Overexpression of isocitrate dehydrogenase mutant proteins renders glioma cells more sensitive to radiation

Mutations in isocitrate dehydrogenase 1 (IDH1) or 2 (IDH2) are found in a subset of gliomas. Among the many phenotypic differences between mutant and wild-type IDH1/2 gliomas, the most salient is that IDH1/2 mutant glioma patients demonstrate markedly improved survival compared with IDH1/2 wild-type glioma patients. To address the mechanism underlying the superior clinical outcome of IDH1/2 mutant glioma patients, we investigated whether overexpression of the IDH1^R132H protein could affect response to therapy in the context of an isogenic glioma cell background. Stable clonal U87MG and U373MG cell lines overexpressing IDH1^WT and IDH1^R132H were generated, as well as U87MG cell lines overexpressing IDH2^WT and IDH2^R172K. In vitro experiments were conducted to characterize baseline growth and migration and response to radiation and temozolomide. In addition, reactive oxygen species (ROS) levels were measured under various conditions. U87MG-IDH1^R132H cells, U373MG-IDH1^R132H cells, and U87MG-IDH2^R172K cells demonstrated increased sensitivity to radiation but not to temozolomide. Radiosensitization of U87MG-IDH1^R132H cells was accompanied by increased apoptosis and accentuated ROS generation, and this effect was abrogated by the presence of the ROS scavenger N-acetyl-cysteine. Interestingly, U87MG-IDH1^R132H cells also displayed decreased growth at higher cell density and in soft agar, as well as decreased migration. Overexpression of IDH1^R132H and IDH2^R172K mutant protein in glioblastoma cells resulted in increased radiation sensitivity and altered ROS metabolism and suppression of growth and migration in vitro. These findings provide insight into possible mechanisms contributing to the improved outcomes observed in patients with IDH1/2 mutant gliomas.     

Gene‐expression signature of tumor recurrence in patients with stage II and III colon cancer treated with 5′fluoruracil‐based adjuvant chemotherapy

Although receiving adjuvant chemotherapy after radical surgery, a disappointing proportion of patients with colorectal cancer will develop tumor recurrence. Probability of relapse is currently predicted from pathological staging, there being a need for additional markers to further select high‐risk patients. This study was aimed to identify a gene‐expression signature to predict tumor recurrence in patients with Stages II and III colon cancer treated with 5′fluoruracil (5FU)‐based adjuvant chemotherapy. Two‐hundred and twenty‐eight patients diagnosed with Stages II–III colon cancer and treated with surgical resection and 5FU‐based adjuvant chemotherapy were included. RNA was extracted from formalin‐fixed, paraffin‐embedded tissue samples and expression of 27 selected candidate genes was analyzed by RT‐qPCR. A tumor recurrence predicting model, including clinico‐pathological variables and gene‐expression profiling, was developed by Cox regression analysis and validated by bootstrapping. The regression analysis identified tumor stage and S100A2 and S100A10 gene expression as independently associated with tumor recurrence. The risk score derived from this model was able to discriminate two groups with a highly significant different probability of tumor recurrence (HR, 2.75; 95%CI, 1.71–4.39; p = 0.0001), which it was maintained when patients were stratified according to tumor stage. The algorithm was also able to distinguish two groups with different overall survival (HR, 2.68; 95%CI, 1.12–6.42; p = 0.03). Identification of a new gene‐expression signature associated with a high probability of tumor recurrence in patients with Stages II and III colon cancer receiving adjuvant 5FU‐based chemotherapy, and its combination in a robust, easy‐to‐use and reliable algorithm may contribute to tailor treatment and surveillance strategies.     

