Rapid diagnosis of IDH1-mutated gliomas by 2-HG detection with gas chromatography mass spectrometry.

The metabolic genes encoding isocitrate dehydrogenase (IDH1, 2) are frequently mutated in gliomas. Mutation of IDH defines a distinct subtype of glioma and predicts therapeutic response. IDH mutation has a remarkable neomorphic activity of converting α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG), which is now commonly referred to as an oncometabolite and biomarker for gliomas. PCR-sequencing (n = 220), immunohistochemistry staining (IHC, n = 220), and gas chromatography mass spectrometry (GC-MS, n = 87) were applied to identify IDH mutation in gliomas, and the sensitivity and specificity of these strategies were compared. PCR-sequencing and IHC staining are reliable for retrospective assessment of IDH1 mutation in gliomas, but both methods usually take 1-2 days, which hinders their application for rapid diagnosis. GC-MS-based methods can detect 2-HG qualitatively and quantitatively, offering information on the IDH1 mutation status in gliomas with the sensitivity and specificity being 100%. Further optimization of the GC-MS based methodology (so called as the mini-column method) enabled us to determine 2-HG within 40 min in glioma samples without complex or time-consuming preparation. Most importantly, the ratio of 2-HG/glutamic acid was shown to be a reliable parameter for determination of mutation status. The mini-column method enables rapid identification of 2-HG, providing a promising strategy for intraoperative diagnosis of IDH1-mutated gliomas in the future.

Correction: Insulin and mTOR Pathway Regulate HDAC3-Mediated Deacetylation and Activation of PGK1.

[This corrects the article DOI: 10.1371/journal.pbio.1002243.].

Insulin and mTOR Pathway Regulate HDAC3-Mediated Deacetylation and Activation of PGK1.

Phosphoglycerate kinase 1 (PGK1) catalyzes the reversible transfer of a phosphoryl group from 1, 3-bisphosphoglycerate (1, 3-BPG) to ADP, producing 3-phosphoglycerate (3-PG) and ATP. PGK1 plays a key role in coordinating glycolytic energy production with one-carbon metabolism, serine biosynthesis, and cellular redox regulation. Here, we report that PGK1 is acetylated at lysine 220 (K220), which inhibits PGK1 activity by disrupting the binding with its substrate, ADP. We have identified KAT9 and HDAC3 as the potential acetyltransferase and deacetylase, respectively, for PGK1. Insulin promotes K220 deacetylation to stimulate PGK1 activity. We show that the PI3K/AKT/mTOR pathway regulates HDAC3 S424 phosphorylation, which promotes HDAC3-PGK1 interaction and PGK1 K220 deacetylation. Our study uncovers a previously unknown mechanism for the insulin and mTOR pathway in regulation of glycolytic ATP production and cellular redox potential via HDAC3-mediated PGK1 deacetylation.

A bronchofiberoscopy-associated outbreak of multidrug-resistant Acinetobacter baumannii in an intensive care unit in Beijing, China.

BACKGROUND: Bronchofiberscopy, a widely used procedure for the diagnosis of various pulmonary diseases within intensive care units, has a history of association with nosocomial infections. Between September and November 2009, an outbreak caused by multidrug-resistant Acinetobacter baumannii (MDR-Ab) was observed in the intensive care unit of a tertiary care hospital in Beijing, China. This study is aimed to describe the course and control of this outbreak and investigate the related risk factors. METHODS: Clinical and environmental sampling, genotyping with repetitive extragenic palindromic polymerase chain reaction (REP-PCR), and case-control risk factor analysis were performed in the current study. RESULTS: During the epidemic period, 12 patients were infected or colonized with MDR-Ab. Sixteen (72.7%) of twenty-two MDR-Ab isolates from the 12 patients and 22 (84.6%) of 26 MDR-Ab isolates from the bronchofiberscope and the healthcare-associated environment were clustered significantly into a major clone (outbreak MDR-Ab strain) by REP-PCR typing. Seven patients carrying the outbreak MDR-Ab strain were defined as the cases. Six of the seven cases (83%) received bronchofiberscopy versus four of the 19 controls (21%) (odds ratio, 22.5; 95% confidence interval, 2.07-244.84; P = 0.005). Several potential administrative and technical problems existed in bronchofiberscope reprocessing. CONCLUSIONS: Bronchofiberscopy was associated with this MDR-Ab outbreak. Infection control precautions including appropriate bronchofiberscope reprocessing and environmental decontamination should be strengthened.

