Identification of Susceptibility Genes Underlying Bovine Respiratory Disease in Xinjiang Brown Cattle Based on DNA Methylation.

DNA methylation is a form of epigenetic regulation, having pivotal parts in controlling cellular expansion and expression levels within genes. Although blood DNA methylation has been studied in humans and other species, its prominence in cattle is largely unknown. This study aimed to methodically probe the genomic methylation map of Xinjiang brown (XJB) cattle suffering from bovine respiratory disease (BRD), consequently widening cattle blood methylome ranges. Genome-wide DNA methylation profiling of the XJB blood was investigated through whole-genome bisulfite sequencing (WGBS). Many differentially methylated regions (DMRs) obtained by comparing the cases and controls groups were found within the CG, CHG, and CHH (where H is A, T, or C) sequences (16,765, 7502, and 2656, respectively), encompassing 4334 differentially methylated genes (DMGs). Furthermore, GO/KEGG analyses showed that some DMGs were involved within immune response pathways. Combining WGBS-Seq data and existing RNA-Seq data, we identified 71 significantly differentially methylated (DMGs) and expressed (DEGs) genes (p < 0.05). Next, complementary analyses identified nine DMGs (LTA, STAT3, IKBKG, IRAK1, NOD2, TLR2, TNFRSF1A, and IKBKB) that might be involved in the immune response of XJB cattle infected with respiratory diseases. Although further investigations are needed to confirm their exact implication in the involved immune processes, these genes could potentially be used for a marker-assisted selection of animals resistant to BRD. This study also provides new knowledge regarding epigenetic control for the bovine respiratory immune process.

Structural Characterization and Cytotoxic Activity Evaluation of Ulvan Polysaccharides Extracted from the Green Algae Ulva papenfussii.

Ulvan, a sulfated heteropolysaccharide with structural and functional properties of interest for various uses, was extracted from the green seaweed Ulva papenfussii. U. papenfussii is an unexplored Ulva species found in the South China Sea along the central coast of Vietnam. Based on dry weight, the ulvan yield was ~15% (w/w) and the ulvan had a sulfate content of 13.4 wt%. The compositional constitution encompassed L-Rhamnose (Rhap), D-Xylose (Xylp), D-Glucuronic acid (GlcAp), L-Iduronic acid (IdoAp), D-Galactose (Galp), and D-Glucose (Glcp) with a molar ratio of 1:0.19:0.35:0.52:0.05:0.11, respectively. The structure of ulvan was determined using High-Performance Liquid Chromatography (HPLC), Fourier Transform Infrared Spectroscopy (FT-IR), and Nuclear Magnetic Resonance spectroscopy (NMR) methods. The results showed that the extracted ulvan comprised a mixture of two different structural forms, namely ("A3s") with the repeating disaccharide [→4)-ß-D-GlcAp-(1→4)-α-L-Rhap 3S-(1→]n, and ("B3s") with the repeating disaccharide [→4)-α-L-IdoAp-(1→4)-α-L-Rhap 3S(1→]n. The relative abundance of A3s, and B3s was 1:1.5, respectively. The potential anticarcinogenic attributes of ulvan were evaluated against a trilogy of human cancer cell lineages. Concomitantly, Quantitative Structure-Activity Relationship (QSAR) modeling was also conducted to predict potential adverse reactions stemming from pharmacological interactions. The ulvan showed significant antitumor growth activity against hepatocellular carcinoma (IC50 ≈ 90 µg/mL), human breast cancer cells (IC50 ≈ 85 µg/mL), and cervical cancer cells (IC50 ≈ 67 µg/mL). The QSAR models demonstrated acceptable predictive power, and seven toxicity indications confirmed the safety of ulvan, warranting its candidacy for further in vivo testing and applications as a biologically active pharmaceutical source for human disease treatment.

Candidate genes for litter size in Xinjiang sheep identified by Specific Locus Amplified Fragment (SLAF) sequencing.