Human and Rat ABC Transporter Efflux of Bisphenol A and Bisphenol A Glucuronide: Interspecies Comparison and Implications for Pharmacokinetic Assessment

Significant interspecies differences exist between human and rodent with respect to absorption, distribution, and excretion of bisphenol A (BPA) and its primary metabolite, BPA-glucuronide (BPA-G). ATP-Binding Cassette (ABC) transporter enzymes play important roles in these physiological processes, and their enzyme localization (apical vs. basolateral) in the plasma membrane allows for different cellular efflux pathways. In this study, we utilized an ATPase assay to evaluate BPA and BPA-G as potential substrates for the human and rat ABC transporters: P-glycoprotein (MDR1), multidrug resistance-associated proteins (MRPs), and breast cancer-resistant protein (BCRP). Based on high ATPase activity, BPA is likely a substrate for rat mdr1b but not for human MDR1 or rat mdr1a. Results indicate that BPA is a potential substrate for rat mrp2 and human MRP2, BCRP, and MRP3. The metabolite BPA-G demonstrated the highest apparent substrate binding affinity for rat mrp2 and human MRP3 but appeared to be a nonsubstrate or potential inhibitor for human MRP2, MDR1, and BCRP and for rat mdr1a, mdr1b, and bcrp. Analysis of ABC transporter amino acid sequences revealed key differences in putative binding site composition that may explain substrate specificity. Collectively, these results suggest that in both rat and human, apical transporters efflux BPA into the bile and/or intestinal lumen. BPA-G would follow a similar pathway in rat; however, in human, due to the basolateral location of MRP3, BPA-G would likely enter systemic and portal blood supplies. These differences between human and rodent ABC transporters may have significant implications for interspecies extrapolation used in risk assessment.     

Alcohol and the human brain: a systematic review of different neuroimaging methods

Background: Imaging techniques have been in widespread use in the scientific community for more than 3 decades. They facilitate noninvasive, in vivo studies of the human brain in both healthy and diseased persons. These brain‐imaging techniques have contributed significantly to our understanding of the effects of alcohol abuse and dependence on structural and functional changes in the human brain. A systematic review summarizing these contributions has not previously been conducted, and this is the goal of the current paper. Methods: The databases PubMed, PsycINFO, and PSYNDEX were searched using central key words. Fulfilling the inclusion criteria were 140 functional and structural imaging studies, together comprising data from more than 7,000 patients and controls. The structural imaging techniques we considered were cranial computerized tomography and various magnetic resonance imaging–based techniques, including voxel‐based morphometry, deformation‐based morphometry, diffusion tensor magnetic resonance imaging, and diffusion‐weighted magnetic resonance imaging. The functional methods considered were magnetic resonance spectroscopy, positron emission tomography, single photon emission computed tomography, and functional magnetic resonance imaging. Results: Results from studies using structural imaging techniques have revealed that chronic alcohol use is accompanied by volume reductions of gray and white matter, as well as microstructural disruption of various white matter tracts. These changes are partially reversible following abstinence. Results from functional imaging methods have revealed metabolic changes in the brain, lower glucose metabolism, and disruptions of the balance of neurotransmitter systems. Additionally, functional imaging methods have revealed increased brain activity in the mesocorticolimbic system in response to alcohol‐themed pictures relative to nondrug‐associated stimuli, which might be of predictive value with regard to relapse. Conclusions: There has been tremendous progress in the development of imaging technologies. Use of these technologies has clearly demonstrated the structural and functional brain abnormalities that can occur with chronic alcohol use. The study of the alcoholic brain provides an heuristic model which furthers our understanding of neurodegenerative changes in general, as well as their partial reversibility with sustained abstinence. Additionally, functional imaging is poised to become an important tool for generating predictions about individual brain functioning, which can then be used as a basis for personalized medicine.     