Prediction of response of collagen-induced arthritis rats to methotrexate: an (1)H-NMR-based urine metabolomic analysis.

Over one half the patients with rheumatoid arthritis (RA) are being treated with methotrexate (MTX). Although well proven, the efficacy of MTX varies in individual patients. This study examined the metabolic biomarkers that can be used to predict the therapeutic effect of MTX by using metabolomic analysis. Rats were immunized with collagen to rapidly cause collagen-induced arthritis (CIA) and then treated with 0.1 mg/kg MTX for 4 weeks. The clinical signs and the histopathological features of CIA were observed to evaluate the therapeutic effects. Urine samples of CIA rats were collected, and analyzed by using 600 M (1)H-nuclear magnetic resonance ((1)H-NMR) for spectral binning after the therapy. The urine spectra were divided into spectral bins, and 20 endogenous metabolites were assigned by Chenomx Suite. Multivariate analyses were performed to identify the spectral pattern of endogenous metabolites related to MTX therapy. The results showed that the clustering of the spectra of the urine samples from the responsive rats (n=20) was different from that from the non-responsive rats (n=11). Multivariate analysis showed difference in metabolic profiles between the responsive and non-responsive rats by using partial least squares-discrimination analysis (PLS-DA) (R(2)=0.812, Q(2)=0.604). In targeted profiling, 13 endogenous metabolites (uric acid, taurine, histidine, methionine, glycine, etc.) were selected as putative biomarkers for predicting therapeutic response to MTX. It was suggested that (1)H-NMR-based metabolomic analysis can be used to predict the therapeutic effect of MTX, and several metabolites were found to be related to the therapeutic effects of MTX.

Virulence of H5N1 virus in mice attenuates after in vitro serial passages.

The virulence of A/Vietnam/1194/2004 (VN1194) in mice attenuated after serial passages in MDCK cells and chicken embryos, because the enriched large-plaque variants of the virus had significantly reduced virulence. In contrast, the small-plaque variants of the virus and the variants isolated from the brain of mice that were infected with the parental virus VN1194 had much higher virulence in mice. The virulence attenuation of serially propagated virus may be caused by the reduced neurotropism in mice. Our whole genome sequence analysis revealed substitutions of a total of two amino acids in PB1, three in PB2, two in PA common for virulence attenuated variants, all or part of which may be correlated with the virulence attenuation and reduced neurotropism of the serially propagated VN1194 in mice. Our study indicates that serial passages of VN1194 in vitro lead to adaptation and selection of variants that have markedly decreased virulence and neurotropism, which emphasizes the importance of direct analysis of original or less propagated virus samples.

Pterostilbene alleviates pulmonary fibrosis by regulating ASIC2.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a serious chronic disease of the respiratory system, but its current treatment has certain shortcomings and adverse effects. In this study, we evaluate the antifibrotic activity of pterostilbene (PTE) using an in vitro IPF model induced by transforming growth factor (TGF)-ß1. METHODS: A549 and alveolar epithelial cells (AECs) were incubated with 10 ng/ml TGF-ß1 to induce lung fibroblast activation. Then, 30 µmol/L of PTE was used to treat these cells. The epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) accumulation, and autophagy in cells were evaluated by western blot. Apoptosis was validated by flow cytometry analysis and western blot. Transcriptome high-throughput sequencing was performed on A549 cells incubated with TGF-ß1 alone or TGF-ß1 and PTE (TGF-ß1 + PTE), and differentially expressed genes in PTE-treated cells were identified. The acid sensing ion channel subunit 2 (ASIC2) overexpression plasmid was used to rescue the protein levels of ASIC2 in A549 and AECs. RESULTS: TGF-ß1 caused EMT and ECM accumulation, and blocked the autophagy and apoptosis of A549 and AECs. Most importantly, 30 µmol/L of PTE inhibited pulmonary fibrosis induced by TGF-ß1. Compared with TGF-ß1, PTE inhibited EMT and ECM accumulation and rescued cell apoptosis and autophagy. The results of transcriptome high-throughput sequencing revealed that PTE greatly reduced the protein level of ASIC2. Compared with the TGF-ß1 + PTE group, the transfection of ASIC2 overexpression plasmid stimulated the EMT and ECM accumulation and inhibited apoptosis and autophagy, suggesting that PTE inhibited pulmonary fibrosis by downregulating ASIC2. CONCLUSIONS: This study suggests that PTE and ASIC2 inhibitors may have potential as IPF treatments in the future.

Evolution of the Shear Band in Cold-Rolling of Strip-Cast Fe-1.3% Si Non-Oriented Silicon Steel.