The aim of this study was to investigate the selection signatures at a genome-wide level in 'Pishan' sheep using Specific Locus Amplified Fragment (SLAF)-seq. Blood samples from 126 ewes were sequenced using SLAF tags, and the ovarian tissues from 8 ewes (Bashbay sheep, a single litter size group (SG group); 'Pishan' sheep, double litter size group (DG group)) were collected to detect expression levels by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Selection signature analysis was performed using global fixation index (Fst) and nucleotide diversity (π) ratio. A total of 1,192,168 high-quality SLAFs were identified. Notably, 2380 candidate regions under selection using two approaches were identified. A total of 2069 genes were identified, which were involved in dopaminergic synapses, thyroid hormone synthesis, ovarian steroidogenesis and thyroid hormone signalling pathways. Furthermore, Growth Differentiation Factor 9 (GDF9), Period Circadian Regulator 2 (PER2), Thyroid Stimulating Hormone Receptor (TSHR), and Nuclear Receptor Coactivator 1 (NCOA1) reside within these regions and pathways. The expression levels of GDF9 and PER2 genes in sheep tissue of the DG group were significantly higher than those in the SG group. These genes are interesting candidates for litter size and provide a starting point for further identification of conservation strategies for 'Pishan' sheep.

The Endo-α(1,3)-Fucoidanase Mef2 Releases Uniquely Branched Oligosaccharides from Saccharina latissima Fucoidans.

Fucoidans are complex bioactive sulfated fucosyl-polysaccharides primarily found in brown macroalgae. Endo-fucoidanases catalyze the specific hydrolysis of α-L-fucosyl linkages in fucoidans and can be utilized to tailor-make fucoidan oligosaccharides and elucidate new structural details of fucoidans. In this study, an endo-α(1,3)-fucoidanase encoding gene, Mef2, from the marine bacterium Muricauda eckloniae, was cloned, and the Mef2 protein was functionally characterized. Based on the primary sequence, Mef2 was suggested to belong to the glycosyl hydrolase family 107 (GH107) in the Carbohydrate Active enZyme database (CAZy). The Mef2 fucoidanase showed maximal activity at pH 8 and 35 °C, although it could tolerate temperatures up to 50 °C. Ca2+ was shown to increase the melting temperature from 38 to 44 °C and was furthermore required for optimal activity of Mef2. The substrate specificity of Mef2 was investigated, and Fourier transform infrared spectroscopy (FTIR) was used to determine the enzymatic activity (Units per µM enzyme: Uf/µM) of Mef2 on two structurally different fucoidans, showing an activity of 1.2 × 10-3 Uf/µM and 3.6 × 10-3 Uf/µM on fucoidans from Fucus evanescens and Saccharina latissima, respectively. Interestingly, Mef2 was identified as the first described fucoidanase active on fucoidans from S. latissima. The fucoidan oligosaccharides released by Mef2 consisted of a backbone of α(1,3)-linked fucosyl residues with unique and novel α(1,4)-linked fucosyl branches, not previously identified in fucoidans from S. latissima.

Stimulation of ORAI1 expression, store-operated Ca2+ entry, and osteogenic signaling by high glucose exposure of human aortic smooth muscle cells.

Diabetes and chronic kidney disease (CKD) both trigger vascular osteogenic signaling and calcification leading to early death by cardiovascular events. Osteogenic signaling involves upregulation of the transcription factors CBFA1, MSX2, and SOX9, as well as alkaline phosphatase (ALP), an enzyme fostering calcification by degrading the calcification inhibitor pyrophosphate. In CKD, osteogenic signaling is triggered by hyperphosphatemia, which upregulates the serum and glucocorticoid-inducible kinase SGK1, a strong stimulator of the Ca2+-channel ORAI1. The channel is activated by STIM1 and accomplishes store-operated Ca2+-entry (SOCE). The present study explored whether exposure of human aortic smooth muscle cells (HAoSMCs) to high extracellular glucose concentrations similarly upregulates ORAI1 and/or STIM1 expression, SOCE, and osteogenic signaling. To this end, HAoSMCs were exposed to high extracellular glucose concentrations (15 mM, 24 h) without or with additional exposure to the phosphate donor ß-glycerophosphate. Transcript levels were estimated using qRT-PCR, protein abundance using Western blotting, ALP activity using a colorimetric assay kit, calcium deposits utilizing Alizarin red staining, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). As a result, glucose enhanced the transcript levels of SGK1 and ORAI1, ORAI2, and STIM2, protein abundance of ORAI1, SOCE, the transcript levels of CBFA1, MSX2, SOX9, and ALPL, as well as calcium deposits. Moreover, glucose significantly augmented the stimulating effect of ß-glycerophosphate on transcript levels of SGK1 and ORAI1, SOCE, the transcript levels of osteogenic markers, as well as calcium deposits. ORAI1 inhibitor MRS1845 (10 µM) significantly blunted the glucose-induced upregulation of the CBFA1 and MSX2 transcript levels. In conclusion, the hyperglycemia of diabetes stimulates expression of SGK1 and ORAI1, thus, augmenting store-operated Ca2+-entry and osteogenic signaling in HAoSMCs.

