Increased interaction between connexin43 and microtubules is critical for glioblastoma stem-like cell maintenance and tumorigenicity.

Glioblastoma (GBM) is the most common primary tumor of the central nervous system. One major challenge in GBM treatment is the resistance to chemotherapy and radiotherapy observed in subpopulations of cancer cells, including GBM stem-like cells (GSCs). These cells hold the ability to self-renew or differentiate following treatment, participating in tumor recurrence. The gap junction protein connexin43 (Cx43) has complex roles in oncogenesis and we have previously demonstrated an association between Cx43 and GBM chemotherapy resistance. Here, we report, for the first time, increased direct interaction between non-junctional Cx43 with microtubules in the cytoplasm of GSCs. We hypothesize that non-junctional Cx43/microtubule complexing is critical for GSC maintenance and survival and sought to specifically disrupt this interaction while maintaining other Cx43 functions, such as gap junction formation. Using a Cx43 mimetic peptide of the carboxyl terminal tubulin-binding domain of Cx43 (JM2), we successfully ablated Cx43 interaction with microtubules in GSCs. Importantly, administration of JM2 significantly decreased GSC survival in vitro , and limited GSC-derived tumor growth in vivo . Together, these results identify JM2 as a novel peptide drug to ablate GSCs in GBM treatment.

A Numerical Procedure for Shakedown Analysis of Thick Cylindrical Vessels with Crossholes under Dual Cyclic Loadings.

A modified numerical procedure for the shakedown analysis of structures under dual cyclic loadings, based on the Abdalla method, is proposed in this paper. Based on the proposed numerical procedure, the shakedown analysis of the thick cylindrical vessels with crossholes (TCVCs) under cyclic internal pressure and cyclic thermal loading was carried out. The effects of material parameters (elastic modulus and thermal expansion coefficient) and crosshole radius on the elastic shakedown limit of TCVCs are discussed and, finally, normalized and formularized. Furthermore, the obtained shakedown limit boundary formulation is compared with FEA results and is verified to evaluate the shakedown behavior of TCVCs under cyclic internal pressure and cyclic thermal loading.

Micromechanical Observation and Numerical Simulation for Local Deformation Evolution of Duplex Stainless Steel.

The characteristics of local strain distribution and evolution of duplex stainless steel during the tensile process were studied using the digital image correlation (DIC) technique. In addition, the finite element inversion of nanoindentation experiments of austenitic and ferrite phases in duplex stainless steel was carried out to obtain the stress-strain response of the two phases. Further, based on the representative volume element (RVE) and the material parameters obtained from the finite element inversion method, the local stress and strain behavior of duplex stainless steel at microscale was simulated numerically. The results fit well with the experiments, showing that the austenite phase is softer than ferrite phase, with the larger strain zone concentrated in the austenite phase and the larger stress zone concentrated in the ferrite phase. The grain boundaries are prone to obvious stress and strain concentrations. The local stress and strain distributions are influenced by the shape and interaction of the grains, while the distribution features become more obvious as the load increases. The research results effectively reveal the two-phase interaction and local failure mechanism of duplex stainless steel, and may provide a reference for material preparation and safety design of related structures.

Characteristics, phenotypes, mechanisms and management of severe asthma.

ABSTRACT: Severe asthma is "asthma which requires treatment with high dose inhaled corticosteroids (ICS) plus a second controller (and/or systemic corticosteroids) to prevent it from becoming 'uncontrolled' or which remains 'uncontrolled' despite this therapy." The state of control was defined by symptoms, exacerbations and the degree of airflow obstruction. Therefore, for the diagnosis of severe asthma, it is important to have evidence for a diagnosis of asthma with an assessment of its severity, followed by a review of comorbidities, risk factors, triggers and an assessment of whether treatment is commensurate with severity, whether the prescribed treatments have been adhered to and whether inhaled therapy has been properly administered. Phenotyping of severe asthma has been introduced with the definition of a severe eosinophilic asthma phenotype characterized by recurrent exacerbations despite being on high dose ICS and sometimes oral corticosteroids, with a high blood eosinophil count and a raised level of nitric oxide in exhaled breath. This phenotype has been associated with a Type-2 (T2) inflammatory profile with expression of interleukin (IL)-4, IL-5, and IL-13. Molecular phenotyping has also revealed non-T2 inflammatory phenotypes such as Type-1 or Type-17 driven phenotypes. Antibody treatments targeted at the T2 targets such as anti-IL5, anti-IL5Rα, and anti-IL4Rα antibodies are now available for treating severe eosinophilic asthma, in addition to anti-immunoglobulin E antibody for severe allergic asthma. No targeted treatments are currently available for non-T2 inflammatory phenotypes. Long-term azithromycin and bronchial thermoplasty may be considered. The future lies with molecular phenotyping of the airway inflammatory process to refine asthma endotypes for precision medicine.

