Isocitrate dehydrogenase 1 mutant R132H sensitizes glioma cells to BCNU-induced oxidative stress and cell death

Isocitrate dehydrogenase 1 (IDH1) decarboxylates isocitrate to α-ketoglutarate (α-KG) leading to generation of NADPH, which is required to regenerate reduced glutathione (GSH), the major cellular ROS scavenger. Mutation of R132 of IDH1 abrogates generation of α-KG and leads to conversion of α-KG to 2-hydroxyglutarate. We hypothesized that glioma cells expressing mutant IDH1 have a diminished antioxidative capacity and therefore may encounter an ensuing loss of cytoprotection under conditions of oxidative stress. Our study was performed with LN229 cells stably overexpressing IDH1 R132H and wild type IDH1 or with a lentiviral IDH1 knockdown. Quantification of GSH under basal conditions and following treatment with the glutathione reductase inhibitor BCNU revealed significantly lower GSH levels in IDH1 R132H expressing cells and IDH1 KD cells compared to their respective controls. FACS analysis of cell death and ROS production also demonstrated an increased sensitivity of IDH1-R132H-expressing cells and IDH1 KD cells to BCNU, but not to temozolomide. The sensitivity of IDH1-R132H-expressing cells and IDH1 KD cells to ROS induction and cell death was further enhanced with the transaminase inhibitor aminooxyacetic acid and under glutamine free conditions, indicating that these cells were more addicted to glutaminolysis. Increased sensitivity to BCNU-induced ROS production and cell death was confirmed in HEK293 cells inducibly expressing the IDH1 mutants R132H, R132C and R132L. Based on these findings we propose that in addition to its established pro-tumorigenic effects, mutant IDH1 may also limit the resistance of gliomas to specific death stimuli, therefore opening new perspectives for therapy.     

Infectious molecular clones from a simian immunodeficiency virus-infected rapid-progressor (RP) macaque: evidence of differential selection of RP-specific envelope mutations in vitro and in vivo

A minor fraction of simian immunodeficiency virus (SIV)-infected macaques progress rapidly to AIDS in the absence of SIV-specific immune responses. Common mutations in conserved residues of env in three SIVsmE543-3-infected rapid-progressor (RP) macaques suggest the evolution of a common viral variant in RP macaques. The goal of the present study was to analyze the biological properties of these variants in vitro and in vivo through the derivation of infectious molecular clones. Virus isolated from a SIVsmE543-3-infected RP macaque, H445 was used to inoculate six naive rhesus macaques. Although RP-specific mutations dominated in H445 tissues, they represented only 10% of the population of the virus stock, suggesting a selective disadvantage in vitro. Only one of these macaques (H635) progressed rapidly to AIDS. Plasma virus during primary infection of H635 was similar to the inoculum. However, RP-specific mutations were apparently rapidly reselected by 4 to 9 weeks postinfection. Terminal plasma from H635 was used as a source of viral RNA to generate seven full-length, infectious molecular clones. With the exception of one clone, which was similar to SIVsmE543-3, clones with RP-specific mutations replicated with delayed kinetics in rhesus peripheral blood mononuclear cells and human T-cell lines. None of the clones replicated in monocyte-derived or alveolar macrophages, and all used CCR5 as their major coreceptor. RP variants appear to be well adapted to replicate in vivo in RP macaques but are at a disadvantage in tissue culture compared to their parent, SIVsmE543-3. Therefore, tissue culture may not provide a good surrogate for replication of RP variants in macaques. These infectious clones will provide a valuable reagent to study the roles of specific viral variants in rapid progression in vivo.     