Multi-dimensional omics characterization in glioblastoma identifies the purity-associated pattern and prognostic gene signatures.

BACKGROUND: The presence of tumor-associated stroma and tumor-infiltrated immune cells have been largely reported across glioblastomas. Tumor purity, defined as the proportion of tumor cells in the tumor, was associated with the genomic and clinicopathologic features of the tumor and may alter the interpretation of glioblastoma biology. METHODS: We use an integrative approach to infer tumor purity based on multi-omic data and comprehensively evaluate the impact of tumor purity on glioblastoma (GBM) prognosis, genomic profiling, and the immune microenvironment in the Cancer Genome Atlas Consortium (TCGA) cohort. RESULTS: We found that low tumor purity was significantly associated with reduced survival time. Additionally, we established a purity-relevant 5-gene signature that was an independent prognostic biomarker and validated it in the TCGA, CGGA and GSE4412 cohort. Moreover, we correlated tumor purity with genomic characteristics and tumor microenvironment. We identified that gamma delta T cells in glioblastoma microenvironment were positively correlated with purity and served as a marker for favorable prognosis, which was validated in both TCGA and CGGA dataset. CONCLUSIONS: We observe the potential confounding effects of tumor purity on GBM clinical and molecular information interpretation. GBM microenvironment could be purity-dependent, which provides new insights into the clinical implications of glioblastoma.

Correction to: A quantitative model based on clinically relevant MRI features differentiates lower grade gliomas and glioblastoma.

The original version of this article, published on 05 February 2020, unfortunately contained a mistake.

A quantitative model based on clinically relevant MRI features differentiates lower grade gliomas and glioblastoma.

OBJECTIVES: To establish a quantitative MR model that uses clinically relevant features of tumor location and tumor volume to differentiate lower grade glioma (LRGG, grades II and III) and glioblastoma (GBM, grade IV). METHODS: We extracted tumor location and tumor volume (enhancing tumor, non-enhancing tumor, peritumor edema) features from 229 The Cancer Genome Atlas (TCGA)-LGG and TCGA-GBM cases. Through two sampling strategies, i.e., institution-based sampling and repeat random sampling (10 times, 70% training set vs 30% validation set), LASSO (least absolute shrinkage and selection operator) regression and nine-machine learning method-based models were established and evaluated. RESULTS: Principal component analysis of 229 TCGA-LGG and TCGA-GBM cases suggested that the LRGG and GBM cases could be differentiated by extracted features. For nine machine learning methods, stack modeling and support vector machine achieved the highest performance (institution-based sampling validation set, AUC > 0.900, classifier accuracy > 0.790; repeat random sampling, average validation set AUC > 0.930, classifier accuracy > 0.850). For the LASSO method, regression model based on tumor frontal lobe percentage and enhancing and non-enhancing tumor volume achieved the highest performance (institution-based sampling validation set, AUC 0.909, classifier accuracy 0.830). The formula for the best performance of the LASSO model was established. CONCLUSIONS: Computer-generated, clinically meaningful MRI features of tumor location and component volumes resulted in models with high performance (validation set AUC > 0.900, classifier accuracy > 0.790) to differentiate lower grade glioma and glioblastoma. KEY POINTS: • Lower grade glioma and glioblastoma have significant different location and component volume distributions. • We built machine learning prediction models that could help accurately differentiate lower grade gliomas and GBM cases. We introduced a fast evaluation model for possible clinical differentiation and further analysis.

Functional Characterization of a New GH107 Endo-α-(1,4)-Fucoidanase from the Marine Bacterium Formosa haliotis.