Expression, Purification, and Preliminary Protection Study of Dehydrin PicW1 From the Biomass of Picea wilsonii.

Dehydrins (DHNs) belong to group II of late embryogenesis-abundant (LEA) proteins, which are up-regulated in most plants during cold, drought, heat, or salinity stress. Despite the importance of dehydrins for the plants to resist abiotic stresses, it is necessary to obtain plant-derived dehydrins from different biomass. Generally, dehydrin PicW1 from Picea wilsonii is involved in Kn-type dehydrin with five K-segments, which has a variety of biological activities. In this work, Picea wilsonii dehydrin PicW1 was expressed in Escherichia coli and purified by chitin-affinity chromatography and size-exclusion chromatography, which showed as a single band by SDS-PAGE. A cold-sensitive enzyme of lactate dehydrogenase (LDH) is used to explore the protective activities of other proteins. Temperature stress assays showed that PicW1 had an effective protective effect on LDH activity, which was better than that of bovine serum albumin (BSA). This study provides insights into the purification and protective activity of K5 DHNs for the advancement of dehydrin structure and function from biomass.

Connexin 43 confers chemoresistance through activating PI3K.

Circumventing chemoresistance is crucial for effectively treating cancer including glioblastoma, a lethal brain cancer. The gap junction protein connexin 43 (Cx43) renders glioblastoma resistant to chemotherapy; however, targeting Cx43 is difficult because mechanisms underlying Cx43-mediated chemoresistance remain elusive. Here we report that Cx43, but not other connexins, is highly expressed in a subpopulation of glioblastoma and Cx43 mRNA levels strongly correlate with poor prognosis and chemoresistance in this population, making Cx43 the prime therapeutic target among all connexins. Depleting Cx43 or treating cells with αCT1-a Cx43 peptide inhibitor that sensitizes glioblastoma to the chemotherapy temozolomide-inactivates phosphatidylinositol-3 kinase (PI3K), whereas overexpression of Cx43 activates this signaling. Moreover, αCT1-induced chemo-sensitization is counteracted by a PI3K active mutant. Further research reveals that αCT1 inactivates PI3K without blocking the release of PI3K-activating molecules from membrane channels and that Cx43 selectively binds to the PI3K catalytic subunit ß (PIK3CB, also called PI3Kß or p110ß), suggesting that Cx43 activates PIK3CB/p110ß independent of its channel functions. To explore the therapeutic potential of simultaneously targeting Cx43 and PIK3CB/p110ß, αCT1 is combined with TGX-221 or GSK2636771, two PIK3CB/p110ß-selective inhibitors. These two different treatments synergistically inactivate PI3K and sensitize glioblastoma cells to temozolomide in vitro and in vivo. Our study has revealed novel mechanistic insights into Cx43/PI3K-mediated temozolomide resistance in glioblastoma and demonstrated that targeting Cx43 and PIK3CB/p110ß together is an effective therapeutic approach for overcoming chemoresistance.

Combination of Type I and Type II MET Tyrosine Kinase Inhibitors as Therapeutic Approach to Prevent Resistance.

