Mammalian Target of Rapamycin (mTOR) Activity Dependent Phospho-Protein Expression in Childhood Acute Lymphoblastic Leukemia (ALL)

Modern treatment strategies have improved the prognosis of childhood ALL; however, treatment still fails in 25–30% of patients. Further improvement of treatment may depend on the development of targeted therapies. mTOR kinase, a central mediator of several signaling pathways, has recently attracted remarkable attention as a potential target in pediatric ALL. However, limited data exists about the activity of mTOR. In the present study, the amount of mTOR activity dependent phospho-proteins was characterized by ELISA in human leukemia cell lines and in lymphoblasts from childhood ALL patients (n = 49). Expression was measured before and during chemotherapy and at relapses. Leukemia cell lines exhibited increased mTOR activity, indicated by phospho-S6 ribosomal protein (p-S6) and phosphorylated eukaryotic initiation factor 4E binding protein (p-4EBP1). Elevated p-4EBP1 protein levels were detected in ALL samples at diagnosis; efficacy of chemotherapy was followed by the decrease of mTOR activity dependent protein phosphorylation. Optical density (OD) for p-4EBP1 (ELISA) was significantly higher in patients with poor prognosis at diagnosis, and in the samples of relapsed patients. Our results suggest that measuring mTOR activity related phospho-proteins such as p-4EBP1 by ELISA may help to identify patients with poor prognosis before treatment, and to detect early relapses. Determining mTOR activity in leukemic cells may also be a useful tool for selecting patients who may benefit from future mTOR inhibitor treatments

Characterisation of bioenergetic pathways and related regulators by multiple assays in human tumour cells

Background: Alterations in cellular metabolism are considered as hallmarks of cancers, however, to recognize these alterations and understand their mechanisms appropriate techniques are required. Our hypothesis was to determine whether dominant bioenergetic mechanism may be estimated by comparing the substrate utilisation with different methods to detect the labelled carbon incorporation and their application in tumour cells. Methods: To define the bioenergetic pathways different metabolic tests were applied: (a) measuring CO2 production from [1-14C]-glucose and [1-14C]-acetate; (b) studying the effect of glucose and acetate on adenylate energy charge; (c) analysing glycolytic and TCA cycle metabolites and the number of incorporated 13C atoms after [U-13C]-glucose/[2-13C]-acetate labelling. Based on [1-14C]-substrate oxidation two selected cell lines out of seven were analysed in details, in which the highest difference was detected at their substrate utilization. To elucidate the relevance of metabolic characterisation the expression of certain regulatory factors, bioenergetic enzymes, mammalian target of rapamycin (mTOR) complexes (C1/C2) and related targets as important elements at the crossroad of cellular signalling network were also investigated. Results: Both [U-13C]-glucose and [1-14C]-substrate labelling indicated high glycolytic capacity of tumour cells. However, the ratio of certain 13C-labelled metabolites showed detailed metabolic differences in the two selected cell lines in further characterisation. The detected differences of GAPDH, β-F1-ATP-ase expression and adenylate energy charge in HT-1080 and ZR-75.1 tumour cells also confirmed the altered metabolism. Moreover, the highly limited labelling of citrate by [2-13C]-acetate-representing a novel functional test in malignant cells-confirmed the defect of TCA cycle of HT-1080 in contrast to ZR-75.1 cells. Noteworthy, the impaired TCA cycle in HT-1080 cells were associated with high mTORC1 activity, negligible protein level and activity of mTORC2, high expression of interleukin-1β, interleukin-6 and heme oxygenase-1 which may contribute to the compensatory mechanism of TCA deficiency. Conclusions: The applied methods of energy substrate utilisation and other measurements represent simple assay system using 13C-acetate and glucose to recognize dominant bioenergetic pathways in tumour cells. These may offer a possibility to characterise metabolic subtypes of human tumours and provide guidelines to find biomarkers for prediction and development of new metabolism related targets in personalized therapy. © 2016 Jeney et al

Comparative constructions of similarity in Northern Samoyedic languages

The purpose of this paper is to analyze the suffixes which are used in Northern Samoyedic languages to build comparative constructions of equality. Depending on the language, the suffixes may perform three functions: word-building, form-building, and inflectional. When they mark the noun, they serve as simulative suffixes and are employed to build object comparison. In the inflectional function, these suffixes mark the verb and are a means of constructing situational comparison. In this case, they signal the formation of a special mood termed the Approximative. This paper provides a detailed description of the Approximative from paradigmatic and syntagmatic perspectives

