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BBA - General Subjects (v.1830, #6)

Editorial Board (pp. i).

Endothelin-1 enhances cell migration through COX-2 up-regulation in human chondrosarcoma by Min Huan Wu; Li-Mien Chen; His-Hsien Hsu; James A. Lin; Yueh-Min Lin; Fuu-Jen Tsai; Chang-Hai Tsai; Chih-Yang Huang; Chih-Hsin Tang (pp. 3355-3364).

Chondrosarcoma is a type of highly malignant tumor with a potent capacity of local invasion and distant metastasis. The effect of endothelin-1 (ET-1) on migration activity in human chondrosarcoma cells is not clearly understood. Here, we found that ET-1 increased the migration and expression of cyclooxygenase (COX)-2 in human chondrosarcoma cells.ET-1-mediated COX-2 expression was assessed by qPCR and Western blot analysis. The mechanisms of action of ET-1 in different signaling pathways were studied using Western blotting. Knockdown of proteins was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of c-Jun to the COX-2 promoter.Human chondrosarcoma tissues had significant expression levels of ET-1 and COX-2, which were higher than that in normal cartilage. Exogenous ET-1 increased cell migration and the expression of COX-2. In addition, COX-2 protein levels and cell migration ability were abolished by ET receptor antagonists. Activation of the mitogen-activated protein kinase (MAPK) and activator protein-1 (AP-1) pathways after ET-1 treatment was demonstrated, and ET-1-induced COX-2 expression and cell migration activity were inhibited by the specific inhibitor and mutant of MAPK and AP-1 cascades. ET-1 increased the binding of c-Jun to the AP-1 element on the COX-2 promoter. Furthermore, knockdown of ET-1 decreased cell metastasis in vitro and in vivo.Our results indicated that ET-1 enhances the cell migration of chondrosarcoma by increasing COX-2 expression through the ET receptors, MAPK, and AP-1 signal transduction pathway.We link high ET-1 and COX-2 expression to chondrosarcoma.•ET-1and COX-2 was highly expressed in chondrosarcoma tissues as compared to normal cartilage.•ET-1 mediates the chondrosarcoma cell migration by up-regulation of COX-2 production.•ET-1 act through the ET receptors, MAPK to induce COX-2 activation.•AP-1 was major key transcription factors of ET-1-induced COX-2 activation.

Keywords: Endothelin-1 (ET-1); Metastasis; Chondrosarcoma; Cyclooxygenase (COX)-2; Activator protein-1 (AP-1)

Positional specifity of acetylxylan esterases on natural polysaccharide: An NMR study by Uhliarikova Iveta Uhliariková; Vrsanska Mária Vršanská; Barry V. McCleary; Peter Biely (pp. 3365-3372).

Microbial degradation of acetylated plant hemicelluloses involves besides enzymes cleaving the glycosidic linkages also deacetylating enzymes. A detailed knowledge of the mode of action of these enzymes is important in view of the development of efficient bioconversion of plant materials that did not undergo alkaline pretreatment leading to hydrolysis of ester linkages.In this work deacetylation of hardwood acetylglucuronoxylan by acetylxylan esterases from Streptomyces lividans (carbohydrate esterase family 4) and Orpinomyces sp. (carbohydrate esterase family 6) was monitored by¹H-NMR spectroscopy.The¹H-NMR resonances of all acetyl groups in the polysaccharide were fully assigned. The targets of both enzymes are 2- and 3-monoacetylated xylopyranosyl residues and, in the case of the Orpinomyces sp. enzyme, also the 2,3-di- O-acetylated xylopyranosyl residues. Both enzymes do not recognize as a substrate the 3- O-acetyl group on xylopyranosyl residues α-1,2-substituted with 4- O-methyl-d-glucuronic acid.The¹H-NMR spectroscopy approach to study positional and substrate specificity of AcXEs outlined in this work appears to be a simple way to characterize catalytic properties of enzymes belonging to various CE families.The results contribute to development of efficient and environmentally friendly procedures for enzymatic degradation of plant biomass.►¹H-NMR signals of the acetyl groups in hardwood acetyglucuronoxylan were assigned. ► The assignment enabled study of the action of acetyxylan esterases on natural substrate. ► Mode of action of Streptomyces lividans and Orpinomyces sp. enzymes (CE4 vs. CE6) was elucidated. ► The results are important in view of further progress of bioconversion of plant biomass.

Keywords: Abbreviations; AcXE; acetylxylan esterase; Xyl; p; d; -xylopyranose or; d; -xylopyranosyl; CE; carbohydrate esteraseAcetylxylan esterase; Positional specificity; NMR; Natural substrate; Acetylglucuronoxylan

The Global Sequence Signature algorithm unveils a structural network surrounding heavy chain CDR3 loop in Camelidae variable domains by Damjana Kastelic; Nicolas Soler; Radovan Komel; Denis Pompon (pp. 3373-3381).

A large fraction of camelid (camels and llamas) antibodies is composed of heavy chain-only homodimers, able to recognise antigens with their variable domain. Events in somatic assembly and maturation of antibodies such as hypermutations and rearrangement of variable loops (CDRs — complementary determining regions) and selection among a wide range of framework variants are generally considered to be random processes.An original algorithmic approach (Global Sequence Signature—GSS) was developed, able to take into account multiple functional and/or local sequence properties to detect scattered evolutionary constraints into sequences.Using the GSS approach, we show that the length of the main hypervariable loop (CDR3) is linked to the nature of 19 surrounding residues on the scaffold. Surprisingly, the relation between CDR3 size and scaffold residues strongly depends on the considered species, illustrating either significant differences in selection mechanisms or functional constraints during antibody maturation.Combined with the statistical coupling analysis (SCA) approach at the level of scaffold residues, this study has unravelled a robust interaction network on antibody structure surrounding the CDR3 loop.In addition to the general applicability of the GSS algorithm, which can bring together functional and sequence data to locate hot spots of constrained evolution, the relationship between CDR3 and scaffold discussed here should be taken into account in protein engineering when designing antibody libraries.► Identification of a Camelidae antibody signature outside of classical hallmarks ► The programme identifies networks of coevolving residues related to sequence properties. ► The length of the CDR3 loop correlates with the nature of its surrounding residues. ► The algorithm constitutes a new analysis tool for protein and antibody design.

Keywords: Abbreviations; CDR; complementarity determining region; FR; framework; GSS; Global Sequence Signature analysis; Ig; immunoglobulin G; MSA; multiple sequence alignment; MDS; multidimensional scaling; PCA; principal component analysis; SCA; statistical coupling analysis; V; _H; heavy chain variable domain of the conventional antibody; V; _H; H; variable domain of a heavy chain antibody; V; _L; light chain variable domain Camelidae; variable domain; Co-evolution; Complementarity determining region 3 (CDR3); V; _H; V; _H; H; Multiple sequence alignment

Role of TRPM2 and TRPV1 cation channels in cellular responses to radiation-induced DNA damage by Kanako Masumoto; Mitsutoshi Tsukimoto; Shuji Kojima (pp. 3382-3390).

Radiation exposure causes DNA damage, and DNA repair systems are essential to rescue damaged cells. Although DNA damage or oxidative stress activates transient receptor potential melastatin 2 (TRPM2) and vanilloid 1 (TRPV1) cation channels, it has not been established whether these TRP channels are involved in cellular responses to radiation-induced DNA damage. Here, we investigated the contribution of TRPM2 and TRPV1 channels to γ-irradiation- and UVB-induced DNA damage responses in human lung cancer A549 cells.A549 cells were irradiated with γ-rays (2.0Gy) or UVB (5–10mJ/cm²). γH2AX foci, ATM activation, 53BP1 accumulation and EGFR expression were evaluated by immunofluorescence staining. Extracellular ATP concentration was measured by luciferin–luciferase assay. Knockdown of TRPM2 and TRPV1 expression was done by siRNA transfection.γ-Irradiation-induced γH2AX focus formation, ATM activation, 53BP1 accumulation and EGFR nuclear translocation, which are all associated with DNA repair, were suppressed by knockdown of TRPM2 and TRPV1 channels in A549 cells. Release of ATP, which mediates DNA damage response-associated activation of P2Y receptors, was suppressed by pre-treatment with catalase or knockdown of TRPM2 channel, but not TRPV1 channel. Similarly, UVB-induced γH2AX focus formation was suppressed in TRPM2- and TRPV1-knockdown cells, while UVB-induced ATP release was blocked in TRPM2- but not TRPV1-knockdown cells.Our results suggest that the activation of TRPM2 channel, which mediates ATP release, and TRPV1 channel plays significant roles in the cellular responses to DNA damage induced by γ-irradiation and UVB irradiation.Our results provide a new insight into the function of TRP channels from the viewpoint of radiation biology.•TRPM2 and TRPV1 channels are involved in γ-radiation-induced DNA damage response.•These channels contribute to radiation-induced translocation of EGFR to nucleus.•TRPM2 channel but not TRPV1 channel mediates ATP release after irradiation.•Results in UVB-irradiated cells were similar to those in γ-irradiated cells.•TRPM2 and TRPV1 cation channels contribute to cellular responses to DNA damage.