Fucoidans from brown macroalgae are sulfated fucose-rich polysaccharides, that have several beneficial biological activities, including anti-inflammatory and anti-tumor effects. Controlled enzymatic depolymerization of the fucoidan backbone can help produce homogeneous, defined fucoidan products for structure-function research and pharmaceutical uses. However, only a few endo-fucoidanases have been described. This article reports the genome-based discovery, recombinant expression in Escherichia coli, stabilization, and functional characterization of a new bacterial endo-α-(1,4)-fucoidanase, Fhf1, from Formosa haliotis. Fhf1 catalyzes the cleavage of α-(1,4)-glycosidic linkages in fucoidans built of alternating α-(1,3)-/α-(1,4)-linked l-fucopyranosyl sulfated at C2. The native Fhf1 is 1120 amino acids long and belongs to glycoside hydrolase (GH) family 107. Deletion of the signal peptide and a 470 amino acid long C-terminal stretch led to the recombinant expression of a robust, minimized enzyme, Fhf1Δ470 (71 kDa). Fhf1Δ470 has optimal activity at pH 8, 37-40 °C, can tolerate up to 500 mM NaCl, and requires the presence of divalent cations, either Ca2+, Mn2+, Zn2+ or Ni2+, for maximal activity. This new enzyme has the potential to serve the need for controlled enzymatic fucoidan depolymerization to produce bioactive sulfated fucoidan oligomers.

Enzyme-Assisted Fucoidan Extraction from Brown Macroalgae Fucus distichus subsp. evanescens and Saccharina latissima.

Fucoidans from brown macroalgae (brown seaweeds) have different structures and many interesting bioactivities. Fucoidans are classically extracted from brown seaweeds by hot acidic extraction. Here, we report a new targeted enzyme-assisted methodology for fucoidan extraction from brown seaweeds. This enzyme-assisted extraction protocol involves a one-step combined use of a commercial cellulase preparation (Cellic®CTec2) and an alginate lyase from Sphingomonas sp. (SALy), reaction at pH 6.0, 40 °C, removal of non-fucoidan polysaccharides by Ca2+ precipitation, and ethanol-precipitation of crude fucoidan. The workability of this method is demonstrated for fucoidan extraction from Fucus distichus subsp. evanescens (basionym Fucus evanescens) and Saccharina latissima as compared with mild acidic extraction. The crude fucoidans resulting directly from the enzyme-assisted method contained considerable amounts of low molecular weight alginate, but this residual alginate was effectively removed by an additional ion-exchange chromatographic step to yield pure fucoidans (as confirmed by 1H NMR). The fucoidan yields that were obtained by the enzymatic method were comparable to the chemically extracted yields for both F. evanescens and S. latissima, but the molecular sizes of the fucoidans were significantly larger with enzyme-assisted extraction. The molecular weight distribution of the fucoidan fractions was 400 to 800 kDa for F. evanescens and 300 to 800 kDa for S. latissima, whereas the molecular weights of the corresponding chemically extracted fucoidans from these seaweeds were 10-100 kDa and 50-100 kDa, respectively. Enzyme-assisted extraction represents a new gentle strategy for fucoidan extraction and it provides new opportunities for obtaining high yields of native fucoidan structures from brown macroalgae.

Gliosarcoma: a clinical and radiological analysis of 48 cases.

OBJECTIVES: To retrospectively review the radiological and clinicopathological features of gliosarcoma (GSM) and differentiate it from glioblastoma multiforme (GBM). METHODS: The clinicopathological data and imaging findings (including VASARI analysis) of 48 surgically and pathologically confirmed GSM patients (group 1) were reviewed in detail, and were compared with that of other glioblastoma (GBM) cases in our hospital (group 2). RESULTS: There were 28 men and 20 women GSM patients with a median age of 52.5 years (range, 24-80 years) in this study. Haemorrhage (n = 21), a salt-and-pepper sign on T2-weighted images (n = 36), unevenly thickened wall (n = 36) even appearing as a paliform pattern (n = 32), an intra-tumoural large feeding artery (n = 32) and an eccentric cystic portion (ECP) (n = 19) were more commonly observed in the GSM group than in GBM patients. Based on our experience, GSM can be divided into four subtypes according to magnetic resonance imaging (MRI) features. When compared to GBM (group 2), there were more patients designated with type III lesions (having very unevenly thickened walls) and IV (solid) lesions among the GSM cases (group 1). On univariate prognostic analysis, adjuvant therapy (radiotherapy, chemotherapy, and radiochemotherapy) and existence of an eccentric cyst region were prognostic factors. However, Cox's regression model showed only adjuvant therapy as a prognostic factor for GSM. CONCLUSIONS: When compared to GBM, certain imaging features are more likely to occur in GSM, which may help raise the possibility of this disease. All GSM patients are recommended to receive adjuvant therapy to achieve a better prognosis with radiotherapy, chemotherapy or radiochemotherapy all as options. KEY POINTS: • Diagnosis of gliosarcoma can be suggested preoperatively by imaging. • Gliosarcoma can be divided into four subtypes based on MRI. • Paliform pattern and ECP tend to present in gliosarcoma more than GBM. • The cystic subtype of gliosarcoma may predict a more dismal prognosis. • All gliosarcoma patients should receive adjuvant therapy to achieve better prognosis.