Conformation-regulated mechanosensory control via titin domains in cardiac muscle

The giant filamentous protein titin is ideally positioned in the muscle sarcomere to sense mechanical stimuli and transform them into biochemical signals, such as those triggering cardiac hypertrophy. In this review, we ponder the evidence for signaling hotspots along the titin filament involved in mechanosensory control mechanisms. On the way, we distinguish between stress and strain as triggers of mechanical signaling events at the cardiac sarcomere. Whereas the Z-disk and M-band regions of titin may be prominently involved in sensing mechanical stress, signaling hotspots within the elastic I-band titin segment may respond primarily to mechanical strain. Common to both stress and strain sensor elements is their regulation by conformational changes in protein domains

Effects of pretreatments of Napier Grass with deionized water, sulfuric acid and sodium hydroxide on pyrolysis oil characteristics

The depletion of fossil fuel reserves has led to increasing interest in liquid bio-fuel from renewable biomass. Biomass is a complex organic material consisting of different degrees of cellulose, hemicellulose, lignin, extractives and minerals. Some of the mineral elements tend to retard conversions, yield and selectivity during pyrolysis processing. This study is focused on the extraction of mineral retardants from Napier grass using deionized water, dilute sodium hydroxide and sulfuric acid and subsequent pyrolysis in a fixed bed reactor. The raw biomass was characterized before and after each pretreatment following standard procedure. Pyrolysis study was conducted in a fixed bed reactor at 600 o�C, 30 �C/min and 30 mL/min N2 flow. Pyrolysis oil (bio-oil) collected was analyzed using standard analytic techniques. The bio-oil yield and characteristics from each pretreated sample were compared with oil from the non-pretreated sample. Bio-oil yield from the raw sample was 32.06 wt% compared to 38.71, 33.28 and 29.27 wt% oil yield recorded from the sample pretreated with sulfuric acid, deionized water and sodium hydroxide respectively. GC–MS analysis of the oil samples revealed that the oil from all the pretreated biomass had more value added chemicals and less ketones and aldehydes. Pretreatment with neutral solvent generated valuable leachate, showed significant impact on the ash extraction, pyrolysis oil yield, and its composition and therefore can be regarded as more appropriate for thermochemical conversion of Napier grass

GABA, glutamine, glutamate oxidation and succinic semialdehyde dehydrogenase expression in human gliomas

Bioenergetic characterisation of malignant tissues revealed that different tumour cells can catabolise multiple substrates as salvage pathways, in response to metabolic stress. Altered metabolism in gliomas has received a lot of attention, especially in relation to IDH mutations, and the associated oncometabolite D-2-hydroxyglutarate (2-HG) that impact on metabolism, epigenetics and redox status. Astrocytomas and oligodendrogliomas, collectively called diffuse gliomas, are derived from astrocytes and oligodendrocytes that are in metabolic symbiosis with neurons; astrocytes can catabolise neuron-derived glutamate and gamma-aminobutyric acid (GABA) for supporting and regulating neuronal functions.Metabolic characteristics of human glioma cell models - including mitochondrial function, glycolytic pathway and energy substrate oxidation - in relation to IDH mutation status and after 2-HG incubation were studied to understand the Janus-faced role of IDH1 mutations in the progression of gliomas/astrocytomas. The metabolic and bioenergetic features were identified in glioma cells using wild-type and genetically engineered IDH1-mutant glioblastoma cell lines by metabolic analyses with Seahorse, protein expression studies and liquid chromatography-mass spectrometry.U251 glioma cells were characterised by high levels of glutamine, glutamate and GABA oxidation. Succinic semialdehyde dehydrogenase (SSADH) expression was correlated to GABA oxidation. GABA addition to glioma cells increased proliferation rates. Expression of mutated IDH1 and treatment with 2-HG reduced glutamine and GABA oxidation, diminished the pro-proliferative effect of GABA in SSADH expressing cells. SSADH protein overexpression was found in almost all studied human cases with no significant association between SSADH expression and clinicopathological parameters (e.g. IDH mutation).Our findings demonstrate that SSADH expression may participate in the oxidation and/or consumption of GABA in gliomas, furthermore, GABA oxidation capacity may contribute to proliferation and worse prognosis of gliomas. Moreover, IDH mutation and 2-HG production inhibit GABA oxidation in glioma cells. Based on these data, GABA oxidation and SSADH activity could be additional therapeutic targets in gliomas/glioblastomas