MET-targeted therapies are clinically effective in MET-amplified and MET exon 14 deletion mutant (METex14) non-small cell lung cancers (NSCLCs), but their efficacy is limited by the development of drug resistance. Structurally distinct MET tyrosine kinase inhibitors (TKIs) (type I/II) have been developed or are under clinical evaluation, which may overcome MET-mediated drug resistance mechanisms. In this study, we assess secondary MET mutations likely to emerge in response to treatment with single-agent or combinations of type I/type II MET TKIs using TPR-MET transformed Ba/F3 cell mutagenesis assays. We found that these inhibitors gave rise to distinct secondary MET mutant profiles. However, a combination of type I/II TKI inhibitors (capmatinib and merestinib) yielded no resistant clones in vitro The combination of capmatinib/merestinib was evaluated in vivo and led to a significant reduction in tumor outgrowth compared with either MET inhibitor alone. Our findings demonstrate in vitro and in vivo that a simultaneous treatment with a type I and type II MET TKI may be a clinically viable approach to delay and/or diminish the emergence of on target MET-mediated drug-resistance mutations.

High-fat diet-induced obesity primes fatty acid ß-oxidation impairment and consequent ovarian dysfunction during early pregnancy.

BACKGROUND: Obesity is associated with many adverse effects on female fertility. Obese women have a higher likelihood of developing ovulatory dysfunction due to dysregulation of the hypothalamic-pituitary-ovarian axis. However, the effect of obesity on ovarian function during early pregnancy needs to be further assessed. METHODS: C57BL6/J mice were given a high-fat diet (HFD) for 12 weeks to induce obesity. An in vitro high-fat model was established by treating the human ovarian granulosa cell line KGN with oleic acid and palmitic acid. Ovarian morphology of obese mice in early pregnancy was assessed by hematoxylin and eosin staining and ovarian function was assessed by enzyme-linked immunosorbent assay, western blotting, and immunohistochemistry. Oil Red O staining and transmission electron microscopy were used to detect fatty acid accumulation. Specific markers relating to the ovarian functional mechanism were assessed by real-time PCR, western blotting, lactate detection, adenosine triphosphate (ATP) detection, biochemical analyses, and enzyme-linked immunosorbent assay. RESULTS: The results of this study showed that during early pregnancy, the number of corpus lutea, serum estradiol and progesterone levels, and the expression of the steroid biosynthesis-related protein CYP19A1 (aromatase), CYP11A1 (cholesterol side chain cleavage enzyme), and StAR (steroidogenic acute regulatory protein), were significantly increased in HFD mice. Mice fed an HFD also showed a significant increase in ovarian lipid accumulation on day 7 of pregnancy. Genes involved in fatty acid synthesis (Acsl4 and Elovl5), and fatty acid uptake and transport (Slc27a4), together with the ß-oxidation rate-limiting enzyme Cpt1a, were significantly upregulated in HFD mice. Specifically, there was abnormal elevation of ATP and aberrant expression of tricarboxylic acid cycle (TCA)- and electron transport chain (ETC)-related genes in the ovaries of pregnant HFD mice. KGN cells treated with etomoxir targeting ß-oxidation of fatty acid showed decreased TCA cycle and ETC related gene expression. The elevation of ATP and estradiol and progesterone levels was reversed. CONCLUSIONS: During early pregnancy, HFD-induced obesity increases fatty acid ß-oxidation, which in turn increases TCA cycle and ETC related gene expression, leading to increased ATP production and ovarian dysfunction.

Transcriptome analysis of genes involved in starch biosynthesis in developing Chinese chestnut (Castanea mollissima Blume) seed kernels.