Keywords: Abbreviations; ADPR; adenine 5′-diphosphoribose; 2-APB; 2-aminoethoxydiphenyl borate; ATM; ataxia telangiectasia mutated; 53BP1; tumor suppressor p53-binding protein 1; CAP; capsaicin; CPZ; capsazepine; DPQ; 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone; EGFR; epithelial growth factor receptor; γH2AX; phosphorylated histone variant H2AX; NAD; nicotinamide adenine dinucleotide; LET; linear energy transfer; PARP; poly(ADP-ribose) polymerase; SSB; single strand break; TRPM2; transient receptor potential melastatin type 2; TRPV1; transient receptor potential vanilloid type 1; UVB; ultraviolet Bγ-Ray; Ultraviolet B; DNA repair; TRPM2 channel; TRPV1 channel; ATP

Mitochondrial NAD dependent aldehyde dehydrogenase either from yeast or human replaces yeast cytoplasmic NADP dependent aldehyde dehydrogenase for the aerobic growth of yeast on ethanol by Abhijit Mukhopadhyay; Baoxian Wei; Henry Weiner (pp. 3391-3398).

In a previous study, we deleted three aldehyde dehydrogenase (ALDH) genes, involved in ethanol metabolism, from yeast Saccharomyces cerevisiae and found that the triple deleted yeast strain did not grow on ethanol as sole carbon source. The ALDHs were NADP dependent cytosolic ALDH1, NAD dependent mitochondrial ALDH2 and NAD/NADP dependent mitochondrial ALDH5. Double deleted strain ΔALDH2+ΔALDH5 or ΔALDH1+ΔALDH5 could grow on ethanol. However, the double deleted strain ΔALDH1+ΔALDH2 did not grow in ethanol.Triple deleted yeast strain was used. Mitochondrial NAD dependent ALDH from yeast or human was placed in yeast cytosol.In the present study we found that a mutant form of cytoplasmic ALDH1 with very low activity barely supported the growth of the triple deleted strain (ΔALDH1+ΔALDH2+ΔALDH5) on ethanol. Finding the importance of NADP dependent ALDH1 on the growth of the strain on ethanol we examined if NAD dependent mitochondrial ALDH2 either from yeast or human would be able to support the growth of the triple deleted strain on ethanol if the mitochondrial form was placed in cytosol. We found that the NAD dependent mitochondrial ALDH2 from yeast or human was active in cytosol and supported the growth of the triple deleted strain on ethanol.This study showed that coenzyme preference of ALDH is not critical in cytosol of yeast for the growth on ethanol.The present study provides a basis to understand the coenzyme preference of ALDH in ethanol metabolism in yeast.► Mutant ALDH1 barely supports the growth of triple ALDH deleted yeast on ethanol. ► NAD dependent mitochondrial ALDH2 replaces cytoplasmic NADP dependent ALDH1 in yeast. ► ALDH1 with mitochondrial leader attached is unable to be imported into mitochondria. ► Localization or coenzyme specificity of ALDH is not essential for aerobic growth of yeast on ethanol.

Keywords: Abbreviations; yALDH1; yeast cytosolic aldehyde dehydrogenase; yALDH2; yeast mitochondrial aldehyde dehydrogenase; ypALDH2; yeast mitochondrial precursor aldehyde dehydrogenase; ypALDH1; yeast cytosolic ALDH1 with mitochondrial leader peptide fused to the N-terminus; hALDH1; human cytosolic aldehyde dehydrogenase; hALDH2; human mitochondrial aldehyde dehydrogenase; hpALDH2; human mitochondrial precursor aldehyde dehydrogenase; hpALDH1; human cytosolic ALDH1 with mitochondrial leader peptide fused to the N-terminusAldehyde dehydrogenase; Co-enzyme specificity; Mitochondria; Aerobic growth

The effects of selenium and the GPx-1 selenoprotein on the phosphorylation of H2AX by A. Jerome-Morais; S. Bera; W. Rachidi; P.H. Gann; A.M. Diamond (pp. 3399-3406).

Significant data supports the health benefits of selenium although supplementation trials have yielded mixed results. GPx-1, whose levels are responsive to selenium availability, is implicated in cancer etiology by human genetic data. Selenium's ability to alter the phosphorylation of the H2AX, a histone protein that functions in the reduction of DNA damage by recruiting repair proteins to the damage site, following exposure to ionizing radiation and bleomycin was investigated.Human cell lines that were either exposed to selenium or were transfected with a GPx-1 expression construct were exposed to ionizing radiation or bleomycin. Phosphorylation of histone H2AX was quantified by flow cytometry and survival by the MTT assay. Phosphorylation of the Chk1 and Chk2 checkpoint proteins was quantified by western blotting.In colon-derived cells, selenium increases GPx-1 and attenuated H2AX phosphorylation following genotoxic exposures while the viability of these cells was unaffected. MCF-7 cells and transfectants that express high GPx-1 levels were exposed to ionizing radiation and bleomycin, and H2AX phosphorylation and cell viability were assessed. GPx-1 increased H2AX phosphorylation and viability following the induction of DNA damage while enhancing the levels of activated Chk1 and Chk2.Exposure of mammalian cells to selenium can alter the DNA damage response and do so by mechanisms that are dependent and independent of its effect on GPx-1.Selenium and GPx-1 may stimulate the repair of genotoxic DNA damage and this may account for some of the benefits attributed to selenium intake and elevated GPx-1 activity.•Phospho-H2AX was used to assess how selenium and selenoproteins may impact DNA damage repair•GPx-1 null and expressing cells were used to evaluate effects of selenium and GPx-1 independently•Both H2AX phosphorylation and cell survival were evaluated•GPx-1 effects on the Chk1 and Chk2 DNA damage repair proteins were observed

Keywords: Selenium; DNA repair; Histone H2AX; Glutathione peroxidase

A novel acylated form of (d-Ala²)GIP with improved antidiabetic potential, lacking effect on body fat stores by Christine M.A. Martin; Nigel Irwin; Peter R. Flatt; Victor A. Gault (pp. 3407-3413).

Rapid enzymatic degradation of the incretin hormone, glucose-dependent insulinotropic polypeptide (GIP), limits therapeutic use of the native peptide for diabetes. However, enzymatically stable analogues of GIP, such as (d-Ala²)GIP, have been generated, but are still susceptible to renal filtration.The present study examines the in vitro and in vivo biological actions of a novel, acylated GIP analogue, (d-Ala²)GIP[Lys³⁷PAL].In BRIN-BD11 cells, (d-Ala²)GIP[Lys³⁷PAL] concentration-dependently stimulated (p<0.05 to p<0.001) insulin secretion at 5.6 and 16.7mM glucose. Intraperitoneal administration of (d-Ala²)GIP[Lys³⁷PAL] to normal mice 8h prior to a glucose load significantly reduced (p<0.05) the overall glycaemic excursion compared to controls, and increased (p<0.001) the insulinotropic response compared to (d-Ala²)GIP and saline treated high fat control mice. Once daily administration of (d-Ala²)GIP[Lys³⁷PAL] for 21days in high fat fed mice did not affect energy intake, body weight or fat deposition. However, circulating blood glucose was significantly lower (p<0.05) accompanied by increased (p<0.05) insulin concentrations by day 21. In addition, (d-Ala²)GIP[Lys³⁷PAL] treatment significantly (p<0.01) reduced the overall glycaemic excursion and increased pancreatic insulin content (p<0.05) and the insulinotropic response (p<0.01) to an exogenous glucose challenge on day 21. Chronic treatment with (d-Ala²)GIP[Lys³⁷PAL] did not result in resistance to the metabolic effects of a bolus injection of native GIP. Finally, insulin sensitivity was significantly improved (p<0.001) in (d-Ala²)GIP[Lys³⁷PAL] treated mice compared to high fat controls.These data confirm that (d-Ala²)GIP[Lys³⁷PAL] is a stable, long-acting potent GIP agonist.(d-Ala²)GIP[Lys³⁷PAL] may be suitable for further evaluation and future clinical development.•(D-Ala²)GIP[Lys³⁷PAL] is a novel stable analogue that activates the GIP receptor.•(D-Ala²)GIP[Lys³⁷PAL] has significant bioactivity in vivo when injected 8h previously.•(D-Ala²)GIP[Lys³⁷PAL] has prominent antidiabetic effects in high fat fed mice.•(D-Ala²)GIP[Lys³⁷PAL] may be suitable for further evaluation and future clinical development in man.

Keywords: Glucose-dependent insulinotropic polypeptide (GIP); Analogue; Acylation; Type 2 diabetes; Glucose tolerance; Insulin secretion

Plasticity of protease gene expression in Helicoverpa armigera upon exposure to multi-domain Capsicum annuum protease inhibitor by Neha S. Mahajan; Manasi Mishra; Vaijayanti A. Tamhane; Vidya S. Gupta; Ashok P. Giri (pp. 3414-3420).

A multi-domain Pin-II type protease inhibitor from Capsicum annuum (CanPI-7) is known to be effective against the insect pest, Helicoverpa armigera. The present study is an attempt to investigate the optimal dose of recombinant CanPI-7 (rCanPI-7) for effective antibiosis to H. armigera and further to characterize the responses of digestive proteases upon rCanPI-7 ingestion.The gut protease activity was assessed biochemically and transcript accumulation pattern for selected trypsin and chymotrypsin genes was analyzed by quantitative Real-Time PCR.The growth retardation upon exposure to rCanPI-7 was more prominent in neonates as compared to third instar larvae. Influence of stage and dosage of rCanPI-7 was conspicuous on the expression and regulation of candidate trypsin and chymotrypsin genes in H. armigera. The transcript accumulation pattern correlated with the protease activity in rCanPI-7 exposed larvae.We conclude that early exposure and specific dose of protease inhibitor are essential for effective antibiosis despite the large diversity and plasticity in the expression of protease genes in H. armigera. Moreover, it is also evident that the regulation and expression of H. armigera gut proteases are specific to the stage of PI exposure.These results highlight the requirement of optimal PI concentration for effective growth retardation and for inhibiting the major gut proteases of H. armigera.Display Omitted•Early exposure of H. armigera to rCanPI-7 crucial for antibiosis•Anti-metabolic effect of rCanPI-7 on H. armigera is dose dependent.•Inhibitor dose dependent regulation of trypsin and chymotrypsin genes revealed.•Stage specific regulation of protease genes observed in response to rCanPI-7

Keywords: Abbreviations; HGP; Helicoverpa armigera; gut protease; PI; Protease inhibitor; HaTry; H; .; armigera; trypsin; HaChy; H. armigera; chymotrypsin; CanPI-7; Capsicum annuum; protease inhibitor-7 Helicoverpa armigera; Trypsin; Chymotrypsin; CanPI-7; Protease inhibitors; Proteases

Effects of estradiol on the endocytic transport of vitamin D carrier protein in hepatocytes by T. Pirani; J. Chen; A. Vieira (pp. 3421-3426).