Cube texture and microstructural evolution of as-cast non-oriented silicon steel (1.3% Si) during cold rolling and annealing were studied. The results showed that the as-cast microstructure with grain size in the range of 100-500 µm had a weak texture. The strong orientation was mainly located at {100} and {110} planes. A significant content of shear-deformed grains oriented with {110}<110> were obtained by cold-rolling, and many regions oriented with Cube texture were distributed in the shear bands. During cold-rolling, the orientation of the shear-deformed microstructure tilted towards the {111}<112> orientation, while the matrix orientation retained {110}<110>. On further cold-rolling, the residual part of {110}<110> experienced shear deformation, forming more shear bands, strengthening the Cube orientation. During annealing, Cube orientation grains nucleated in the shear bands leading to strong Cube texture, and corresponding B50 was 1.83T/1.79T.

Tumor-derived neomorphic mutations in ASXL1 impairs the BAP1-ASXL1-FOXK1/K2 transcription network.

Additional sex combs-like 1 (ASXL1) interacts with BRCA1-associated protein 1 (BAP1) deubiquitinase to oppose the polycomb repressive complex 1 (PRC1)-mediated histone H2A ubiquitylation. Germline BAP1 mutations are found in a spectrum of human malignancies, while ASXL1 mutations recurrently occur in myeloid neoplasm and are associated with poor prognosis. Nearly all ASXL1 mutations are heterozygous frameshift or nonsense mutations in the middle or to a less extent the C-terminal region, resulting in the production of C-terminally truncated mutant ASXL1 proteins. How ASXL1 regulates specific target genes and how the C-terminal truncation of ASXL1 promotes leukemogenesis are unclear. Here, we report that ASXL1 interacts with forkhead transcription factors FOXK1 and FOXK2 to regulate a subset of FOXK1/K2 target genes. We show that the C-terminally truncated mutant ASXL1 proteins are expressed at much higher levels than the wild-type protein in ASXL1 heterozygous leukemia cells, and lose the ability to interact with FOXK1/K2. Specific deletion of the mutant allele eliminates the expression of C-terminally truncated ASXL1 and increases the association of wild-type ASXL1 with BAP1, thereby restoring the expression of BAP1-ASXL1-FOXK1/K2 target genes, particularly those involved in glucose metabolism, oxygen sensing, and JAK-STAT3 signaling pathways. In addition to FOXK1/K2, we also identify other DNA-binding transcription regulators including transcription factors (TFs) which interact with wild-type ASXL1, but not C-terminally truncated mutant. Our results suggest that ASXL1 mutations result in neomorphic alleles that contribute to leukemogenesis at least in part through dominantly inhibiting the wild-type ASXL1 from interacting with BAP1 and thereby impairing the function of ASXL1-BAP1-TF in regulating target genes and leukemia cell growth.

Hydrogel 3D printing with the capacitor edge effect.

Recent decades have seen intense developments of hydrogel applications for cell cultures, tissue engineering, soft robotics, and ionic devices. Advanced fabrication techniques for hydrogel structures are being developed to meet user-specified requirements. Existing hydrogel 3D printing techniques place substantial constraints on the physical and chemical properties of hydrogel precursors as well as the printed hydrogel structures. This study proposes a novel method for patterning liquids with a resolution of 100 µm by using the capacitor edge effect. We establish a complete hydrogel 3D printing system combining the patterning and stacking processes. This technique is applicable to a wide variety of hydrogels, overcoming the limitations of existing techniques. We demonstrate printed hydrogel structures including a hydrogel scaffold, a hydrogel composite that responds sensitively to temperature, and an ionic high-integrity hydrogel display device. The proposed technique offers great opportunities in rapid prototyping hydrogel devices using multiple compositions and complex geometries.

SIRT5 deficiency suppresses mitochondrial ATP production and promotes AMPK activation in response to energy stress.

Sirtuin 5 (SIRT5) is a member of the NAD+-dependent sirtuin family of protein deacylase that catalyzes removal of post-translational modifications, such as succinylation, malonylation, and glutarylation on lysine residues. In light of the SIRT5's roles in regulating mitochondrion function, we show here that SIRT5 deficiency leads to suppression of mitochondrial NADH oxidation and inhibition of ATP synthase activity. As a result, SIRT5 deficiency decreases mitochondrial ATP production, increases AMP/ATP ratio, and subsequently activates AMP-activated protein kinase (AMPK) in cultured cells and mouse hearts under energy stress conditions. Moreover, Sirt5 knockout attenuates transverse aortic constriction (TAC)-induced cardiac hypertrophy and cardiac dysfunction in mice, which is associated with decreased ATP level, increased AMP/ATP ratio and enhanced AMPK activation. Our study thus uncovers an important role of SIRT5 in regulating cellular energy metabolism and AMPK activation in response to energy stress.

