Dose-dependent effects of Allopurinol on human foreskin fibroblast cell and human umbilical vein endothelial cell under hypoxia

Allopurinol, an inhibitor of xanthine oxidase, has been used in clinical trials of patients with cardiovascular and chronic kidney disease. These are two pathologies with extensive links to hypoxia and activation of the transcription factor hypoxia inducible factor (HIF) family. Here we analysed the effects of allopurinol treatment in two different cellular models, and their response to hypoxia. We explored the dose-dependent effect of allopurinol on Human Foreskin Fibroblasts (HFF) and Human Umbilical Vein Endothelial Cells (HUVEC) under hypoxia and normoxia. Under normoxia and hypoxia, high dose allopurinol reduced the accumulation of HIF-1α protein in HFF and HUVEC cells. Allopurinol had only marginal effects on HIF-1α mRNA level in both cellular systems. Interestingly, allopurinol effects over the HIF system were independent of prolyl-hydroxylase activity. Finally, allopurinol treatment reduced angiogenesis traits in HUVEC cells in an in vitro model. Taken together these results indicate that high doses of allopurinol inhibits the HIF system and pro-angiogenic traits in cells

Mammalian Comparative Sequence Analysis of the Agrp Locus

Agouti-related protein encodes a neuropeptide that stimulates food intake. Agrp expression in the brain is restricted to neurons in the arcuate nucleus of the hypothalamus and is elevated by states of negative energy balance. The molecular mechanisms underlying Agrp regulation, however, remain poorly defined. Using a combination of transgenic and comparative sequence analysis, we have previously identified a 760 bp conserved region upstream of Agrp which contains STAT binding elements that participate in Agrp transcriptional regulation. In this study, we attempt to improve the specificity for detecting conserved elements in this region by comparing genomic sequences from 10 mammalian species. Our analysis reveals a symmetrical organization of conserved sequences upstream of Agrp, which cluster into two inverted repeat elements. Conserved sequences within these elements suggest a role for homeodomain proteins in the regulation of Agrp and provide additional targets for functional evaluation

Integration of first-trimester assessment in the ultrasound staging of placenta accreta spectrum disorders.

OBJECTIVE: To explore the role of early first trimester ultrasound at 5-7 postmenstrual weeks of gestation in predicting sonographic staging of placenta accreta spectrum (PAS) and to elucidate whether integrating first trimester assessment with ultrasound staging of PAS can predict surgical outcome in women at risk for PAS. METHODS: Secondary analysis of prospectively collected data of women who had at least one previous caesarean delivery (CD) or uterine surgery and placenta previa for whom early (5-7 weeks of gestation) ultrasound images could be retrieved. The relationship between gestational sac position and prior CD scar was assessed using classifications by Cali et al. (cross-over COS), Kaelin Agten et al. ("on the scar" vs "in the niche" implantation) and Timor-Tritsch et al. ("above the line" vs "below the line" implantation) by two different examiners blinded to the final diagnosis and clinical outcome. Primary aim of the study was to explore the strength of association and predictive accuracy of first trimester ultrasound in predicting PAS stage. Secondary aim was to elucidate whether integration of first trimester ultrasound with PAS staging can predict surgical outcome. Logistic regression and area under the curve analyses were used to analyse the data. RESULTS: One hundred and eighty-seven women were included. Of these ,79.6% (95% CI 67.1-88.2) had COS1, 94.4% (95% CI 84.9-98.1) "in the niche" and 92.6% (95% CI 82.4-97.1) "below the line" implantation confirmed to be affected by PAS3 in the third trimester of pregnancy. On multivariate logistic regression analysis, COS1 (OR: 7.9 (95% CI 4.0-15.5; p<0.001), "in the niche" (OR: 29.1, 95% CI 8.1-104; p<0.001) and "below the line" (OR: 38.1, 95% CI 12.1-121; p<0.001) implantations, however, neither parity (p= 0.4), nor the number of prior CDs (p= 0.5) were independently associated with PAS3. When translating these figures in a diagnostic model, either COS1 (AUC: 0.94, 95% CI 0.91-0.97), or implantation "in the niche" (AUC: 0.92, 95% CI 0.89-0.96) or "below the line" (AUC: 0.92, 95% CI 0.88-0.96) had a high predictive accuracy for PAS3. Adverse surgical outcome was more common in women with COS1 (p<0.001), implantation "in the niche" (p<0.001) and "below the line" (p<0.001) then those without them.) On multivariate logistic regression analysis, ultrasound diagnosis of PAS3 (OR: 4.3, 95% CI 2.1-17.3), COS1 (OR: 7.9, 95% CI 4.0-15.5; p<0.001), "in the niche" (OR: 29.1, 95% CI 8.1-104; p<0.001) and "below the line" (OR: 7.9, 95% CI 4.0-15.5; p<0.001) implantations were independently associated with adverse surgical outcome. When combining the three imaging methods, we identified, an area we call "high-risk-for-PAS Triangle" which may enable an easy visual perception and application of the three methods to prognosticate the risk for CSP and PAS, although it requires validation in further large prospective studies. CONCLUSION: Early first trimester sonographic assessment of pregnancies after CDs can reliably predict ultrasound staging of possible PAS. Integrating first with second and third trimester ultrasound can stratify surgical risk of women affected by PAS. This article is protected by copyright. All rights reserved