The possible modulation of receptor-mediated endocytosis (RME) by sex steroids is not well understood, especially in terms of the different receptor–ligand systems and cell types that may exhibit such regulation. The main objective of the current study was to examine the short-term effects of 17β-estradiol (E2) on RME of an extracellular carrier protein for calciferols, vitamin D-binding protein (DBP).Murine male and female primary hepatocytes were treated for 30min in the absence (controls) or presence of Ε2 (1μM). Labeled DBP was then added, and its endocytosis was measured after an incubation of 10min at 37°C using standard ELISA techniques. To obtain further insight into potential molecular mechanisms, fulvestrant and 17α-ethinyl estradiol (EE) were also analyzed. And as part of comparative analyses, a second nutrient carrier protein, vitamin A-binding protein (RBP), was also analyzed.The results provide the first evidence for an estradiol-dependent stimulation of DBP endocytosis ( p<0.05 relative to controls without Ε2). This stimulation, however, was only observed in female hepatocytes. Uptake of RBP was enhanced to a similar extent as DBP by estradiol. In normal (non-estradiol treated) male and female hepatocytes such changes in DBP or RBP endocytosis were not observed. Both fulvestrant and EE exhibited a significant ( p<0.05), but incomplete, inhibition of Ε2-dependent stimulation of endocytosis.The results provide novel evidence for Ε2 effects on endocytic transport; and for gender-related differences in E2-enhanced transport. These Ε2 effects may be partly dependent on estrogen receptors; but possible, additional or alternative mechanisms are also proposed.Endocytic transport is a fundamental function whose regulation has implications for cell signaling, growth, survival, differentiation, and death. This study helps delineate a possible endocrine regulatory pathway involving modulation of endocytosis by a steroid hormone. It also provides a potential, new relation between different hormonal regulators, e.g., estradiol effects on cellular assimilation of calciferols.► We examine the effects of estradiol on endocytosis of vitamin D-binding protein. ► Stimulation of such transport by estradiol (E2) occurs in female murine hepatocytes. ► Fulvestrant, an estrogen receptor antagonist, only partly inhibits this stimulation. ► Study helps delineate possible endocrine control mechanisms involving endocytosis.

Keywords: Estradiol; Endocytosis; Nutrient-carrier proteins; Hepatocytes; Vitamin D-binding protein; Fulvestrant

Enhanced antimicrobial activity of novel synthetic peptides derived from vejovine and hadrurin by Sanchez-Vasquez Lorenzo Sánchez-Vásquez; Silva-Sanchez Jesus Silva-Sanchez; Jimenez-Vargas Juana Maria Jiménez-Vargas; Rodriguez-Romero Adela Rodríguez-Romero; Munoz-Garay Carlos Muñoz-Garay; Rodriguez Maria C. Rodríguez; Georgina B. Gurrola; Lourival D. Possani (pp. 3427-3436).

Microbial antibiotic resistance is a challenging medical problem nowadays. Two scorpion peptides displaying antibiotic activity: hadrurin and vejovine were taken as models for the design of novel shorter peptides with similar activity.Using the standard Fmoc-based solid phase synthesis technique of Merrifield twelve peptides (18 to 29 amino acids long) were synthesized, purified and assayed against a variety of multi-drug resistant Gram-negative bacteria from clinical isolates. Hemolytic and antiparasitic activities of the peptides and their possible interactions with eukaryotic cells were verified. Release of the fluorophore calcein from liposomes treated with these peptides was measured.A peptide with sequence GILKTIKSIASKVANTVQKLKRKAKNAVA), and three analogs: Δ(Α29), Δ(K12-Q18; Ν26−Α29), and K4N Δ(K12-Q18; Ν26−Α29) were shown to inhibit the growth of Gram-negative ( E. coli ATCC25922) and Gram-positive bacteria ( S. aureus), as well as multi-drug resistant (MDR) clinical isolated. The antibacterial and antiparasitic activities were found with peptides at 0.78 to 25μM and 5 to 25μM concentration, respectively. These peptides have low cytotoxic and hemolytic activities at concentrations significantly exceeding their minimum inhibitory concentrations (MICs), showing values between 40 and 900μM for their EC₅₀, compared to the parent peptides vejovine and hadrurin that at the same concentration of their MICs lysed more than 50% of human erythrocytes cells.These peptides promise to be good candidates to combat infections caused by Gram-negative bacteria from nosocomial infections.Our results confirm that well designed synthetic peptides can be an alternative for solving the lack of effective antibiotics to control bacterial infections.► Twelve peptides were synthesized based on two antimicrobial venom components. ► Hadrurin and vejovine were used to design novel active but shorter peptides (18mers). ► Four of them showed good antimicrobial activity at micromolar range concentration. ► A variety of bacteria resistant to antibiotics were sensitive to these peptides. ► Procaryotic microorganisms were better targets than eukaryotic cells.

Keywords: Abbreviations; AMPs; antimicrobial peptides; ATCC; American type culture collection; CD; circular dichroism; CFU; colony forming units; CLSI; Clinical and Laboratory Standards Institute; DIPEA; N,N-diisopropylethylamine; DMEM; Dulbecco's modified eagle's medium; DMF; N,N-dimethylformamide; Fmoc; 9-fluorenylmethoxicarbonyl; HBTU; 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt; N-hydroxibenzotriazole hydrate; MIC; minimal inhibitory concentration; MDR; multi-drug resistant; MTS; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H- tetrazolium); POPC; L-α-phosphatidylcholine; POPG; 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol); RP-HPLC; reverse-phase high-performance liquid chromatography; SUVs; small unilamellar vesicles; TFA; trifluoroacetic acidAntibiotic; Antimicrobial peptide; Calcein release; Cell viability; MDR clinical isolate; Plasmodium berghei

Biosilica aging: From enzyme-driven gelation via syneresis to chemical/biochemical hardening by Xiaohong Wang; Schroder Heinz C. Schröder; Schlossmacher Ute Schloßmacher; Lei Jiang; Michael Korzhev; Muller Werner E.G. Müller (pp. 3437-3446).

The distinguished property of the siliceous sponge spicules is their enzyme (silicatein)-catalyzed biosilica formation. The enzymatically formed, non-structured biosilica product undergoes a molding, syneresis, and hardening process to form the species-specifically shaped, hard structured skeletal spicules. Besides of silicatein, a silicatein-associated protein, silintaphin-2, is assumed to be involved in the process of biosilica formation in vivo.Biosilica has been synthesized enzymatically and determined quantitatively. In addition, the subsequent hardening/aging steps have been followed by spectroscopic and electron microscopic analyses.The young spicules, newly formed in sponge cell aggregates, comprise high concentrations of sodium (~1w/w%) and potassium (0.3%). During aging the two alkali metals are removed from the spicules by 80%. In parallel, water is withdrawn from the biosilica deposits. A protein, the silicatein-α interactor silintaphin-2, comprises clusters rich in the anionic amino acids aspartic acid [D] and glutamic acid [E]. The very acidic peptide was found to significantly enhance silica polymerization. This peptide also caused a strong aggregation of silicatein/biosilica particles.The observations are explained by sodium ion removal from the initially formed biosilica deposits to the acidic amino acids in silintaphin-2. The crucial amino acids facilitating/forcing the silicatein-mediated biosilica reaction are D and E.The data presented here provide a reaction mechanism that at neutral pH the extent of biosilica formation can be strongly intensified by the removal of cations. The results contribute to an understanding of the structuring process taking place during the formation of the solid spicule rods.Display Omitted► Sponge skeleton/biosilica is synthesized by silicatein at physiological conditions. ► During aging of biosilica alkali metal ions are removed from biosilica. ► Silicatein-associated protein silintaphin-2 accepts the metal ions. ► Presence of silintaphin-2 intensifies silicatein-mediated biosilica formation. ► Reaction mechanism presented explains the structuring process of biosilica.

Keywords: Biosilica; Silicatein; Silintaphin-2; Siliceous sponge spicule; Aging of (bio)silica

Decreased reticuloendothelial system clearance and increased blood half-life and immune cell labeling for nano- and micron-sized superparamagnetic iron-oxide particles upon pre-treatment with Intralipid by Li Liu; T. Kevin Hitchens; Qing Ye; Yijen Wu; Brent Barbe; Devin E. Prior; Wendy F. Li; Fang-Cheng Yeh; Lesley M. Foley; Daniel J. Bain; Chien Ho (pp. 3447-3453).