Chinese chestnut (Castanea mollissima Blume) seed kernels (CCSK) with high quality and quantity of starch has emerged as a potential raw material for food industry, but the molecular regulatory mechanism of starch accumulation in developing CCSK is still unclear. In this study, we firstly analyzed the fruit development, starch accumulation, and microscopic observation of dynamic accumulation of starch granules of developing CCSK from 10 days after flowering (DAF) to 100 DAF, of which six representative CCSK samples (50-100 DAF) were selected for transcriptome sequencing analysis. Approximately 40 million valid reads were obtained, with an average length of 124.95 bp, which were searched against a reference genome, returning 38,146 unigenes (mean size = 1164.19 bp). Using the DESeq method, 1968, 1573, 1187, 1274, and 1494 differentially expressed unigenes were identified at 60:50, 70:60, 80:70, 90:80 and 100:90 DAF, respectively. The relationship between the unigene transcriptional profiles and starch dynamic patterns in developing CCSK was comparatively analyzed, and the specific unigenes encoding for metabolic enzymes (SUSY2, PGM, PGI, GPT, NTT, AGP3, AGP2, GBSS1, SS1, SBE1, SBE2.1, SBE2.2, ISA1, ISA2, ISA3, and PHO) were characterized to be involved potentially in the biosynthesis of G-1-P, ADPG, and starch. Finally, the temporal transcript profiles of genes encoding key enzymes (susy2, pgi2, gpt1, agp2, agp3, gbss1, ss1, sbe1, sbe2.1, sbe2.2, isa1, isa2, isa3, and pho) were validated by quantitative real-time PCR (qRT-PCR). Our findings could help to reveal the molecular regulatory mechanism of starch accumulation in developing CCSK and may also provide potential candidate genes for increasing starch content in Chinese chestnut or other starchy crops.

Ligustilide alleviates the insulin resistance, lipid accumulation, and pathological injury with elevated phosphorylated AMPK level in rats with diabetes mellitus.

BACKGROUND: Diabetes mellitus (DM) is one of the major risk factors of disability and death worldwide. Despite of the protective role of ligustilide (LIG) in many cell types, we aimed to investigate whether LIG could be a potential to treat DM. METHODS: Sprague Dawley rats were randomly assigned to five groups. Rats except control were raised on a high-fat diet (HFD). Streptozotocin was intraperitoneally injected into HFD-fed rats to construct DM model. Rats in the LIG intervention groups received intraperitoneal injection of LIG (10, 20, and 40 mg/kg) post-induction of DM. Blood glucose, plasma insulin (p-insulin), adiponectin, HbA1C%, obesity index, HOMA-IR, and biochemical parameters were estimated. Histopathological analysis and apoptosis in liver and kidney, along with proliferation and apoptosis of islet ß-cells, were analyzed. Expression of CPT-1 and ACC, and phosphorylation of Nrf2 and AMPKα1, were finally assessed. RESULTS: DM-induced alterations were all relived by LIG intervention. In brief, obesity index, glucose level, P-insulin content, HbA1C, and HOMA-IR were lowered while adiponectin level was elevated. Meanwhile, levels of TC, TG, ALT, and AST were decreased in the LIG intervention groups, along with up-regulated CPT-1 level and down-regulated ACC level. Pathological changes in liver and kidney tissues were alleviated, and apoptotic cells were reduced by LIG treatment. For islet ß-cells, LIG up-regulated Ki67 and c-Myc expression, and mitigated ratios of Bax/Bcl-2 and cleaved cas3(9)/cas3(9). Finally, LIG could promote phosphorylation of Nrf2 and AMPKα1. CONCLUSIONS: LIG alleviated the insulin resistance, lipid accumulation, and pathological injury with the activation of AMPK pathway in DM rats.

Food and feed trade has greatly impacted global land and nitrogen use efficiencies over 1961-2017.

International trade of agricultural products has complicated and far-reaching impacts on land and nitrogen use efficiencies. We analysed the productivity of cropland and livestock and associated use of feed and fertilizer efficiency for over 240 countries, and estimated these countries' cumulative contributions to imports and exports of 190 agricultural products for the period 1961-2017. Crop trade has increased global land and partial fertilizer nitrogen productivities in terms of protein production, which equalled savings of 2,270 Mha cropland and 480 Tg synthetic fertilizer nitrogen over the analysed period. However, crop trade decreased global cropland productivity when productivity is expressed on an energy (per calorie) basis. Agricultural trade has generally moved towards optimality, that is, has increased global land and nitrogen use efficiencies during 1961-2017, but remains at a relatively low level. Overall, mixed impacts of trade on resource use indicate the need to rethink trade patterns and improve their optimality.