Ribosomal oxygenases are structurally conserved from prokaryotes to humans

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components1,2 and in the hydroxylation of transcription factors3 and splicing factor proteins4. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA5,6,7 and ribosomal proteins8 have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy9,10,11,12. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans8 raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone Nε-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases

Optoelectronic characterization of CuInGa(S)2 thin films grown by spray pyrolysis for photovoltaic application

[EN] Copper-indium gallium disulfide (CIGS) is a good absorber for photovoltaic application. Thin films of CIGS were prepared by spray pyrolysis on glass substrates in the ambient atmosphere. The films were characterized by different techniques, such as structural, morphological, optical and electrical properties of CIGS films were analyzed by X-ray diffraction (XRD), scanning electron microscopy (SEM), atomic force microscopy (AFM), spectrophotometer and Hall effect, respectively. After optimization, the deposited films structure, grain size, and crystallinity became more important with an increase of annealing time at 370 degrees C for 20 min. Transmission electron microscopy (TEM) analysis shows that the interface sheets are well crystallized and the inter planer distance are 0.25 nm, 0.28 nm, and 0.36 nm. The atomic force microscopy (AFM) observation shows that the grain size and roughness can be tolerated by optimizing the annealing time. The strong absorbance and low transmittance were observed for the prepared films with a suitable energy bandgap about 1.46 eV. The Hall effect measurement system examined that CIGS films exhibited optimal electrical properties, resistivity, carrier mobility, and carrier concentration which were determined to be 4.22 x 10(6) omega cm, 6.18 x 10(2) cm(2) V-1 S-1 and 4.22 x 10(6) cm(-3), respectively. The optoelectronic properties of CIGS material recommended being used for the photovoltaic application.Prof. Bouchaib HARTITI, The Senior Associate at ICTP, is very grateful to ICTP for permanent support. Prof. Mohamed Ebn Touhami, Director of the University Center for Analysis, Expertise, Transfer of Technology and Incubation, Kenitra, Morocco, is very grateful to CUA2TI for financial support. Thanks to Doctor Diogo M.F. Santos for the supervision of Amal Bouich's work during her research in CeFEMA research center. The authors also thank researchers from CeFEMA (IST-ULisboa, Portugal) and CUA2TI (FS-Kenitra Morocco) for their help.Bouich, A.; Hartiti, B.; Ullah, S.; Ullah, H.; Ebn Touhami, M.; Santos, DMF.; Marí, B. (2019). Optoelectronic characterization of CuInGa(S)2 thin films grown by spray pyrolysis for photovoltaic application. Applied Physics A. 125(8):1-9. https://doi.org/10.1007/s00339-019-2874-4S191258T. Feurer, P. Reinhard, E. Avancini, B. Bissig, J. Löckinger, P. Fuchs, S. Buecheler, Progress in thin film CIGS photovoltaics–Research and development, manufacturing, and applications. Prog. Photovolt. Res. Appl. 25(7), 645–667 (2017)A. Zegadi, M.A. Slifkin, M. Djamin, A.E. Hill, R.D. Tomlinson, A photoacoustic study of CuInxGa1− xSe2 alloys. Phys. Status Solidi (A) 133(2), 