Superparamagnetic iron-oxide nanoparticles are useful as contrast agents for anatomical, functional and cellular MRI, drug delivery agents, and diagnostic biosensors. Nanoparticles are generally cleared by the reticuloendothelial system (RES), in particular taken up by Kupffer cells in the liver, limiting particle bioavailability and in-vivo applications. Strategies that decrease the RES clearance and prolong the circulation residence time of particles can improve the in-vivo targeting efficiency.Intralipid 20.0%, an FDA approved nutritional supplement, was intravenously administered in rats at the clinical dose (2g/kg) 1h before intravenous injection of ultra-small superparamagnetic iron-oxide (USPIO) or micron-sized paramagnetic iron-oxide (MPIO) particles. Blood half-life, monocyte labeling efficiency, and particle biodistribution were assessed by magnetic resonance relaxometry, flow cytometry, inductively-coupled plasma MS, and histology.Pre-treatment with Intralipid resulted in a 3.1-fold increase in USPIO blood half-life and a 2-fold increase in USPIO-labeled monocytes. A 2.5-fold increase in MPIO blood half-life and a 5-fold increase in MPIO-labeled monocytes were observed following Intralipid pre-treatment, with a 3.2-fold increase in mean iron content up to 2.60pg Fe/monocyte. With Intralipid, there was a 49.2% and 45.1% reduction in liver uptake vs. untreated controls at 48h for USPIO and MPIO, respectively.Intralipid pre-treatment significantly decreases initial RES uptake and increases in-vivo circulation and blood monocyte labeling efficiency for nano- and micron-sized superparamagnetic iron-oxide particles.Our findings can have broad applications for imaging and drug delivery applications, increasing the bioavailability of nano- and micron-sized particles for target sites other than the liver.Display Omitted► Intralipid targets the liver Kupffer cells and blunts nanoparticle clearance. ► Intralipid results in an ~50% decrease in liver uptake of USPIO and MPIO particles. ► Intralipid produces an ~3-fold increase in blood half-life of the particles. ► Intralipid causes a 2 to 5-fold increase in labeling efficiency of blood monocytes. ► Nanoparticles can target more diverse sites or organs, other than the liver.

Keywords: Abbreviations; BN; Brown Norway; FDA; Food and Drug Administration; FITC; fluorescein isothiocyanate; ICP-MS; inductively coupled plasma-mass spectrometry; MPIO; micron-sized superparamagnetic iron-oxide; MRI; magnetic resonance imaging; PBS; phosphate-buffered-saline; ppm; part per million; PEG; polyethylene glycol; r; ₂; transverse relaxivity; R; ₂; transverse relaxation rate; RES; reticuloendothelial system; USPIO; ultra-small superparamagnetic iron-oxideNanoparticles; Intralipid; RES clearance; Blood half-life; Labeling of immune cells

Bacterial membrane disrupting dodecapeptide SC4 improves survival of mice challenged with Pseudomonas aeruginosa by Ruud P.M. Dings; Judith R. Haseman; Dan B. Leslie; Mike Luong; David L. Dunn; Kevin H. Mayo (pp. 3454-3457).

Dodecapeptide SC4 is a broad-spectrum bactericidal agent that functions by disintegrating bacterial membranes and neutralizing endotoxins. For insight into which SC4 amino acids are functionally important, we assessed Gram-negative bactericidal effects in structure–activity relationship experiments. Subsequently, SC4 was tested in a murine bacteremia model to combine and compare the efficacy with Zosyn, a first-line antibiotic against Pseudomonas aeruginosa ( P. aeruginosa).SC4 alanine-scanning analogs and their activities on were tested on P. aeruginosa. Survival studies in P. aeruginosa challenged mice were executed to monitor overall efficacy of SC4 and Zosyn, as a single modality and also as combination treatment. ELISAs were used to measure blood serum levels of selected inflammatory cytokines during treatment.Cationic residues were found to play a crucial role in terms of bactericidal activity against P. aeruginosa. In vivo, while only 9% (3/34) of control animals survived to day two and beyond, 44% (12/27) to 41% (14/34) of animals treated with SC4 or Zosyn, respectively, survived beyond one week. Combination treatment of SC4 and Zosyn demonstrated improved survival, i.e. 60% (12/20). The TNFα, IL-1, and IL-6 serum levels were attenuated in each treatment group compared to the control group.These data show that combination treatment of SC4 and Zosyn is most effective at killing P. aeruginosa and attenuating inflammatory cytokine levels in vivo.Combination treatment of SC4 and Zosyn may be useful in the clinic as a more effective antibiotic therapy against Gram-negative infectious diseases.This figure illustrates functionally important amino acid residues in dodecapeptide SC4 that are crucial to bactericidal effects on P. aeruginosa. Key residues are highlighted in purple on the sequence of SC4 (left, N-terminus at the top). In the folded structure of SC4, these residues are all aligned on one side of the helix (right).Display Omitted► Dodecapeptide SC4 is highly effective against Pseudomonas aeruginosa ( P.a.). ► SC4 attenuates inflammatory cytokine levels in serum. ► Combination treatment of SC4 and Zosyn is most effective at killing P.a. in vivo.

Keywords: Peptide; Bactericidal; Lipopolysaccharide; Endotoxin neutralizing

P-glycoprotein (Mdr1a/1b) and breast cancer resistance protein (Bcrp) decrease the uptake of hydrophobic alkyl triphenylphosphonium cations by the brain by Carolyn M. Porteous; David K. Menon; Franklin I. Aigbirhio; Robin A.J. Smith; Michael P. Murphy (pp. 3458-3465).

Mitochondrial dysfunction contributes to degenerative neurological disorders, consequently there is a need for mitochondria-targeted therapies that are effective within the brain. One approach to deliver pharmacophores is by conjugation to the lipophilic triphenylphosphonium (TPP) cation that accumulates in mitochondria driven by the membrane potential. While this approach has delivered TPP-conjugated compounds to the brain, the amounts taken up are lower than by other organs.To discover why uptake of hydrophobic TPP compounds by the brain is relatively poor, we assessed the role of the P-glycoprotein (Mdr1a/b) and breast cancer resistance protein (Bcrp) ATP binding cassette (ABC) transporters, which drive the efflux of lipophilic compounds from the brain thereby restricting the uptake of lipophilic drugs. We used a triple transgenic mouse model lacking two isoforms of P-glycoprotein (Mdr1a/1b) and the Bcrp.There was a significant increase in the uptake into the brain of two hydrophobic TPP compounds, MitoQ and MitoF, in the triple transgenics following intra venous (IV) administration compared to control mice. Greater amounts of the hydrophobic TPP compounds were also retained in the liver of transgenic mice compared to controls. The uptake into the heart, white fat, muscle and kidneys was comparable between the transgenic mice and controls.Efflux of hydrophobic TPP compounds by ABC transporters contributes to their lowered uptake into the brain and liver.These findings suggest that strategies to bypass ABC transporters in the BBB will enhance delivery of mitochondria-targeted antioxidants, probes and pharmacophores to the brain.► Brain accumulation of triphenylphosphonium cations is decreased by ABC transporters. ► ABC-transporter inactivation increases brain uptake of triphenylphosphonium cations. ► Bypassing ABC transporters may increase the effectiveness of mitochondrial therapies.

Keywords: Abbreviations; ABC proteins; ATP binding cassette proteins; BBB; blood–brain barrier; Bcrp; breast cancer resistance protein; CsA; cyclosporin A; IP; intra peritoneal; IV; intra venous; Mdr1; multi drug resistance 1; MitoF; 11-fluoroundecyltriphenylphosphonium mesylate; MitoQ; [10-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)decyl]triphenylphosphonium mesylate; MPTP; 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; TPB; tetraphenylborate; TPP; triphenylphosphonium cation; ROS; reactive oxygen species; TPMP; methyltriphenylphosphoniumMitochondria; Lipophilic cation; Blood–brain barrier; ABC transporters; MitoQ

Alteration of cell membrane proteoglycans impairs FSH receptor/Gs coupling and ERK activation through PP2A-dependent mechanisms in immature rat Sertoli cells by Guénaëlle Levallet; Pierre-Jacques Bonnamy; Jérôme Levallet (pp. 3466-3475).

During the pre-pubertal life, the cessation of Sertoli cell proliferation and the onset of differentiation are associated with a shift in the FSH-mediated signaling leading to inhibition of the ERK-mitogenic pathway and to a concomitant sensitization of cAMP/PKA pathway.To highlight the role of cell proteoglycans (PGs) in the shift of FSH signaling, both FSH-induced cAMP production and ERK1/2 inactivation were studied in untreated and sodium chlorate PG-depleted cultured Sertoli cells from 20day-old rats.Depletion of cell membrane PGs by sodium chlorate reduced FSH-, but not cholera toxin-stimulated cAMP production as well as basal ERK phosphorylation through an okadaic acid (OA)-sensitive mechanism. Involvement of PP2A was further substantiated by a marked decrease in membrane- associated PP2A activity under SC conditions and by the OA-induced restoration of PKA-dependent ERK inactivation in SC-treated cells.In 20-day-old rat Sertoli cells, transmembrane cell PGs, through tethering/activation of PP2A activity exerts regulatory control on both FSH receptor/Gs coupling and ERK phosphorylation.Besides their antiproliferative roles, cell PGs such as syndecan-1, could be involved in the increase in cAMP response to FSH occurring in Sertoli cells at the time of transition between proliferative and differentiated states.•FSH response is studied in proteoglycans-depleted 20day-old Sertoli cells.•Alteration of cell membrane proteoglycans reduces the FSH receptor/Gs coupling.•Cell PGs are involved in the recruitment/activation of a PP2A-like activity.•Cell PGs, through retention of PP2A activity, stabilize FSHR/Gs coupling.•Depletion of Cell PGs decreases basal ERK phosphorylation.