Endometrial pyruvate kinase M2 is essential for decidualization during early pregnancy.

Embryo implantation is essential for normal pregnancy. Decidualization is known to facilitate embryo implantation and maintain pregnancy. Uterine stromal cells undergo transformation into decidual cells after embryo attachment to the endometrium. Pyruvate kinase M2 (PKM2) is a rate limiting enzyme in the glycolysis process which catalyzes phosphoenolpyruvic acid into pyruvate. However, little is known regarding the role of PKM2 during endometrial decidualization. In this study, PKM2 was found to be mainly located in the uterine glandular epithelium and luminal epithelium on day 1 and day 4 of pregnancy and strongly expressed in the decidual zone after embryo implantation. PKM2 was dramatically increased with the onset of decidualization. Upon further exploration, PKM2 was found to be more highly expressed at the implantation sites than at the inter-implantation sites on days 5 to 7 of pregnancy. PKM2 expression was also significantly increased after artificial decidualization both in vivo and in vitro. After PKM2 expression was knocked down by siRNA, the number of embryo implantation sites in mice on day 7 of pregnancy was significantly reduced, and the decidualization markers BMP2 and Hoxa10 were also obviously downregulated in vivo and in vitro. Downregulated PKM2 could also compromise cell proliferation in primary endometrial stromal cells and in Ishikawa cells. The migration rate of Ishikawa cells was also obviously suppressed by si-PKM2 according to the wound healing assay. In conclusion, PKM2 might play an important role in decidualization during early pregnancy, and cell proliferation might be one pathway for PKM2 regulated decidualization.

Changes in Metabolome and Nutritional Quality of Lycium barbarum Fruits from Three Typical Growing Areas of China as Revealed by Widely Targeted Metabolomics.

This study aimed at assessing the climatic factors influencing the wolfberry fruit morphology, and the composition of its nutritious metabolites. The cultivar Ningqi1, widely grown in Northwest China was collected from three typical ecological growing counties with contrasting climatic conditions: Ningxia Zhongning (NF), Xinjiang Jinghe (XF) and Qinghai Nomuhong (QF). During the ripening period, 45 fruits from different plantations at each location were sampled. A total of 393 metabolites were detected in all samples through the widely targeted metabolomics approach and grouped into 19 known classes. Fruits from QF were the biggest followed by those from XF and NF. The altitude, relative humidity and light intensity had negative and strong correlations with most of the metabolites, suggesting that growing wolfberry in very high altitudes and under high light intensity is detrimental for the fruit nutritional quality. Soil moisture content is highly and negatively correlated with vitamins, organic acids and carbohydrates while moderately and positively correlated with other classes of metabolites. In contrast, air and soil temperatures exhibited positive correlation with majority of the metabolites. Overall, our results suggest high soil and air temperatures, low altitude and light intensity and moderate soil moisture, as the suitable conditions to produce Lycium fruits with high content of nutritious metabolites.

Author Correction: A large-scale RNA interference screen identifies genes that regulate autophagy at different stages.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

[Closed reduction combined with Taylor three-dimensional space stent fixation for supracondylar femoral fracture in children].

OBJECTIVE: To explore the effectiveness and safety of closed reduction combined with Taylor three-dimensional space stent fixation in treatment of supracondylar femoral fractures in children. METHODS: Between July 2008 and July 2016, 20 patients with supracondylar femoral fractures were treated with closed reduction combined with Taylor three-dimensional space stent fixation. There were 14 males and 6 females, with an average age of 10.3 years (range, 6-14 years). The cause of injury was traffic accident in 5 cases, falling from high place in 6 cases, and falling in 9 cases. All fractures were closed fractures. Among them, 12 cases were flexion type and 8 cases were straight type. According to AO classification, 12 cases were rated as type A1 and 8 cases as type A2. The fractures were over 0.5-5.0 cm (mean, 2.5 cm) of the epiphysis line. The time from injury to surgery was 2-8 days (mean, 3.5 days). Postoperative knee joint function was evaluated based on the Kolment evaluation criteria. RESULTS: All children were followed up 6-24 months (mean, 18.1 months). There was no complication such as nail infection, vascular nerve injury, external fixation looseing, fracture displacement, or re-fracture. All fractures healed and the fracture healing time was 4-6 weeks with an average of 4.5 weeks. The stent removal time was 8-12 weeks (mean, 9.5 weeks). The gait and knee function recovered, and there was no abnormality of the epiphysis. At last follow-up, the knee joint function were excellent in 18 cases and good in 2 cases according to the Kolment evaluation criteria, and the excellent and good rate was 100%. CONCLUSION: Closed reduction combined with Taylor three-dimensional space stent fixation is an effective treatment for the children with supracondylar femoral fractures, with small trauma and rapid recovery. It can avoid damaging the tarsal plate, be high fracture healing rate, and promote the recovery of limb function.

