outer retina. In rats, however, we report that they were not effective. Even with direct proteasomal inhibition in rats, the effects upon transduction were only minimal and restricted to the inner retina. Even tyrosine capsid mutant rAAVs in rats had a transduction profile similar to wtAAV. Thus, the differences between mouse and rat, in both eye size and the inner limiting membrane, compromise the efficiency of AAV vectors penetration from the vitreous into the retina, and impact the efficacy of strategies developed to enhance intravitreal retinal rAAV transduction. Further improvement of strategies, then are required.

Determination of Endogenous Concentrations of Uracil and Dihydrouracil in Dried Saliva Spots by LC-MS/MS: Method Development, Validation, and Clinical Application.

BACKGROUND: The aim of this study was to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the measurement of uracil (U) and dihydrouracil (UH2) concentrations in dried saliva spots (DSSs), for the evaluation of dihydropyrimidine dehydrogenase (DPD) enzyme activity. RESULTS: Nine 18-mm diameter DSS discs were extracted with acetate:isopropyl alcohol (85:15, vol/vol) and analyzed by LC-MS/MS. The assay was linear in the range of 10-1000 ng·mL, with accuracy between 89% and 112% and precision between 5.7% and 13%. The metabolic ratio [UH2]/[U] was stable in DSS for up to 9 days at 45°C. Concentrations of U and UH2, as well as the metabolic ratio, were highly concordant between matrices. Using a metabolic ratio classification cutoff of 1.16 for the identification of slow DPD metabolizers, 98.7% concordance was achieved between SS and saliva. CONCLUSIONS: DSS samples could be a useful alternative for DPD activity screening, particularly in locations with limited access to highly equipped laboratories.

Official International Association for Therapeutic Drug Monitoring and Clinical Toxicology Guideline: Development and Validation of Dried Blood Spot-Based Methods for Therapeutic Drug Monitoring.

Dried blood spot (DBS) analysis has been introduced more and more into clinical practice to facilitate Therapeutic Drug Monitoring (TDM). To assure the quality of bioanalytical methods, the design, development and validation needs to fit the intended use. Current validation requirements, described in guidelines for traditional matrices (blood, plasma, serum), do not cover all necessary aspects of method development, analytical- and clinical validation of DBS assays for TDM. Therefore, this guideline provides parameters required for the validation of quantitative determination of small molecule drugs in DBS using chromatographic methods, and to provide advice on how these can be assessed. In addition, guidance is given on the application of validated methods in a routine context. First, considerations for the method development stage are described covering sample collection procedure, type of filter paper and punch size, sample volume, drying and storage, internal standard incorporation, type of blood used, sample preparation and prevalidation. Second, common parameters regarding analytical validation are described in context of DBS analysis with the addition of DBS-specific parameters, such as volume-, volcano- and hematocrit effects. Third, clinical validation studies are described, including number of clinical samples and patients, comparison of DBS with venous blood, statistical methods and interpretation, spot quality, sampling procedure, duplicates, outliers, automated analysis methods and quality control programs. Lastly, cross-validation is discussed, covering changes made to existing sampling- and analysis methods. This guideline of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology on the development, validation and evaluation of DBS-based methods for the purpose of TDM aims to contribute to high-quality micro sampling methods used in clinical practice.

rAAV8-733-Mediated Gene Transfer of CHIP/Stub-1 Prevents Hippocampal Neuronal Death in Experimental Brain Ischemia.

Brain ischemia is a major cause of adult disability and death, and it represents a worldwide health problem with significant economic burden for modern society. The identification of the molecular pathways activated after brain ischemia, together with efficient technologies of gene delivery to the CNS, may lead to novel treatments based on gene therapy. Recombinant adeno-associated virus (rAAV) is an effective platform for gene transfer to the CNS. Here, we used a serotype 8 rAAV bearing the Y733F mutation (rAAV8-733) to overexpress co-chaperone E3 ligase CHIP (also known as Stub-1) in rat hippocampal neurons, both in an oxygen and glucose deprivation model in vitro and in a four-vessel occlusion model of ischemia in vivo. We show that CHIP overexpression prevented neuronal degeneration in both cases and led to a decrease of both eIF2α (serine 51) and AKT (serine 473) phosphorylation, as well as reduced amounts of ubiquitinated proteins following hypoxia or ischemia. These data add to current knowledge of ischemia-related signaling in the brain and suggest that gene therapy based on the role of CHIP in proteostasis may provide a new venue for brain ischemia treatment.

Characterization of imatinib mesylate formulations distributed in South American countries: Determination of genotoxic impurities by UHPLC-MS/MS and dissolution profile.

Imatinib mesylate (IM) is an anti-neoplasic drug used for the treatment of cancer. Recent new guidelines specify daily doses and concentration limits for genotoxic impurities (GTIs) in pharmaceutical final products. Therefore, in this work an analytical method using UHPLC-MS/MS was developed, validated and applied to characterize IM tablets for two GTIs: N-(2-methyl-5-aminophenyl)-4-(3-pyridyl)-2-pyrimidine amine (Imp. 1), and N-[4-methyl-3-(4-methyl-3-yl-pyrimidin-2-ylamino)-phenyl]-4- chloromethyl benzamide (Imp. 2), simultaneously. Additionally, dissolution data of IM tablets were compared using a methodology recommended by the US Food and Drug Administration. The UHPLC method utilized an Acquity BEH C18 (150 × 2.1 mm, 1.7 µm) maintained at 40°C. The mobile phase consisted of ammonium formate 0.063% (phase A) and acetonitrile plus 0.05% formic acid (phase B) in gradient elution. A sensitive method for determination of previously mentioned GTIs in IM tablets was successfully developed and applied. Overall, the formulations analyzed in this work showed low levels of Imp. 1 and Imp. 2. However, the sample named D1 showed very high levels of Imp. 1 and failed to meet the requirements established by the US Food and Drug Administration for dissolution data. Periodic verification of GTIs in pharmaceutical formulations is important to minimize safety risks, so analytical methods to determine it need be available and implemented in routine analysis.

Cellular effects of thirdhand tobacco smoke from smokers' homes.

After the cigarette is extinguished, many toxic compounds remain in the environment and accumulate in the air or on surfaces. This exposure is termed thirdhand smoke (THS) and its risks are poorly known. The aim of the study was to evaluate the cellular effects of THS from smokers' homes. Papers were placed in nine smoker's home and three nonsmoker's homes. An area equivalent to the paper size was cleaned with a cotton wipe. A549, Hep-2 and 3T3 cells were exposed to THS for 24 h and cellular functions were assessed by MTT, neutral red (NR) reuptake and trypan blue exclusion assays. High levels of nicotine were found in samples from smokers' homes. Cellular proliferation was similar in almost all samples after THS exposure. Few changes in the cellular functions were observed, mainly higher mitochondrial activity, in paper samples. We are able to detect markers of THS collected from smokers' homes, but a clear evidence of cellular toxicity cannot be demonstrated by the present assays. This is the first study to evaluate the cellular effects of THS samples collected from smokers' homes.