Sequencing, expression, and functional analyses support the candidacy of Ncf2 in susceptibility to Salmonella typhimurium infection in wild-derived mice

A recessive Salmonella Typhimurium susceptibility locus (immunity to Typhimurium (Ity3) was reported previously on distal mouse chromosome 1 using a cross between C57BL/6J and wild-derived MOLF/Ei mice. This quantitative trait locus is located in a genomic region spanning 84 Mb, rich in candidate genes for which a role in host resistance to Salmonella infection is either known or can be envisioned. In this study, we report the evaluation of neutrophil cytosolic factor 2 (Ncf2) as a candidate Salmonella susceptibility gene for Ity3. Ncf2 encodes p67phox, a subunit of the multiprotein enzyme complex NADPH oxidase, known to be responsible for the generation of superoxides. Congenic mice carrying the Ity3 region from MOLF/Ei, B6.MOLF-Ity/Ity3 were more susceptible to infection compared with control mice heterozygous at Ity3, B6.MOLF-Ity/Ity3(MOLF/B6), confirming the existence of a recessive Salmonella susceptibility locus on distal chromosome 1. Spleen Ncf2 expression levels were lower in infected congenic mice homozygous for the MOLF/Ei allele at Ity3 compared with mice heterozygous at Ity3. C57BL/6J and MOLF/Ei Ncf2 sequence comparisons revealed one nonconservative amino acid change (R394Q) in the functional and highly conserved Phox and Bem1 domain of the protein. Functional analysis revealed that the MOLF/Ei allele had reduced PMA- and Salmonella-induced superoxide induction as compared with their wild-type counterparts ex vivo. The R394Q substitution seems to occur on an amino acid involved in electrostatic interactions with p40phox, crucial in its activation. Moreover, a human mutation in the corresponding R395W, resulting in chronic granulatomous disease, is known to lead to reduced superoxide levels. These results support the candidacy of Ncf2 as the gene underlying Ity3.     

Chibby interacts with NBPF1 and clusterin, two candidate tumor suppressors linked to neuroblastoma

The NBPF genes are members of a gene family that underwent a remarkable increase in their copy number during recent primate evolution. The NBPF proteins contain 5 to 40 copies of a domain known as the NBPF repeat or DUF1220. Very little is known about the function of these domains or about the NBPF proteins. We performed a yeast two-hybrid screening with the aminoterminal domain of NBPF11 and found that Chibby, a documented repressor of Wnt signaling, interacts with multiple NBPF proteins. More specifically, a coiled-coil region in the NBPF proteins interacts with the coiled-coil domain in the carboxyterminal region of Chibby. Nonetheless, this interaction did not influence the repressor function of Chibby in a TOPFLASH reporter assay. Using Chibby as bait in a new yeast two-hybrid screening, we identified clusterin as a binding protein. Chibby and clusterin were co-immunoprecipitated with NBPF1, suggesting the formation of a tri-molecular complex. Although we have not pinpointed the role of these mutual interactions, the possible formation of a macromolecular complex of three candidate tumor suppressor proteins, including the enigmatic NBPF1, points at important functional implications.     

Structural Insight into Layer Gliding and Lattice Distortion in Layered Manganese Oxide Electrodes for Potassium‐Ion Batteries

Potassium‐ion batteries (PIBs) are an emerging, affordable, and environmentally friendly alternative to lithium‐ion batteries, with their further development driven by the need for suitably performing electrode materials capable of reversibly accommodating the relatively large K⁺. Layer‐structured manganese oxides are attractive as electrodes for PIBs, but suffer from structural instability and sluggish kinetics of K⁺ insertion/extraction, leading to poor rate capability. Herein, cobalt is successfully introduced at the manganese site in the K_xMnO₂ layered oxide electrode material and it is shown that with only 5% Co, the reversible capacity increases by 30% at 22 mA g^‐1 and by 92% at 440 mA g^‐1. In operando synchrotron X‐ray diffraction reveals that Co suppresses Jahn–Teller distortion, leading to more isotropic migration pathways for K⁺ in the interlayer, thus enhancing the ionic diffusion and consequently, rate capability. The detailed analysis reveals that additional phase transitions and larger volume change occur in the Co‐doped material as a result of layer gliding, with these associated with faster capacity decay, despite the overall capacity remaining higher than the pristine material, even after 500 cycles. These results assert the importance of understanding the detailed structural evolution that underpins performance that will inform the strategic design of electrode materials for high‐performance PIBs.     