Suppression of STING Associated with LKB1 Loss in KRAS-Driven Lung Cancer.

KRAS-driven lung cancers frequently inactivate TP53 and/or STK11/LKB1, defining tumor subclasses with emerging clinical relevance. Specifically, KRAS-LKB1 (KL)-mutant lung cancers are particularly aggressive, lack PD-L1, and respond poorly to immune checkpoint blockade (ICB). The mechanistic basis for this impaired immunogenicity, despite the overall high mutational load of KRAS-mutant lung cancers, remains obscure. Here, we report that LKB1 loss results in marked silencing of stimulator of interferon genes (STING) expression and insensitivity to cytoplasmic double-strand DNA (dsDNA) sensing. This effect is mediated at least in part by hyperactivation of DNMT1 and EZH2 activity related to elevated S-adenylmethionine levels and reinforced by DNMT1 upregulation. Ectopic expression of STING in KL cells engages IRF3 and STAT1 signaling downstream of TBK1 and impairs cellular fitness, due to the pathologic accumulation of cytoplasmic mitochondrial dsDNA associated with mitochondrial dysfunction. Thus, silencing of STING avoids these negative consequences of LKB1 inactivation, while facilitating immune escape. SIGNIFICANCE: Oncogenic KRAS-mutant lung cancers remain treatment-refractory and are resistant to ICB in the setting of LKB1 loss. These results begin to uncover the key underlying mechanism and identify strategies to restore STING expression, with important therapeutic implications because mitochondrial dysfunction is an obligate component of this tumor subtype.See related commentary by Corte and Byers, p. 16.This article is highlighted in the In This Issue feature, p. 1.

Casein Kinase 1 Epsilon Regulates Glioblastoma Cell Survival.

Glioblastoma is the most common malignant brain cancer with a dismal prognosis. The difficulty in treating glioblastoma is largely attributed to the lack of effective therapeutic targets. In our previous work, we identified casein kinase 1 ε (CK1ε, also known as CSNK1E) as a potential survival factor in glioblastoma. However, how CK1ε controls cell survival remains elusive and whether targeting CK1ε is a possible treatment for glioblastoma requires further investigation. Here we report that CK1ε was expressed at the highest level among six CK1 isoforms in glioblastoma and enriched in high-grade glioma, but not glia cells. Depletion of CK1ε remarkably inhibited the growth of glioblastoma cells and suppressed self-renewal of glioblastoma stem cells, while having limited effect on astrocytes. CK1ε deprivation activated ß-catenin and induced apoptosis, which was further counteracted by knockdown of ß-catenin. The CK1ε inhibitor IC261, but not PF-4800567, activated ß-catenin and blocked the growth of glioblastoma cells and glioblastoma stem cells. Congruently, IC261 elicited a robust growth inhibition of human glioblastoma xenografts in mice. Together, our results demonstrate that CK1ε regulates the survival of glioblastoma cells and glioblastoma stem cells through ß-catenin signaling, underscoring the importance of targeting CK1ε as an effective treatment for glioblastoma.

Overcoming Resistance to Dual Innate Immune and MEK Inhibition Downstream of KRAS.