Prevalence of sleep problems in Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive genetic disorder, characterized by multiple congenital anomalies, and intellectual disability. It is caused by a genetically inherited deficiency of the enzyme 7-dehydrocholesterol-delta-7-reductase, which results in increased serum levels of 7-dehydrocholesterol (7-DHC), and decreased levels of cholesterol. This study assesses the prevalence of sleep problems in patients with SLOS. The study group comprised 18 subjects with SLOS, ages 2-31 years (median 10.7 ± 8.5 years). Parents completed several questionnaires (Intake Demographic Form; Pediatric Sleep Questionnaire; Pediatric Daytime Sleepiness Scale). The SLOS subjects had symptoms of sleep-disordered breathing (50% snoring; 66.7% mouth breathing), problems with sleep onset [difficulty falling asleep (61.1%) sleep onset time >30 min (62%)], sleep maintenance [wake up screaming (61.1%), waking up more than twice (44.4%), having trouble falling back to sleep (66.7%), waking up early in the morning (61.1%), and restless sleep (50%)]. The subjects with SLOS needed parents in the room to fall asleep (50%), watch TV or listen to music to fall asleep (44.4%), and described bed sharing (33.3%), indicating sleep-anxiety and sleep-associations. Symptoms of excessive-daytime-sleepiness were frequently reported [un-refreshed in the morning (38.9%), daytime sleepiness (44.4%), and daytime naps (55.6%)]. Parents frequently observed difficulty of organizing tasks (66.7%), and easy distractibility (88.9%). Sleep problems such as sleep-disordered breathing, sleep-related anxiety and sleep associations, disturbed sleep patterns at night, and excessive daytime sleepiness are frequent in children with SLOS.     

A multicentre study of antifungal strategies and outcome of Candida spp. peritonitis in intensive-care units

Information on the species causing Candida peritonitis, their in vitro susceptibility, antifungal strategies in this setting and patient outcome is still scarce. AmarCand was a prospective, non-interventional study in 271 adult intensive-care unit (ICU) patients with proven invasive Candida infection who received systemic antifungal therapy (France, 2005–2006). Of these ICU patients, 93 (median age 65 years, simplified acute physiology score II 52) had Candida peritonitis, including 73 nosocomial peritonitis, 53 concomitant bacterial peritoneal infections and 26 candidaemias. Candida species were C. albicans (n = 63/108 isolates, 58%), C. glabrata (n = 22, 20%), C. krusei (n = 9), C. kefyr (n = 5), C. parapsilosis (n = 3), C. tropicalis (n = 3), C. ciferii (n = 2) and C. lusitaniae (n = 1). Of tested isolates, 28% were fluconazole-resistant or susceptible dose-dependent (C. albicans 3/32, C. glabrata 9/14, C. krusei 4/4). Empiric antifungal treatment was started 1 day (median) after peritonitis diagnosis, with fluconazole (n = 72 patients), caspofungin (n = 12), voriconazole (n = 3), amphotericin B (n = 2), or a combination (n = 4). Following susceptibility testing, empiric antifungal treatment was judged inadequate in 9/45 (20%) patients and modified in 30 patients (fluconazole was replaced by caspofungin (n = 14) or voriconazole (n = 4)). Mortality in ICU was 38% (35/93) and was not influenced by type of Candida species, fluconazole susceptibility, time to treatment, candidaemia, nosocomial acquisition, or concomitant bacterial infection. No specific factors for death were identified. In summary, a high proportion of fluconazole-resistant or susceptible dose-dependent strains was cultured. These results confirm the high mortality rates of Candida peritonitis and plead for additional investigation in this population. Antifungal treatment for severe cases of Candida peritonitis in ICU patients remains the standard care.     

Retro peptide-hybrids as selective inhibitors of the Dengue virus NS2B-NS3 protease

New chemotherapeutics against Dengue virus and related flaviviruses are of growing interest in antiviral drug discovery. The viral serine protease NS2B-NS3 is a promising target for the development of such agents. Drug-like inhibitors of this protease with high affinity to the target are not available at the moment. The present work describes the discovery of new retro di- and tripeptide hybrids that do not necessarily require an electrophilic "warhead" to achieve affinities in the low micromolar range. The most active sequence in this series is the tripeptide R-Arg-Lys-Nle-NH(2). By variation of the N-terminal groups (R) it could be shown that the previously described arylcyanoacrylamide moiety is a preferable group in this position. Retro tripeptide hybrids were found to be more active and more selective than retro dipeptide hybrids. A significant selectivity towards the Dengue virus protease could be shown in a counterscreen with thrombin and the West Nile virus protease. Alternative sequences to R-Arg-Lys-Nle-NH(2) did not have higher affinities towards the Dengue virus protease, similar to retro-inverse sequences with D-lysine and D-arginine residues. The results of a competition assay with the known inhibitor aprotinin indicate that the N-terminal arylcyanoacrylamide residue of this compound class binds near the catalytic center of the enzyme.