Inhibition of Collagen Related Peptide Induced Platelet Activation and Apoptosis by Ceritinib.

BACKGROUND/AIMS: The anaplastic lymphoma (tyrosine) kinase (ALK) inhibitor ceritinib triggers apoptosis of tumor cells and eryptosis of erythrocytes. Blood platelets may similarly enter a state resembling apoptosis, which could be triggered by activation with collagen related peptide (CRP). CRP-induced platelet apoptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the platelet surface and cell shrinkage, preceded by externalization of Ca2+ channel Orai1, increase of cytosolic Ca2+-activity ([Ca2+]i), formation of reactive oxygen species (ROS), and caspase activation. The present study explored whether ceritinib triggers platelet apoptosis and/or modifies the CRP induced apoptosis. METHODS: Platelets isolated from wild-type mice were exposed for 30 minutes to ceritinib (1.5 µg/ml) without or with 2.5 - 15 min pretreatment with CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, ROS abundance from 2',7'-dichlorodihydrofluorescein diacetate fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbß3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. RESULTS: In the absence of CRP, ceritinib slightly, but significantly decreased [Ca2+]i without significantly modifying the other measured parameters. CRP significantly increased [Ca2+]i, ROS abundance, P-selectin abundance, activated αIIbß3 integrin, annexin-V-binding, caspase activity as well as aggregation and decreased cell volume, all effects significantly blunted in the presence of ceritinib. CONCLUSIONS: The present observations uncover a novel, unexpected effect of ceritinib, i.e. inhibition of CRP-induced platelet activation and apoptosis.

Sonidegib, a Novel Inhibitor of Suicidal Erythrocyte Death.

BACKGROUND/AIMS: The Hedgehog pathway disrupting drug sonidegib is used in the treatment of basal cell carcinoma. Side effects of sonidegib include anemia, which could result either from impaired erythropoiesis or from loss of erythrocytes e.g. due to suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Eryptosis is stimulated by cell stress, including energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether sonidegib exerts an effect on eryptosis. METHODS: Human erythrocytes have been treated with energy depletion (glucose withdrawal for 48 hours), hyperosmotic shock (addition of 550 mM sucrose for 6 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca2+ ionophore ionomycin (1 µM for 60 min) in absence and presence of sonidegib (2-6 µg/ ml). After treatment flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, and cell volume from forward scatter. Hemolysis was estimated from the hemoglobin concentration in the supernatant. RESULTS: In the absence of cell stress exposure to sonidegib did not significantly modify annexin-V-binding or forward scatter, but triggered hemolysis. Energy depletion, hyperosmotic shock, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. Sonidegib significantly blunted the effect of energy depletion, hyperosmotic shock, and oxidative stress, but not of ionomycin on annexin-V-binding. Sonidegib further significantly blunted the effect of energy depletion, but not of hyperosmotic shock, oxidative stress, and ionomycin on forward scatter. CONCLUSIONS: Sonidegib is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion, hyperosmotic shock and oxidative stress.

NFAT5-sensitive Orai1 expression and store-operated Ca2+ entry in megakaryocytes.