Super tough magnetic hydrogels for remotely triggered shape morphing.

Despite their potential in various fields such as soft robots, drug delivery and biomedical engineering, magnetic hydrogels have always been limited by their poor mechanical properties. Here a universal soaking strategy has been presented to synthesize tough magnetic nanocomposite (NC) hydrogels. We can simultaneously solve two common issues for magnetic hydrogels: the poor mechanical properties and poor distribution of magnetic particles. The toughness of the magnetic NC hydrogel achieves approximately 11 000 J m-2. The outstanding properties of tough magnetic hydrogels will enable myriad applications. Here we demonstrate a new application for remotely triggered shape morphing. Heterogeneous structures based on magnetic hydrogels are shown to evolve into bio-inspired three-dimensional (3D) shapes (lotus flowers) from 2D-structured sheets. The self-folding of the structure is controlled by the magnetothermal effect in an alternating magnetic field. The capability to control the shape morphing of a multi-material system by a magnetic field may emerge as a new general strategy for programming complex soft structures.

SNIP1 Recruits TET2 to Regulate c-MYC Target Genes and Cellular DNA Damage Response.

The TET2 DNA dioxygenase regulates gene expression by catalyzing demethylation of 5-methylcytosine, thus epigenetically modulating the genome. TET2 does not contain a sequence-specific DNA-binding domain, and how it is recruited to specific genomic sites is not fully understood. Here we carried out a mammalian two-hybrid screen and identified multiple transcriptional regulators potentially interacting with TET2. The SMAD nuclear interacting protein 1 (SNIP1) physically interacts with TET2 and bridges TET2 to bind several transcription factors, including c-MYC. SNIP1 recruits TET2 to the promoters of c-MYC target genes, including those involved in DNA damage response and cell viability. TET2 protects cells from DNA damage-induced apoptosis dependending on SNIP1. Our observations uncover a mechanism for targeting TET2 to specific promoters through a ternary interaction with a co-activator and many sequence-specific DNA-binding factors. This study also reveals a TET2-SNIP1-c-MYC pathway in mediating DNA damage response, thereby connecting epigenetic control to maintenance of genome stability.

Niu-Huang-Shen suppresses hepatocellular carcinoma cell growth and metastasis by regulating Yap1 expression.

Hepatocellular carcinoma (HCC) is one of the most common cancers types. Niu-Huang-Shen (NHS), a Chinese medicine, has been reported to exert antipyretic, anti-inflammatory and vasodilatation effects. However, whether NHS has inhibitory effects on HCC cell phenotypes has remained elusive. In the present study, Cell Counting Kit-8, colony formation, fluorescence-activated cell sorting and Transwell assays were used to evaluate the effect of NHS on cell proliferation, migration and invasion. The results indicated that NHS suppressed cell proliferation and invasion, inhibited cell apoptosis, and induced cell cycle arrest. In addition, NHS significantly suppressed the mRNA and protein expression of Yes-associated protein (YAP). It was concluded that NHS downregulated YAP expression and inhibited the Hippo signaling pathway as well as HCC cell growth and invasion. NHS may be a novel potential therapeutic for HCC patients.

D-2-hydroxyglutarate is essential for maintaining oncogenic property of mutant IDH-containing cancer cells but dispensable for cell growth.

Cancer-associated isocitrate dehydrogenase (IDH) 1 and 2 mutations gain a new activity of reducing α-KG to produce D-2-hydroxyglutarate (D-2-HG), which is proposed to function as an oncometabolite by inhibiting α-KG dependent dioxygenases. We investigated the function of D-2-HG in tumorigenesis using IDH1 and IDH2 mutant cancer cell lines. Inhibition of D-2-HG production either by specific deletion of the mutant IDH1-R132C allele or overexpression of D-2-hydroxyglutarate dehydrogenase (D2HGDH) increases α-KG and related metabolites, restores the activity of some α-KG-dependent dioxygenases, and selectively alters gene expression. Ablation of D-2-HG production has no significant effect on cell proliferation and migration, but strongly inhibits anchorage independent growth in vitro and tumor growth in xenografted mouse models. Our study identifies a new activity of oncometabolite D-2-HG in promoting tumorigenesis.