Despite extensive efforts, oncogenic KRAS remains resistant to targeted therapy. Combined downstream RAL-TBK1 and MEK inhibition induces only transient lung tumor shrinkage in KRAS-driven genetically engineered mouse models (GEMMs). Using the sensitive KRAS;LKB1 (KL) mutant background, we identify YAP1 upregulation and a therapy-induced secretome as mediators of acquired resistance. This program is reversible, associated with H3K27 promoter acetylation, and suppressed by BET inhibition, resensitizing resistant KL cells to TBK1/MEK inhibition. Constitutive YAP1 signaling promotes intrinsic resistance in KRAS;TP53 (KP) mutant lung cancer. Intermittent treatment with the BET inhibitor JQ1 thus overcomes resistance to combined pathway inhibition in KL and KP GEMMs. Using potent and selective TBK1 and BET inhibitors we further develop an effective therapeutic strategy with potential translatability to the clinic.

A large-scale RNA interference screen identifies genes that regulate autophagy at different stages.

Dysregulated autophagy is central to the pathogenesis and therapeutic development of cancer. However, how autophagy is regulated in cancer is not well understood and genes that modulate cancer autophagy are not fully defined. To gain more insights into autophagy regulation in cancer, we performed a large-scale RNA interference screen in K562 human chronic myeloid leukemia cells using monodansylcadaverine staining, an autophagy-detecting approach equivalent to immunoblotting of the autophagy marker LC3B or fluorescence microscopy of GFP-LC3B. By coupling monodansylcadaverine staining with fluorescence-activated cell sorting, we successfully isolated autophagic K562 cells where we identified 336 short hairpin RNAs. After candidate validation using Cyto-ID fluorescence spectrophotometry, LC3B immunoblotting, and quantitative RT-PCR, 82 genes were identified as autophagy-regulating genes. 20 genes have been reported previously and the remaining 62 candidates are novel autophagy mediators. Bioinformatic analyses revealed that most candidate genes were involved in molecular pathways regulating autophagy, rather than directly participating in the autophagy process. Further autophagy flux assays revealed that 57 autophagy-regulating genes suppressed autophagy initiation, whereas 21 candidates promoted autophagy maturation. Our RNA interference screen identifies identified genes that regulate autophagy at different stages, which helps decode autophagy regulation in cancer and offers novel avenues to develop autophagy-related therapies for cancer.

PIK3CB/p110ß is a selective survival factor for glioblastoma.

Background: Glioblastoma (GBM) is difficult to treat. Phosphoinositide 3-kinase (PI3K) is an attractive therapeutic target for GBM; however, targeting this pathway to effectively treat GBM is not successful because the roles of PI3K isoforms remain to be defined. The aim of this study is to determine whether PIK3CB/p110ß, but not other PI3K isoforms, is a biomarker for GBM recurrence and important for cell survival. Methods: Gene expression and clinical relevance of PI3K genes in GBM patients were analyzed using online databases. Expression/activity of PI3K isoforms was determined using immunoblotting. PI3K genes were inhibited using short hairpin RNAs or isoform-selective inhibitors. Cell viability/growth was assessed by the MTS assay and trypan blue exclusion assay. Apoptosis was monitored using the caspase activity assay. Mouse GBM xenograft models were used to gauge drug efficacy. Results: PIK3CB/p110ß was the only PI3K catalytic isoform that significantly correlated with high incidence rate, risk, and poor survival of recurrent GBM. PIK3CA/p110α, PIK3CB/p110ß, and PIK3CD/p110δ were differentially expressed in GBM cell lines and primary tumor cells derived from patient specimens, whereas PIK3CG/p110γ was barely detected. PIK3CB/p110ß protein levels presented a stronger association with the activities of PI3K signaling than other PI3K isoforms. Blocking p110ß deactivated PI3K signaling, whereas inhibition of other PI3K isoforms had no effect. Specific inhibitors of PIK3CB/p110ß, but not other PI3K isoforms, remarkably suppressed viability and growth of GBM cells and xenograft tumors in mice, with minimal cytotoxic effects on astrocytes. Conclusions: PIK3CB/p110ß is a biomarker for GBM recurrence and selectively important for GBM cell survival.