DNA methylation of loci within ABCG1 and PHOSPHO1 in blood DNA is associated with future type 2 diabetes risk

Identification of subjects with a high risk of developing type 2 diabetes (T2D) is fundamental for prevention of the disease. Consequently, it is essential to search for new biomarkers that can improve the prediction of T2D. The aim of this study was to examine whether 5 DNA methylation loci in blood DNA (ABCG1, PHOSPHO1, SOCS3, SREBF1, and TXNIP), recently reported to be associated with T2D, might predict future T2D in subjects from the Botnia prospective study. We also tested if these CpG sites exhibit altered DNA methylation in human pancreatic islets, liver, adipose tissue, and skeletal muscle from diabetic vs. non-diabetic subjects. DNA methylation at the ABCG1 locus cg06500161 in blood DNA was associated with an increased risk for future T2D (OR = 1.09, 95% CI = 1.02–1.16, P-value = 0.007, Q-value = 0.018), while DNA methylation at the PHOSPHO1 locus cg02650017 in blood DNA was associated with a decreased risk for future T2D (OR = 0.85, 95% CI = 0.75–0.95, P-value = 0.006, Q-value = 0.018) after adjustment for age, gender, fasting glucose, and family relation. Furthermore, the level of DNA methylation at the ABCG1 locus cg06500161 in blood DNA correlated positively with BMI, HbA1c, fasting insulin, and triglyceride levels, and was increased in adipose tissue and blood from the diabetic twin among monozygotic twin pairs discordant for T2D. DNA methylation at the PHOSPHO1 locus cg02650017 in blood correlated positively with HDL levels, and was decreased in skeletal muscle from diabetic vs. non-diabetic monozygotic twins. DNA methylation of cg18181703 (SOCS3), cg11024682 (SREBF1), and cg19693031 (TXNIP) was not associated with future T2D risk in subjects from the Botnia prospective study.     

Parameters of Metabolic Syndrome and Its Relationship with Zincemia and Activities of Superoxide Dismutase and Glutathione Peroxidase in Obese Women

Alterations in antioxidant defense in obese people with metabolic syndrome can contribute to oxidative stress. This study assessed the relationship between the parameters of metabolic syndrome and the zincemia, activity of superoxide dismutase, and glutathione peroxidase enzymes in obese women. Seventy-three premenopausal women, aged between 20 and 50 years, were divided into two groups: case group, composed of obese (n = 37), and control group, composed of no obese (n = 36). Analyses of zinc intake, parameters of metabolic syndrome, plasma, and erythrocyte zinc, and activities of superoxide dismutase and glutathione peroxidase were carried out. The mean values of body mass index of obese women and control group were 34.5 ± 3.4 and 21.7 ± 1.9 kg/m², respectively (p < 0.05). In the study, body mass index, waist circumference, and zinc intake were higher in obese women than control group (p < 0.05). The plasma zinc and activity of superoxide dismutase did not show significant differences between obese and controls (p > 0.05). The values of erythrocyte zinc was 36.4 ± 15.0 μg/gHb and 45.4 ± 14.3 μg/gHb and of glutathione peroxidase was 46.4 ± 19.4 U/gHb and 36.7 ± 13.6 U/gHb in obese women and controls, respectively (p < 0.05). The study shows that there are alterations in biochemical parameters of zinc in obese women, with low zinc concentrations in erythrocytes. Regression analysis demonstrates that the erythrocyte zinc and activity of superoxide dismutase enzyme is influenced by components of the metabolic syndrome, and the plasmatic glucose, body mass index, and waist circumference have a negative correlation with this enzyme.     

Restricted aeroallergen access to airway mucosal dendritic cells in vivo limits allergen-specific CD4+ T cell proliferation during the induction of inhalation tolerance