Keywords: Abbreviations; ERK; Extracellular signal-regulated kinases; FBS; Fetal bovine serum; HSPG; Heparan sulfate Proteoglycans; MAPK; mitogen-activated protein kinase; OA; okadaic acid; PGs; proteoglycans; PP2A; protein phosphatase 2A; SC; sodium chlorateSertoli cell; FSH signaling; Heparan sulfate proteoglycan; Protein phosphatase 2A; Syndecan-1

Biochemical and pharmacological properties of a new thrombin-like serine protease (Russelobin) from the venom of Russell's Viper ( Daboia russelii russelii) and assessment of its therapeutic potential by Ashis K. Mukherjee; Stephen P. Mackessy (pp. 3476-3488).

Snake venoms are rich sources of bioactive molecules, and several venom-derived proteins have entered clinical trials for use in ischemic disorders; however, late-stage failure of a recent drug candidate due to low in vivo efficacy demonstrated the need for new sources of fibrinogenolytic drug candidates.A 51.3kDa thrombin-like serine protease (Russelobin) purified from the venom of Russell's Viper ( Daboia russelii russelii) was subjected to extensive biochemical characterization, including N-terminal sequencing, substrate specificity, kinetic and inhibitor assays, glycosylation analysis and stability assays. Toxicity and pathology analyses were conducted in NSA mice.Russelobin has extensive N-terminus identity with a beta-fibrinogenase-like serine proteinase precursor from Daboia russelii siamensis venom, a mass of 51.3kDa and contains extensive N-linked oligosaccharides. Serine protease inhibitors and heparin significantly decreased activity, with much lower inhibition by DTT, antithrombin-III and α₂-macroglobulin. Russelobin preferentially released FPA and slowly released FPB from human fibrinogen, forming a labile fibrin clot readily hydrolyzed by plasmin. The partially deglycosylated enzyme showed significantly lower activity toward fibrinogen and less resistance against neutralization by plasma α₂MG and antithrombin-III. Russelobin was non-cytotoxic, non-lethal and produced no histopathologies in mice, and it demonstrated in vivo dose-dependent defibrinogenating activity.Russelobin is an A/B fibrinogenase with high specificity toward fibrinogen, both in vitro and in vivo. Extensive glycosylation appears to protect the molecule against endogenous protease inhibitors, prolonging its in vivo efficacy.Due to its low toxicity, stability and activity as a defibrinogenating agent, Russelobin shows high potential for cardiovascular drug development.► First report of high mass thrombin-like serine protease from Russell's Viper venom ► Preferentially releases FPA from fibrinogen, producing hypofibrinogenemia ► Extensive post-translational glycosylation protecting against endogenous inhibitors ► Non-toxic to mice and shows in vitro and in vivo defibrinogenating activity ► High potential for use as a defibrinogenating drug and/or for drug design

Keywords: Antithrombotic agent; Cardiovascular drug; Fibrinogenolysis; Fibrinopeptide; Insulin B-chain cleavage; Mass spectrometry

Disulfide reduction abolishes tissue factor cofactor function by Jolanta Krudysz-Amblo; Mark E. Jennings II; Tyler Knight; Dwight E. Matthews; Kenneth G. Mann; Saulius Butenas (pp. 3489-3496).

Tissue factor (TF), an in vivo initiator of blood coagulation, is a transmembrane protein and has two disulfides in the extracellular domain. The integrity of one cysteine pair, Cys186–Cys209, has been hypothesized to be essential for an allosteric “decryption” phenomenon, presumably regulating TF procoagulant function, which has been the subject of a lengthy debate. The conclusions of published studies on this subject are based on indirect evidences obtained by the use of reagents with potentially oxidizing/reducing properties.The status of disulfides in recombinant TF_1–263 and natural placental TF in their non-reduced native and reduced forms was determined by mass-spectrometry. Functional assays were performed to assess TF cofactor function.In native proteins, all four cysteines of the extracellular domain of TF are oxidized. Reduced TF retains factor VIIa binding capacity but completely loses the cofactor function.The reduction of TF disulfides (with or without alkylation) eliminates TF regulation of factor VIIa catalytic function in both membrane dependent FX activation and membrane independent synthetic substrate hydrolysis.Results of this study advance our knowledge on TF structure/function relationships.► The reduction of tissue factor (TF) disulfides abolishes TF cofactor function. ► Reduced TF binds to factor VIIa but does not increase factor VIIa activity. ► Treatment of reduced TF with PDI does not restore TF function.

Keywords: Abbreviations; TF; tissue factor; FVII; factor VII; FVIIa; factor VIIa; Cys; cysteine; FX; factor X; FXase; extrinsic factor Xase; PS; phosphatidylserine; PC; phosphotidylcholine; PCPS; synthetic vesicles of (1,2-dioleoyl-; sn; -glycero-3-phosphocholine) and (1,2-dioleoyl-; sn; -glycero-3-phospho-L-serine); PDI; protein disulfide isomerase; LPS; lipopolysaccharide; pTF; placental tissue factor; rTF; _1–263; recombinant tissue factor 1–263; mAb; monoclonal antibody; HRP; horse radish peroxidase; BSA; bovine serum albumin; TMB; tetramethylbenzidine; FPRnbs; D-FPR-ANSNH-C; ₄; H; ₉; •2HCl; CHAPS; 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate; IAA; iodoacetamide; DTT; dithiothreitol; GSH; reduced glutathione; GSSG; oxidized glutathione; EDTA; ethylenediaminetetraacetic acid disodium salt dihydrate; PEG; polyethylene glycol; NR/NA; non-reduced/non-alkylated; R/NA; reduced/non-alkylated; R/A; reduced/alkylated; NR/A; non-reduced/alkylated; MS; mass spectrometry; LC; liquid chromatography; FIU; fluorescence intensity units; Ig; immunoglobulinTissue factor; Disulfides; Mass-spectrometry; Extrinsic factor Xase; Fluorogenic assay

High-throughput analytical gel filtration screening of integral membrane proteins for structural studies by Low Christian Löw; Per Moberg; Esben M. Quistgaard; Hedren Marie Hedrén; Fatma Guettou; Jens Frauenfeld; Lars Haneskog; Pär Nordlund (pp. 3497-3508).

Structural studies of integral membrane proteins (IMPs) are often hampered by difficulties in producing stable homogenous samples for crystallization. To overcome this hurdle it has become common practice to screen large numbers of target proteins to find suitable candidates for crystallization. For such an approach to be effective, an efficient screening strategy is imperative. To this end, strategies have been developed that involve the use of green fluorescent protein (GFP) fusion constructs. However, these approaches suffer from two drawbacks; proteins with a translocated C-terminus cannot be tested and scale-up from analytical to preparative purification is often non-trivial and may require re-cloning.Here we present a screening approach that prioritizes IMP targets based on three criteria: expression level, detergent solubilization yield and homogeneity as determined by high-throughput small-scale immobilized metal affinity chromatography (IMAC) and automated size-exclusion chromatography (SEC).To validate the strategy, we screened 48 prokaryotic IMPs in two different vectors and two Escherichia coli strains. A set of 11 proteins passed all preset quality control checkpoints and was subjected to crystallization trials. Four of these crystallized directly in initial sparse matrix screens, highlighting the robustness of the strategy.We have developed a rapid and cost efficient screening strategy that can be used for all IMPs regardless of topology. The analytical steps have been designed to be a good mimic of preparative purification, which greatly facilitates scale-up.The screening approach presented here is intended and expected to help drive forward structural biology of membrane proteins.► Screening approach to prioritize integral membrane proteins for structural studies ► Rapid and cost efficient screening strategy for all IMPs regardless of topology ► Automated analytical gel filtration as quality control tool ► Strategy validation by analysis of 48 different IMPs

Keywords: Abbreviations; IMP; integral membrane protein; GFP; green fluorescent protein; FSEC; fluorescence-detected size-exclusion chromatography; IMAC; immobilized metal affinity chromatography purification; SEC; size-exclusion chromatography; GF; gel filtration; AGF; analytical gel filtration; E. coli; Escherichia coli; LIC; ligation-independent cloning; TB; terrific broth; LB; Luria Bertani; IPTG; isopropyl-ß-; d; -thiogalactopyranoside; OD; _600nm; optical density at 600; nm; HRP; horseradish peroxidase; DDM; dodecyl-β-; d; -maltoside; LDAO; N,N-dimethyldodecylamine-N-oxide; CYMAL-5; 5-cyclohexyl-1-pentyl-β-; d; -maltoside; FC12; fos-choline-12; SDS–PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; TEV; tobacco etch virus; GpA; glycophorin A; MATE; multidrug and toxic compound extrusion; BASS; bile acid sodium symporter; POT; proton dependent oligopeptide transporter; BisTris; bis(2-hydroxyethyl)-amino-tris(hydroxymethyl)-methane; Tris; tris(hydroxymethyl)-methane; BSA; bovine serum albumin; TCEP; tris(2-carboxyethyl)phosphine; DTT; dithiothreitol; EDTA; ethylenediaminetetraacetic acidStructural genomics; Membrane protein structure; Membrane protein over-expression; Analytical gel filtration; Detergent screen

An antifungal peptide from Coffea canephora seeds with sequence homology to glycine-rich proteins exerts membrane permeabilization and nuclear localization in fungi by Umberto Zottich; Maura Da Cunha; André O. Carvalho; Germana B. Dias; Nádia Casarin; Ilka M. Vasconcelos; Valdirene M. Gomes (pp. 3509-3516).