533–540 (1992)T.H. Sajeesh, A.R. Warrier, C.S. Kartha, K.P. Vijayakumar, Optimization of parameters of chemical spray pyrolysis technique to get n and p-type layers of SnS. Thin Solid Films 518(15), 4370–4374 (2010)J. Liu, D. Zhuang, H. Luan, M. Cao, M. Xie, X. Li, Preparation of Cu (In, Ga) Se2 thin film by sputtering from Cu (In, Ga) Se2 quaternary target. Progr. Nat. Sci. Mater. Int. 23(2), 133–138 (2013)M.I. Hossain, Fabrication and characterization of CIGS solar cells with In2 S3 buffer layer deposited by PVD technique. Chalcogenide Lett. 9(5), 185–191 (2012)M.A. Mughal, R. Engelken, R. Sharma, Progress in indium (III) sulfide (In2S3) buffer layer deposition techniques for CIS, CIGS, and CdTe-based thin film solar cells. Sol. Energy 120, 131–146 (2015)M. Powalla, M. Cemernjak, J. Eberhardt, F. Kessler, R. Kniese, H.D. Mohring, B. Dimmler, Large-area CIGS modules: Pilot line production and new developments. Sol. Energy Mater Sol. Cells 90(18–19), 3158–3164 (2006)M.E. Calixto, P.J. Sebastian, R.N. Bhattacharya, R. Noufi, Compositional and optoelectronic properties of CIS and CIGS thin films formed by electrodeposition. Sol. Energy Mater. Sol. Cells 59(1–2), 75–84 (1999)S. Jung, S. Ahn, J.H. Yun, J. Gwak, D. Kim, K. Yoon, Effects of Ga contents on properties of CIGS thin films and solar cells fabricated by co-evaporation technique. Curr. Appl. Phys. 10(4), 990–996 (2010)S. R. Ovshinsky, X. Deng, R. Young, U.S. Patent No. 5,231,047. Washington, DC: U.S. Patent and Trademark Office (1993).M. Kaelin, D. Rudmann, A.N. Tiwari, Low cost processing of CIGS thin film solar cells. Sol. Energy 77(6), 749–756 (2004)Fangdan Jiang, Jiayou Feng, Effect of temperature on selenization process of metallic Cu–In alloy precursors. Thin Solid Films 515(4), 1950–1955 (2006)S. Shirakata, Y. Kannaka, H. Hasegawa, T. Kariya, S. Isomura, Properties of Cu (In, Ga) Se2 thin films prepared by chemical spray pyrolysis. Jpn. J. Appl. Phys. 38(9R), 4997 (1999)Y.K. Kumar, G.S. Babu, P.U. Bhaskar, V.S. Raja, Effect of starting-solution pH on the growth of Cu2ZnSnS4 thin films deposited by spray pyrolysis. Phys. Status Solidi (A) 206(7), 1525–1530 (2009)M. Ajili, M. Castagné, N.K. Turki, Characteristics of CuIn1− xGaxS2 thin films synthesized by chemical spray pyrolysis. J. Lumin. 150, 1–7 (2014)B.J. Babu, S. Velumani, A. Kassiba, R. Asomoza, J.A. Chavez-Carvayar, J. Yi, Deposition and characterization of graded Cu (In1-xGax) Se2 thin films by spray pyrolysis. Mater. Chem. Phys. 162, 59–68 (2015)S.F. Varol, G. Babür, G. Çankaya, U. Kölemen, Synthesis of sol–gel derived nano-crystalline ZnO thin films as TCO window layer: effect of sol aging and boron. RSC Adv. 4(100), 56645–56653 (2014)J.A. Frantz, R.Y. Bekele, V.Q. Nguyen, J.S. Sanghera, A. Bruce, S.V. Frolov, I.D. Aggarwal, Cu (In, Ga) Se2 thin films and devices sputtered from a single target without additional selenization. Thin Solid Films 519(22), 7763–7765 (2011)C. Calderón, G. Gordillo, P. Bartolo-Pérez, F. Mesa, Effect of the deposition conditions on the optical, morphological and compositional properties of CuIn1− xGaxSe2 thin films prepared by a multistage process. 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Rapid induction of p21WAF1 but delayed down-regulation of Cdc25A in the TGF-β-induced cell cycle arrest of gastric carcinoma cells