Chronic innocuous aeroallergen exposure attenuates CD4(+) T cell-mediated airways hyperresponsiveness in mice; however, the mechanism(s) remain unclear. We examined the role of airway mucosal dendritic cell (AMDC) subsets in this process using a multi-OVA aerosol-induced tolerance model in sensitized BALB/c mice. Aeroallergen capture by both CD11b(lo) and CD11b(hi) AMDC and the delivery of OVA to airway draining lymph nodes by CD8α(-) migratory dendritic cells (DC) were decreased in vivo (but not in vitro) when compared with sensitized but nontolerant mice. This was functionally significant, because in vivo proliferation of OVA-specific CD4(+) T cells was suppressed in airway draining lymph nodes of tolerized mice and could be restored by intranasal transfer of OVA-pulsed and activated exogenous DC, indicating a deficiency in Ag presentation by endogenous DC arriving from the airway mucosa. Bone marrow-derived DC Ag-presenting function was suppressed in multi-OVA tolerized mice, and allergen availability to airway APC populations was limited after multi-OVA exposure, as indicated by reduced OVA and dextran uptake by airway interstitial macrophages, with diffusion rather than localization of OVA across the airway mucosal surface. These data indicate that inhalation tolerance limits aeroallergen capture by AMDC subsets through a mechanism of bone marrow suppression of DC precursor function coupled with reduced Ag availability in vivo at the airway mucosa, resulting in limited Ag delivery to lymph nodes and hypoproliferation of allergen-specific CD4(+) T cells.     

Effects of Sensory Behavioral Tasks on Pain Threshold and Cortical Excitability

Background/Objective

Transcutaneous electrical stimulation has been proven to modulate nervous system activity, leading to changes in pain perception, via the peripheral sensory system, in a bottom up approach. We tested whether different sensory behavioral tasks induce significant effects in pain processing and whether these changes correlate with cortical plasticity.

Methodology/Principal Findings

This randomized parallel designed experiment included forty healthy right-handed males. Three different somatosensory tasks, including learning tasks with and without visual feedback and simple somatosensory input, were tested on pressure pain threshold and motor cortex excitability using transcranial magnetic stimulation (TMS). Sensory tasks induced hand-specific pain modulation effects. They increased pain thresholds of the left hand (which was the target to the sensory tasks) and decreased them in the right hand. TMS showed that somatosensory input decreased cortical excitability, as indexed by reduced MEP amplitudes and increased SICI. Although somatosensory tasks similarly altered pain thresholds and cortical excitability, there was no significant correlation between these variables and only the visual feedback task showed significant somatosensory learning.

Conclusions/Significance

Lack of correlation between cortical excitability and pain thresholds and lack of differential effects across tasks, but significant changes in pain thresholds suggest that analgesic effects of somatosensory tasks are not primarily associated with motor cortical neural mechanisms, thus, suggesting that subcortical neural circuits and/or spinal cord are involved with the observed effects. Identifying the neural mechanisms of somatosensory stimulation on pain may open novel possibilities for combining different targeted therapies for pain control.     

Human liver epigenetic alterations in non-alcoholic steatohepatitis are related to insulin action

Both genetic and lifestyle factors contribute to the risk of non-alcoholic steatohepatitis (NASH). Additionally, epigenetic modifications may also play a key role in the pathogenesis of NASH. We therefore investigated liver DNA methylation, as a marker for epigenetic alterations, in individuals with simple steatosis and NASH, and further tested if these alterations were associated with clinical phenotypes. Liver biopsies obtained from 95 obese individuals (age: 49.5 ± 7.7 years, BMI: 43 ± 5.7 kg/m², type 2 diabetes [T2D]: 35) as a wedge biopsy during a Roux-en-Y gastric bypass operation were investigated. Thirty-four individuals had a normal liver phenotype, 35 had simple steatosis, and 26 had NASH. Genome-wide DNA methylation pattern was analyzed using the Infinium HumanMethylation450 BeadChip. mRNA expression was analyzed from 42 individuals using the HumanHT-12 Expression BeadChip. We identified 1,292 CpG sites representing 677 unique genes differentially methylated in liver of individuals with NASH (q < 0.001), independently of T2D, age, sex, and BMI. Focusing on the top-ranking 30 and another 37 CpG sites mapped to genes enriched in pathways of metabolism (q = 0.0036) and cancer (q = 0.0001) all together, 59 NASH-associated CpG sites correlated with fasting insulin levels independently of age, fasting glucose, or T2D. From these, we identified 30 correlations between DNA methylation and mRNA expression, for example LDHB (r = ?0.45, P = 0.003). We demonstrated that NASH, more than simple steatosis, associates with differential DNA methylation in the human liver. These epigenetic alterations in NASH are linked with insulin metabolism.