The superfamily of glycine-rich proteins (GRPs) corresponds to a large and complex group of plant proteins that may be involved in many developmental and physiological processes such as RNA biogenesis, stress tolerance, pollen hydration and plant-pathogen interactions, showing defensive activity against fungi, bacteria and viruses.In this study, the peptides from Coffea canephora seeds were extracted according to the methods of Egorov et al. (2005). The purified peptide was submitted for amino acid sequencing and antimicrobial activity measurement.The purified peptide with a molecular weight of 7kDa, named Cc-GRP, was observed to display homology to GRPs. The Cc-GRP–fungi interaction led to morphological changes and membrane permeability, including the formation of pseudohyphae, which were visualized with the aid of SYTOX green dye. Additionally, Cc-GRP also prevented colony formation by yeasts. Antifungal assays of Fusarium oxysporum and Colletotrichum lindemuthianum, observed by light microscopy, showed that the two molds exhibited morphological changes after the growth assay. Cc-GRP coupled to FITC and its subsequent treatment with DAPI revealed the presence of the peptide in the cell wall, cell surface and nucleus of F. oxysporum.In this work we purified, characterized and evaluated the in vitro effect on fungi of a new peptide from coffee, named Cc-GRP, which is involved in the plant defense system against pathogens by acting through a membrane permeabilization mechanism and localized in the nuclei of fungal cells. We also showed, for the first time, the intracellular localization of Cc-GRP during antimicrobial assay.• Cc-GRP shows molecular weight of 7kDa and display homology to glycine rich proteins family.• Cc-GRP–fungi interaction led to yeast membrane permeability.• Cc-GRP exhibited strong antifungal activity against Candida albicans and C. tropicalis yeasts.• Cc-GRP promoted morphological changes in Fusarium oxysporum and Colletotrichum lindemuthianum fungi.•The intracellular localization of a GRP, in nucleus, was showed for the first time.

Keywords: Glycine-rich peptide; Coffee; Antimicrobial peptides; AMPs; Defense plant

Non-invasive label-free monitoring the cardiac differentiation of human embryonic stem cells in-vitro by Raman spectroscopy by Flavius C. Pascut; Spandan Kalra; Vinoj George; Nathan Welch; Chris Denning; Ioan Notingher (pp. 3517-3524).

Online label-free monitoring of in-vitro differentiation of stem cells remains a major challenge in stem cell research. In this paper we report the use of Raman micro-spectroscopy (RMS) to measure time- and spatially-resolved molecular changes in intact embryoid bodies (EBs) during in-vitro cardiogenic differentiation.EBs formed by aggregation of human embryonic stem cells (hESCs) were cultured in defined medium to induce differentiation towards cardiac phenotype and maintained in purpose-built micro-bioreactors on the Raman microscope for 5days (between days 5 and 9 of differentiation) and spatially-resolved spectra were recorded at 24h intervals.The Raman spectra showed that the onset of spontaneous beating of EBs at day 7 coincided with an increase in the intensity of the Raman bands at 1340cm−¹, 1083cm−¹, 937cm−¹, 858cm−¹, 577cm−¹ and 482cm−¹. The spectral maps corresponding to these bands had a high positive correlation with the expression of the cardiac-specific α-actinin obtained by immuno-fluorescence imaging of the same EBs. The spectral markers obtained here are also in agreement with previous studies performed on individual live hESC-derived CMs.The intensity profile of these Raman bands can be used for label-free in-situ monitoring of EBs to estimate the efficacy of cardiogenic differentiation.As the acquisition of the time-course Raman spectra did not affect the viability or the differentiation potential of the hESCs, this study demonstrates the feasibility of using RMS for on-line non-invasive continuous monitoring of such processes inside bioreactor culture systems.► Label-free Raman spectroscopy measurements of stem cells were conducted during differentiation. ► Stem cells are maintained in physiological conditions within micro-bioreactors. ► Bands assigned to nucleic acids, proteins, lipids and carbohydrates can be detected. ► The abundance of cardiomyocytes within embryoid bodies can be assessed. ► This technique is suitable for monitoring stem cell bioprocesses.

Keywords: Embryoid body; Human embryonic stem cell; Differentiation; Cardiomyocyte; Raman spectroscopy

Probing non-enzymatic glycation of type I collagen: A novel approach using Raman and infrared biophotonic methods by Marie Guilbert; Georges Said; Teddy Happillon; Valérie Untereiner; Roselyne Garnotel; Pierre Jeannesson; Ganesh D. Sockalingum (pp. 3525-3531).

Non-enzymatic glycation is the main post-translational modification of long-life proteins observed during aging and physiopathological processes such as diabetes and atherosclerosis. Type I collagen, the major component in matrices and tissues, represents a key target of this spontaneous reaction which leads to changes in collagen biomechanical properties and by this way to tissue damages.The current study was performed on in vitro glycated type I collagens using vibrational microspectroscopies, FT-IR and Raman, to highlight spectral features related to glycation effect.We report a conservation of the triple-helical structure of type I collagen and noticeable variations in the exposure of proline upon glycation. Our data also show that the carbohydrate band can be a good spectroscopic marker of the glycation level, correlating well with the fluorescent AGEs formation with sugar addition.These non-invasive and label-free methods can shed new light on the spectral features of glycated collagens and represent an effective tool to study changes in the extracellular matrix observed in vivo during aging or on the advent of a pathological situation.► Glycated collagen was studied by direct, rapid, and label-free biophotonic methods. ► FT-IR and Raman provide complementary molecular information on glycation effects. ► The level of glycation can be evaluated in a semi-quantitative manner. ► Spectroscopic markers of glycation correlate with AGEs-fluorescence quantification.

Keywords: Type I collagen; Glycation; Advanced Glycation End products; Fourier-transform infrared microspectroscopy; Raman microspectroscopy

New insights into deleterious impacts of in vivo glycation on albumin antioxidant activities by Jennifer Baraka-Vidot; Alexis Guerin-Dubourg; Fanny Dubois; Bertrand Payet; Emmanuel Bourdon; Philippe Rondeau (pp. 3532-3541).

Albumin constitutes the most abundant circulating antioxidant and prevents oxidative damages. However, in diabetes, this plasmatic protein is exposed to several oxidative modifications, which impact on albumin antioxidant properties.Most studies dealing on albumin antioxidant activities were conducted on in vitro modified protein. Here we tried to decipher whether reduced antioxidant properties of albumin could be evidenced in vivo. For this, we compared the antioxidant properties of albumin purified from diabetic patients to in vitro models of glycated albumin.Both in vivo and in vitro glycated albumins displayed impaired antioxidant activities in the free radical-induced hemolysis test. Surprisingly, the ORAC method (Oxygen Radical Antioxidant Capacity) showed an enhanced antioxidant activity for glycated albumin. Faced with this paradox, we investigated antioxidant and anti-inflammatory activities of our albumin preparations on cultured cells (macrophages and adipocytes). Reduced cellular metabolism and enhanced intracellular oxidative stress were measured in cells treated with albumin from diabetics. NF-kB –mediated gene induction was higher in macrophages treated with both type of glycated albumin compared with cells treated with native albumin. Anti inflammatory activity of native albumin is significantly impaired after in vitro glycation and albumin purified from diabetics significantly enhanced IL6 secretion by adipocytes. Expression of receptor for advanced glycation products is significantly enhanced in glycated albumin-treated cells.Our results bring new evidences on the deleterious impairments of albumin important functions after glycation and emphasize the importance of in vivo model of glycation in studies relied to diabetes pathology.► Enhanced glycative modification of albumin in diabetes. ► Effect of in vitro and in vivo glycated albumin on macrophages and adipocytes. ► Impaired anti-oxidant/inflammatory properties of albumin purified from diabetics.

Keywords: Abbreviations; AGE; advanced glycation end-products; HSA; human serum albumin; HSA-ND and HSA-D; purified human serum albumin from non-diabetic and diabetic patients, respectively; HbA; _1C; glycated hemoglobin level; PBS; phosphate-buffered salineAlbumin; Glycation; Advanced glycation end-products; Diabetes; Antioxidant; Inflammation

Age-dependent guanine oxidation in DNA of different brain regions of Wistar rats and prematurely aging OXYS rats by Evgeniya A. Sattarova; Olga I. Sinitsyna; Elena A. Vasyunina; Alexander B. Duzhak; Nataliya G. Kolosova; Dmitry O. Zharkov; Georgy A. Nevinsky (pp. 3542-3552).

Oxidative damage to the cell, including the formation of 8-oxoG, has been regarded as a significant factor in carcinogenesis and aging. An inbred prematurely aging rat strain (OXYS) is characterized by high sensitivity to oxidative stress, lipid peroxidation, protein oxidation, DNA rearrangements, and pathological conditions paralleling several human degenerative diseases including learning and memory deterioration.We have used monoclonal antibodies against a common pre-mutagenic base lesion 8-oxoguanine (8-oxoG) and 8-oxoguanine DNA glycosylase (OGG1) in combination with indirect immunofluorescence microscopy and image analysis to follow the relative amounts and distribution of 8-oxoG and OGG1 in various cells of different brain regions from OXYS and control Wistar rats.It was shown that 8-oxoG increased with age in mature neurons, nestin- and glial fibrillary acidic protein (GFAP)-positive cells of hippocampus and frontal cortex in both strains of rats, with OXYS rats always displaying statistically significantly higher levels of oxidative DNA damage than Wistar rats. The relative content of 8-oxoG and OGG1 in nestin- and GFAP-positive cells was higher than in mature neurons in both Wistar and OXYS rats. However, there was no significant interstrain difference in the content of OGG1 for all types of cells and brain regions analyzed, and no difference in the relative content of 8-oxoG between different brain regions.Oxidation of guanine may play an important role in the development of age-associated decrease in memory and learning capability of OXYS rats.The findings are important for validation of the OXYS rat strain as a model of mammalian aging.► An inbred prematurely aging OXYS and control Wistar rats were used. ► OXYS is characterized by high sensitivity to oxidative stress, learning and memory deterioration. ► We have analyzed base lesion 8-oxoguanine in different brain regions of these rats. ► 8-OxoG increased in neurons, nestin- and (GFAP)-positive cells of hippocampus and frontal cortex. ► OXYS rats demonstrate significantly higher levels of oxidative brain DNA damage than Wistar rats.