The transcription factor nuclear factor of activated T cells 5 (NFAT5) is up-regulated in several clinical disorders, including dehydration. NFAT5-sensitive genes include serum and glucocorticoid-inducible kinase 1 (SGK1). The kinase is a powerful regulator of Orai1, a Ca2+ channel accomplishing store-operated Ca2+ entry (SOCE). Orai1 is stimulated after intracellular store depletion by the Ca2+ sensors stromal interaction molecule 1 (STIM1), or STIM2, or both. In the present study, we explored whether nuclear factor of activated T cell (NFAT)-5 influences Ca2+ signaling in megakaryocytes. To this end, human megakaryocytic (MEG-01) cells were transfected with NFAT5 or with siNFAT5. Platelets and megakaryocytes were isolated from wild-type mice with either access to water ad libitum or dehydration by 36 h of water deprivation. Transcript levels were determined with quantitative RT-PCR and protein abundance by Western blot analysis and flow cytometry, cytosolic (intracellular) Ca2+ concentration ([Ca2+]i) by fura-2-fluorescence. SOCE was estimated from the increase of [Ca2+]i following readdition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). Platelet degranulation was estimated from P-selectin abundance and integrin activation from αIIbß3 integrin abundance determined by flow cytometry. As a result, NFAT5 transfection or exposure to hypertonicity (+40 mM NaCl) of MEG-01 cells increased Orai1, Orai2, STIM1, and STIM2 transcript levels. Orai1 transcript levels were decreased by NFAT5 silencing. NFAT5 transfection and IκB inhibitor BMS 345541 (5 µM) increased SOCE, whereas NFAT5 silencing and SGK1 inhibitor GSK650394 (10 µM) decreased SOCE. In the mice, dehydration increased NFAT5 and Orai1 protein abundance in megakaryocytes and NFAT5, Orai1, and Orai2 abundance in platelets. Dehydration further augmented the degranulation and integrin activation by thrombin and collagen-related peptide. In summary, NFAT5 is a powerful regulator of Orai1-expression and SOCE in megakaryocytes.-Sahu, I., Pelzl, L., Sukkar, B., Fakhri, H., al-Maghout, T., Cao, H., Hauser, S., Gutti, R., Gawaz, M., Lang, F. NFAT5-sensitive Orai1 expression and store-operated Ca2+ entry in megakaryocytes.

Novel Enzyme Actions for Sulphated Galactofucan Depolymerisation and a New Engineering Strategy for Molecular Stabilisation of Fucoidan Degrading Enzymes.

Fucoidans from brown macroalgae have beneficial biomedical properties but their use as pharma products requires homogenous oligomeric products. In this study, the action of five recombinant microbial fucoidan degrading enzymes were evaluated on fucoidans from brown macroalgae: Sargassum mcclurei, Fucus evanescens, Fucus vesiculosus, Turbinaria ornata, Saccharina cichorioides, and Undaria pinnatifida. The enzymes included three endo-fucoidanases (EC 3.2.1.-GH 107), FcnA2, Fda1, and Fda2, and two unclassified endo-fucoglucuronomannan lyases, FdlA and FdlB. The oligosaccharide product profiles were assessed by carbohydrate-polyacrylamide gel electrophoresis and size exclusion chromatography. The recombinant enzymes FcnA2, Fda1, and Fda2 were unstable but were stabilised by truncation of the C-terminal end (removing up to 40% of the enzyme sequence). All five enzymes catalysed degradation of fucoidans containing α(1→4)-linked l-fucosyls. Fda2 also degraded S. cichorioides and U. pinnatifida fucoidans that have α(1→3)-linked l-fucosyls in their backbone. In the stabilised form, Fda1 also cleaved α(1→3) bonds. For the first time, we also show that several enzymes catalyse degradation of S. mcclurei galactofucan-fucoidan, known to contain α(1→4) and α(1→3) linked l-fucosyls and galactosyl-ß(1→3) bonds in the backbone. These data enhance our understanding of fucoidan degrading enzymes and their substrate preferences and may assist development of enzyme-assisted production of defined fuco-oligosaccharides from fucoidan substrates.

Genetic epidemiology and risk factors for brain tumors.

This report summarized current knowledge and findings relevant to environmental and genetic risk factors in brain tumors, with a particular focus on glioma. To date, the established risk factors for brain tumors are family history and ionizing radiation exposure; whereas there is an inverse association between tumors and other factors such as history of allergies, atopic conditions, chickenpox, and varicella zoster virus infection. To identify inherited genetic variants impacting susceptibility of brain tumors, large scale familial linkage-scan pedigree analysis, population-based candidate genes, and genome-wide association study were performed. More recently, next generation exome and whole genome sequencing studies were also conducted.