Transforming growth factor-beta (TGF-beta) is a multifunctional polypeptide that inhibits cellular proliferation in most epithelial cells. cdk4 and several cyclin-dependent kinase (cdk) inhibitors (p15(INK4B), p21(WAFI/Cip1) and p27(Kip1)) have been implicated in the TGF-beta-induced cell cycle arrest. More recently, down-regulation of Cdc25A, a cdk activator, was additionally suggested as a mechanism underlying growth inhibition by TGF-beta. The existence of diverse cellular mediators, of TGF-beta, however, raises the question of whether their involvement might occur in a redundant manner or coordinately in a certain cell type. Using two TGF-beta-sensitive gastric carcinoma cell lines (SNU-16 and -620), we addressed the contributory roles of several cdk inhibitors, and of cdk4 and Cdc25A, in TGF-beta-induced cell cycle arrest by comparing their temporal expression pattern in response to TGF-beta. Among the cdk inhibitors examined, p21 mRNA was most rapidly (in less than 1 h) and prominently induced by TGF-beta. In contrast, p15 mRNA was more slowly induced than p21 in SNU-620: cells, and not expressed in SNU-16 cells harbouring homozygous deletion of p15. Western blotting results confirmed the rapid increase of p21 while opposite patterns of p27 expression were observed in the two cell lines. The down-regulation of Cdc25A mRNA occurred, but was more delayed than that of p15 or p21. Until G1 arrest was established, changes in the protein levels of both Cdc25A and cdk4 were marginal. Co-immunoprecipitation with anti-cdk4 antibody showed that induced p21 associates with cdk4, and that its kinase activity is reduced by TGF-beta, which kinetically correlates closely with G1 arrest following TGF-beta treatment of both cell lines. These results suggest that in certain human epithelial cells, p21 may play an early role in TGF-beta-induced cell cycle arrest, and its cooperation with other cdk inhibitors is different depending on cell type. Delayed down-regulation of Cdc25A and cdk4 may contribute to cell adaptation to the quiescent state in the two gastric carcinoma cell lines studied

Potent and selective chemical probe of hypoxic signaling downstream of HIF-α hydroxylation via VHL inhibition

Chemical strategies to using small molecules to stimulate hypoxia inducible factors (HIFs) activity and trigger a hypoxic response under normoxic conditions, such as iron chelators and inhibitors of prolyl hydroxylase domain (PHD) enzymes, have broad-spectrum activities and off-target effects. Here we disclose VH298, a potent VHL inhibitor that stabilizes HIF-α and elicits a hypoxic response via a different mechanism, that is the blockade of the VHL:HIF-α protein-protein interaction downstream of HIF-α hydroxylation by PHD enzymes. We show that VH298 engages with high affinity and specificity with VHL as its only major cellular target, leading to selective on-target accumulation of hydroxylated HIF-α in a concentration- and time-dependent fashion in different cell lines, with subsequent upregulation of HIF-target genes at both mRNA and protein levels. VH298 represents a high-quality chemical probe of the HIF signalling cascade and an attractive starting point to the development of potential new therapeutics targeting hypoxia signalling

Quantitative Proteomics Identifies the Myb-Binding Protein p160 as a Novel Target of the von Hippel-Lindau Tumor Suppressor

Background: The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of a ubiquitin ligase complex, which is best understood as a negative regulator of hypoxia inducible factor (HIF). VHL ubiquitinates and degrades the a subunits of HIF, and this is proposed to suppress tumorigenesis and tumor angiogenesis. However, several lines of evidence suggest that there are unidentified substrates or targets for VHL that play important roles in tumor suppression. Methodology/Principal Findings: Employing quantitative proteomics, we developed an approach to systematically identify the substrates of ubiquitin ligases and using this method, we identified the Myb-binding protein p160 as a novel substrate of VHL. Conclusions/Significance: A major barrier to understanding the functions of ubiquitin ligases has been the difficulty in pinpointing their ubiquitination substrates. The quantitative proteomics approach we devised for the identification of VHL substrates will be widely applicable to other ubiquitin ligases

Target gene selectivity of hypoxia-inducible factor-α in renal cancer cells is conveyed by post-DNA-binding mechanisms

Inactivation of the von Hippel–Lindau tumour suppressor in renal cell carcinoma (RCC) leads to failure of proteolytic regulation of the α subunits of hypoxia-inducible factor (HIF), constitutive upregulation of the HIF complex, and overexpression of HIF target genes. However, recent studies have indicated that in this setting, upregulation of the closely related HIF-α isoforms, HIF-1α and HIF-2α, have contrasting effects on tumour growth, and activate distinct sets of target genes. To pursue these findings, we sought to elucidate the mechanisms underlying target gene selectivity for HIF-1α and HIF-2α. Using chromatin immunoprecipitation to probe binding to hypoxia response elements in vivo, and expression of chimaeric molecules bearing reciprocal domain exchanges between HIF-1α and HIF-2α molecules, we show that selective activation of HIF-α target gene expression is not dependent on selective DNA-binding at the target locus, but depends on non-equivalent C-terminal portions of these molecules. Our data indicate that post-DNA binding mechanisms that are dissimilar for HIF-1α and HIF-2α determine target gene selectivity in RCC cells