Keywords: Abbreviations; Abs; antibodies; DAPI; 4′,6-diamidino-2-phenylindole; GFAP; glial fibrillary acidic protein; OGG1; 8-oxoguanine DNA glycosylase; 8-oxoG; 8-oxoguanine; PBS; phosphate-buffered salinePremature aging; OXYS rats; Brain; Guanine oxidation; Immunofluorescence

Chronic hypoxia leads to a glycolytic phenotype and suppressed HIF-2 signaling in PC12 cells by Alexander V. Zhdanov; Ruslan I. Dmitriev; Anna V. Golubeva; Svetlana A. Gavrilova; Dmitri B. Papkovsky (pp. 3553-3569).

Along with other regulators of cell metabolism, hypoxia-inducible factors HIF-1 and HIF-2 differentially regulate cell adaptation to hypoxia. Switches in HIF-1/HIF-2 signaling in chronic hypoxia have not been fully investigated.Proliferation, viability, apoptosis, neuronal and bioenergetic markers, mitochondrial function, respiration, glycolysis, HIF signalling, responses to O₂ and glucose deprivation (OGD) were examined using tumor PC12 and SH-SY5Y cells continuously grown at 3% O₂.Hypoxic PC12 cells ( H-cells) exhibit reduced proliferation and histone H4 acetylation, NGF-independent differentiation, activation of AMPK, inhibition of Akt, altered mitochondria and response to NGF. Cellular cytochrome c is increased with no effect on apoptosis. Reduction in respiration has minor effect on cellular ATP which is maintained through activated uptake (GLUT1) and utilization (HK2, PFK2) of glucose. H-cells exhibit resistance to OGD linked to increased glycogen stores. HIF-2alpha protein is decreased without changes in mRNA. Unlike HIF-1alpha, HIF-2alpha is not stabilized pharmacologically or by O₂ deprivation. Capacity for HIF-2alpha stabilization is partly restored when H-cells are cultured at normoxia. In low-respiring SH-SY5Y cells cultured under the same conditions HIF-2alpha stabilization and energy budget are not affected.In chronically hypoxic PC12 cells glycolytic energy budget, increased energy preservation and low susceptibility to OGD are observed. HIF-2alpha no longer orchestrates adaptive responses to anoxia.Demonstrated switch in HIF-1/HIF-2 signaling upon chronic hypoxia can facilitate cell survival in energy crisis, by regulating balance between energy saving and decrease in proliferation, on one hand and active cell growth and tumor expansion, on the other.► Chronic hypoxia (CH) affects levels of neuronal markers and reduces proliferation of PC12 cells. ► CH alters mitochondrial shape and turnover and leads to cytochrome c increase. ► Reduction in O₂ consumption rate is coupled with activated AMPK and glycolysis. ► Glycogen production and resistance to O₂ and glucose deprivation in hypoxic cells are increased. ► Unlike HIF-1, HIF-2 no longer regulates response to acute O₂ deprivation in CH cells.

Keywords: Chronic hypoxia; Cell energy budget; HIF-2; Mitochondria; Oxygen and glucose deprivation; PC12 cells

The allosteric modulation of thyroxine-binding globulin affinity is entropy driven by Ariel A. Petruk; María S. Labanda; Alvarez Rosa M.S. Álvarez; Marcelo A. Marti (pp. 3570-3577).

Thyroxine-binding globulin (TBG) is a non-inhibitory member of the serpin family of proteins whose main structural element is the reactive center loop (RCL), that, upon cleavage by proteases, is inserted into the protein core adopting a β-strand conformation (stressed to relaxed transition, S-to-R). After S-to-R transition thyroxine (T4) affinity decreases. However, crystallographic studies in the presence or absence of the hormone in different states are unable to show significant differences in the structure and interactions of the binding site. Experimental results also suggest the existence of several S states (differing in the number of inserted RCL residues), associated with a differential affinity.To shed light into the molecular basis that regulates T4 affinity according to the degree of RCL insertion in TBG, we performed extended molecular dynamics simulations combined with several thermodynamic analysis of the T4 binding to TBG in three different S states, and in the R state.Our results show that, despite T4 binding in the protein by similar interactions in all states, a good correlation between the degree of RCL insertion and the binding affinity, driven by a change in TBG conformational entropy, was observed.TBG allosteric regulation is entropy driven. The presence of multiple S states may allow more efficient T4 release due to protease activity.The presented results are clear examples of how computer simulation methods can reveal the thermodynamic basis of allosteric effects, and provide a general framework for understanding serpin allosteric affinity regulation.•T4 binding to TBG is modulated by the degree of reactive center loop insertion.•MD reveals entropy driven affinity regulation in TBG.•TBG loop insertion determines TBG conformational entropy.•Multiple conformational states allow more efficient T4 release.

Keywords: Serpin family; Thyroxine-binding globulin; Allostery; Conformational entropy; Generalized Born surface analysis; Molecular dynamics

Transducing properties of a pre-structured α-helical DPT-peptide containing a short canine adenovirus type 2 E4orf4 PP2A₁-binding sequence by A. Galioot; A.N. Godet; V. Maire; P.B. Falanga; X. Cayla; B. Baron; P. England; A. Garcia (pp. 3578-3583).

Induction of the death pathway resulting from the specific interaction of the PP2A₁ phosphatase with adenoviral E4orf4 protein is a promising approach for cancer therapy. With the aim of deregulating tumor pathways, and mimicking E4orf4 anti-cancer signal, we have previously proposed the DPT technology concept, based on design of specific PP1/PP2A interacting penetrating peptides.Using biochemical, structural and cell survival experiments, we have characterized new DPT-peptides containing short PP2A binding sequences.We identified overlapping sequences, located within the N-terminal domain E4orf4_23-46 of canine adenoviral E4orf4 protein, that interact with the PP2A-Bα subunit of PP2A₁ holoenzyme. We characterized DPT-E4orf4₄ and TAT-E4orf4₄, two bi-partite cell penetrating peptides containing the 12 PP2A₁ binding residues of the canine type 2 E4orf4_27-38 sequence, respectively fused to the DPT-sh1 and TAT shuttle sequences. Surprisingly DPT-E4orf4₄, in contrast to inactive TAT-E4orf4₄, adopted a well defined α-helical structure and co-precipitated PP2A₁ from HeLa cell extracts. DPT-E4orf4₄ also internalized streptavidin-HRP and inhibited survival of HeLa cells more efficiently than TAT, TAT-E4orf4₄ or the previously published anti-tumor TAT-derived peptide shepherdin. DPT-E4orf4₄ also efficiently inhibited the survival of human adherent transformed cells, including wild type and p53 mutated colonic HCT116 cells, without affecting survival of human non-transformed fibroblasts.We characterized the transducing properties of a new α-helical DPT-E4orf4₄ peptide containing a short PP2A-interacting sequence from canine Adenoviral E4orf4 protein.Our results suggest that α-helical structured DPT peptides specifically interacting with PP2A could be a valuable anti-cancer drug design scaffold.•Canine adenoviral E4orf4 protein contains a new PP2A₁-interacting domain•DPT-E4orf4₄ is a new α-helical PP2A₁-interacting peptide•DPT-E4orf4₄ is a biologically active peptide

Keywords: PP2A; E4orf4; DPT-peptides; Cell-death; Cancer

Transcriptional and post-translational regulation of Bim is essential for TGF-β and TNF-α-induced apoptosis of gastric cancer cell by Huyen Trang Ha Thi; Hee-Sun Lim; Jooyoung Kim; Young-Mi Kim; Hye-Youn Kim; Suntaek Hong (pp. 3584-3592).

Tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) are well known as central signaling molecules in natural antitumor mechanisms. However, some cancer cells are resistant to TNF-α or TGF-β-induced death signaling. Herein, we investigated synergistic activities of TGF-β and TNF-α and molecular mechanisms involved in apoptosis of gastric cancer cells.SNU620, a human gastric carcinoma cell line was tested for cell viability by treatment of TGF-β in combination with TNF-α. Cell apoptosis, proliferation, caspase activation and gene expression were tested using flow cytometry, Western blot, MTT assay, luciferase assay and real-time qRT-PCR analysis. Knockdown of target genes were performed using lentiviral shRNA system.TGF-β sensitizes SNU620 cells undergoing TNF-α-induced caspase-dependent apoptosis. TNF-α and TGF-β synergistically induced the degradation of poly(ADP–ribose) polymerase (PARP) and caspase cascade activation. We also confirmed that c-Jun NH₂-terminal kinase (JNK) and Smad3 play critical roles in the apoptotic pathway. In addition, a pro-apoptotic protein Bim was critical for apoptosis and was regulated by TGF-β and TNF-α at the transcriptional and post-translational levels. Expression of Bim was induced at the transcriptional level by Smad3 while Bim protein stability was maintained by a JNK-mediated pathway.By understanding the synergistic activation of TGF-β and TNF-α in apoptosis, we may have a chance to identify good therapeutic approaches for the treatment of cancers that are resistant to death signals.Our results indicate that TGF-β and TNF-α act in concert to activate apoptosis in gastric cancer cell through crosstalk between Smad and JNK signaling pathways.•TGF-β sensitizes gastric cancer cell in TNF-α-induced caspase-dependent apoptosis.•Bim is a key mediator of TGF-β and TNF-α-induced apoptosis in gastric cancer cell.•Smad3 enhances the expression of Bim via transcriptional regulation.•JNK signaling stabilizes the Bim protein at post-translational step.•Crosstalk between Smad3 and JNK pathway is important for cancer cell death.