Triggering of Eryptosis, the Suicidal Erythrocyte Death by Mammalian Target of Rapamycin (mTOR) inhibitor Temsirolimus.

BACKGROUND/AIMS: The mammalian target of rapamycin (mTOR) inhibitor temsirolimus is utilized for the treatment of malignancy. Temsirolimus is at least in part effective by triggering suicidal tumor cell death. The most common side effect of temsirolimus treatment is anemia. At least in theory, the anemia following temsirolimus treatment could result from stimulation of eryptosis, the suicidal erythrocyte death. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the orchestration of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, as well as activation of staurosporine and chelerythrine sensitive protein kinase C, SB203580 sensitive p38 kinase, D4476 sensitive casein kinase 1, and zVAD sensitive caspases. The purpose of the present study was to test whether temsirolimus influences eryptosis and, if so, to shed light on the signaling involved. METHODS: Flow cytometry was employed to estimate cell volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was determined from hemoglobin concentration in the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to temsirolimus (5 - 20 µg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Temsirolimus significantly increased Fluo3-fluorescence, DCFDA fluorescence and ceramide abundance at the erythrocyte surface. The effect of temsirolimus on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+ and by addition of staurosporine (1 µM) or chelerythrine (10 µM) but not significantly modified by addition of SB203580 (2 µM), D4476 (10 µM), or zVAD (10 µM). Chelerythrine (10 µM) further significantly blunted the effect of temsirolimus on DCFDA fluorescence but not ceramide formation. Removal of extracellular Ca2+ had no effect on temsirolimus induced ROS formation or ceramide abundance. CONCLUSIONS: Temsirolimus triggers eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress, ceramide and activation of staurosporine/Chelerythrine sensitive kinase(s).

Inhibition of Erythrocyte Cell Membrane Scrambling by ASP3026.

BACKGROUND/AIMS: The anaplastic lymphoma kinase (ALK) inhibitor ASP3026 is in clinical development for the treatment of ALK expressing non-small cell lung carcinoma (NSCLC). ASP3026 is in part effective by inducing apoptosis of tumor cells. Erythrocytes lack mitochondria and nuclei, key organelles in the execution of apoptosis, but are nevertheless able to enter suicidal death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Eryptosis is triggered by cell stress, such as energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether ASP3026 impacts on eryptosis. METHODS: Human erythrocytes have been exposed to energy depletion (glucose withdrawal for 48 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca2+ loading with Ca2+ ionophore ionomycin (1 µM for 60 min) in absence and presence of ASP3026 (1-4 µg/ml). Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, and cell volume from forward scatter. RESULTS: Treatment with ASP3026 alone did not significantly modify annexin-V-binding or forward scatter. Energy depletion, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. ASP3026 significantly blunted the effect of energy depletion and oxidative stress, but not of ionomycin on annexin-V-binding. ASP3026 did not significantly influence the effect of any maneuver on forward scatter. CONCLUSIONS: ASP3026 is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion and oxidative stress.

Triggering of Suicidal Erythrocyte Death by Exemestane.

BACKGROUND/AIMS: The steroidal aromatase inactivator exemestane blocks estrogen biosynthesis and is thus employed for the prevention and treatment of breast cancer. Exemestane is in part effective by stimulation of suicidal cell death or apoptosis. Side effects of exemestane treatment include anemia. At least in theory, exemestane induced anemia could be secondary to stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, several kinases and caspases. The present study explored, whether exemestane is able to trigger eryptosis and, if so, to shed some light on the signaling involved. METHODS: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCF fluorescence, and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to exemestane (≥ 10 µg/ml) significantly increased the percentage of annexin-V-binding cells without significantly modifying forward scatter. Exemestane significantly increased Fluo3-fluorescence (10 and 20, but not 40 µg/ml), DCF fluorescence (40 µg/ml), and ceramide abundance (40 µg/ml). The effect of exemestane (40 µg/ml) on annexin-V-binding was significantly blunted by antioxidant N-acetylcysteine (1mM), but was not significantly modified by removal or increase of extracellular Ca2+, by p38 kinase inhibitor SB203580 (2 µM), casein kinase inhibitor D4476 (10 µM) and caspase inhibitor zVAD (10 µM). CONCLUSIONS: Exemestane triggers phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by enhanced [Ca2+]i, oxidative stress, and increased ceramide abundance.