Keywords: Abbreviations; TNF-α; tumor necrosis factor-α; TGF-β; transforming growth factor-β; JNK; c-Jun NH; ₂; -terminal kinase; PARP; poly(ADP–ribose) polymerase; NF-κB; nuclear factor-κB; CHX; cycloheximide; TNFR1; TNF receptor 1; RIP; receptor-interacting protein; TRAF2; TNF receptor-associated factor 2; MAPK; mitogen-activated protein kinase; ERK; extracellular signal-regulated kinase; SBE; Smad-binding elementTumor necrosis factor-α; Transforming growth factor-β; Smad3; c-Jun NH; ₂; -terminal kinase; Bim; Apoptosis

Activation of H⁺-ATPase by glucose in Saccharomyces cerevisiae involves a membrane serine protease by Alexis Nazareno Campetelli; Noelia Edith Monesterolo; Gabriela Previtali; Verónica Silvina Santander; Marina Rafaela Amaiden; Carlos Angel Arce; Javier Valdez-Taubas; César Horacio Casale (pp. 3593-3603).

Glucose induces H⁺-ATPase activation in Saccharomyces cerevisiae. Our previous study showed that (i) S. cerevisiae plasma membrane H⁺-ATPase forms a complex with acetylated tubulin (AcTub), resulting in inhibition of the enzyme activity; (ii) exogenous glucose addition results in the dissociation of the complex and recovery of the enzyme activity.We used classic biochemical and molecular biology tools in order to identify the key components in the mechanism that leads to H⁺-ATPase activation after glucose treatment.We demonstrate that glucose-induced dissociation of the complex is due to pH-dependent activation of a protease that hydrolyzes membrane tubulin. Biochemical analysis identified a serine protease with a kDa of 35–40 and an isoelectric point between 8 and 9. Analysis of several knockout yeast strains led to the detection of Lpx1p as the serine protease responsible of tubulin proteolysis. When lpx1Δ cells were treated with glucose, tubulin was not degraded, the AcTub/H⁺-ATPase complex did not undergo dissociation, and H⁺-ATPase activation was significantly delayed.Our findings indicate that the mechanism of H⁺-ATPase activation by glucose involves a decrease in the cytosolic pH and consequent activation of a serine protease that hydrolyzes AcTub, accelerating the process of the AcTub/H⁺-ATPase complex dissociation and the activation of the enzyme.Our data sheds light into the mechanism by which acetylated tubulin dissociates from the yeast H⁺-ATPase, identifying a degradative step that remained unknown. This finding also proposes an indirect way to pharmacologically regulate yeast H⁺-ATPase activity and open the question about mechanistic similarities with other higher eukaryotes.•A degradative step is required for plasma membrane H⁺-ATPase activation by glucose.•Lpx1p degrades tubulin from the Tubulin-H⁺-ATPase complex.•Tubulin-H⁺-ATPase complex dissociation by Lpx1p results in H⁺-ATPase activation.•Tubulin degradation by Lpx1p is an early event in the H⁺-ATPase activation mechanism.•Tubulin proteolysis by Lpx1p is an essential step to reach H⁺-ATPase fast activation.

Keywords: Abbreviations; AcTub; acetylated tubulin; Ig; immunoglobulin; mAb; monoclonal antibody; pH; _e; extracellular pH; pH; _i; intracellular pH; PMSF; phenyl methyl sulfonyl fluorideYeast H; ⁺; -ATPase; Acetylated tubulin; pH regulation; Proteolysis

Acetyl-11-keto-β-boswellic acid (AKBA) inhibits human gastric carcinoma growth through modulation of the Wnt/β-catenin signaling pathway by Yu-Sheng Zhang; Ji-Zhen Xie; Julia-Li Zhong; Yuan-Yuan Li; Rui-Qi Wang; Yi-Zhuo Qin; Hong-Xiang Lou; Zu-Hua Gao; Xian-Jun Qu (pp. 3604-3615).

Acetyl-11-keto-beta-boswellic acid (AKBA) is a derivative of boswellic acid, an active component of Boswellia serrata gum resin. We examined the effect of AKBA on human gastric carcinoma growth and explored the underlying molecular mechanisms.Inhibition of cancer cell growth was estimated by colorimetric and clonogenic assays. Cell cycle distribution was analyzed by flow cytometry and apoptosis determined using Annexin V-FITC/PI staining and DNA ladder quantification. After three weeks of oral AKBA administration in nude mice bearing cancer xenografts, animals were sacrificed and xenografts removed for TUNEL staining and western blot analysis.AKBA exhibited anti-cancer activity in vitro and in vivo. With oral application in mice, AKBA significantly inhibited SGC-7901 and MKN-45 xenografts without toxicity. This effect might be associated with its roles in cell cycle arrest and apoptosis induction. The results also showed activation of p21^Waf1/Cip1 and p53 in mitochondria and increased cleaved caspase-9, caspase-3, and PARP and Bax/Bcl-2 ratio after AKBA treatment. Further analysis suggested that these effects might arise from AKBA's modulation of the aberrant Wnt/β-catenin signaling pathway. Upon AKBA treatment, β-catenin expression in nuclei was inhibited, and membrane β-catenin was activated. In the same sample, active GSK3β was increased and its non-active form decreased. Levels of cyclin D1, PCNA, survivin, c-Myc, MMP-2, and MMP-7, downstream targets of Wnt/β-catenin, were inhibited.AKBA effects on human gastric carcinoma growth were associated with its activity in modulating the Wnt/β-catenin signaling pathway.AKBA could be useful in the treatment of gastric cancers.•AKBA inhibits human gastric carcinoma growth through apoptosis induction.•Oral AKBA in mice significantly delays growth of SGC-7901 and MKN-45 xenografts.•AKBA inhibits cancer growth without toxicity to mice.•AKBA activity is associated with modulation of the Wnt/β-catenin signaling pathway.

Keywords: Gastric carcinoma; Acetyl-11-keto-β-boswellic acid (AKBA); Inhibitory effect; Apoptosis; Wnt/β-catenin

Selenium-binding protein 1: Its physiological function, dependence on aryl hydrocarbon receptors, and role in wasting syndrome by 2,3,7,8-tetrachlorodibenzo- p-dioxin by Sayuri Tsujimoto; Takumi Ishida; Tomoki Takeda; Yuji Ishii; Yuko Onomura; Kiyomi Tsukimori; Shinji Takechi; Tadatoshi Yamaguchi; Hiroshi Uchi; Satoshi O. Suzuki; Midori Yamamoto; Masaru Himeno; Masutaka Furue; Hideyuki Yamada (pp. 3616-3624).

Selenium-binding protein 1 (Selenbp1) is suggested to play a role in tumor suppression, and may be involved in the toxicity produced by dioxin, an activator of aryl hydrocarbon receptors (AhR). However, the mechanism or likelihood is largely unknown because of the limited information available about the physiological role of Selenbp1.To address this issue, we generated Selenbp1-null [Selenbp1 (−/−)] mice, and examined the toxic effect of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in this mouse model.Selenbp1 (−/−) mice exhibited only a few differences from wild-type mice in their apparent phenotypes. However, a DNA microarray experiment showed that many genes including Notch1 and Cdk1, which are known to be enhanced in ovarian carcinoma, are also increased in the ovaries of Selenbp1 (−/−) mice. Based on the different responses to TCDD between C57BL/6J and DBA/2J strains of mice, the expression of Selenbp1 is suggested to be under the control of AhR. However, wasting syndrome by TCDD occurred equally in Selenbp1 (−/−) and (+/+) mice.The above pieces of evidence suggest that 1) Selenbp1 suppresses the expression of tumor-promoting genes although a reduction in Selenbp1 alone is not very serious as far as the animals are concerned; and 2) Selenbp1 induction by TCDD is neither a pre-requisite for toxicity nor a protective response for combating TCDD toxicity.Selenbp1 (−/−) mice exhibit little difference in their apparent phenotype and responsiveness to dioxin compared with the wild-type. This may be due to the compensation of Selenbp1 function by a closely-related protein, Selenbp2.•We generated Se-binding protein 1-null mice for clarifying its physiological role.•The null mice obtained exhibited little change in their apparent phenotypes.•However, the expression of many cancer-related genes in the ovary was changed.•Wasting syndrome caused by dioxin occurred equally both in KO and wild-type mice.

Keywords: Abbreviations; AhR; aryl hydrocarbon receptor; ALT; alanine aminotransferase; AST; aspartate aminotransferase; bp; base pair; Cyp; cytochrome P450; GPx; glutathione peroxidase; RT-PCR; reverse transcription-polymerase chain reaction; Selenbp; selenium-binding protein; TCDD; 2,3,7,8-tetrachlorodibenzo-; p; -dioxin; TBARS; thiobarbituric acid-reactive substancesSelenium-binding protein 1; Knockout mouse; Ovarian cancer; 2,3,7,8-Tetrachlorodibenzo-; p; -dioxin; Aryl hydrocarbon receptor; Wasting syndrome

Preclinical evaluation of zoledronate using an in vitro mimetic cellular model for breast cancer metastatic bone disease by P.G. Dedes; I. Kanakis; Ch. Gialeli; A.D. Theocharis; T. Tsegenidis; D. Kletsas; G.N. Tzanakakis; N.K. Karamanos (pp. 3625-3634).

The interactions between metastatic breast cancer cells and host cells of osteoclastic lineage in bone microenvironment are essential for osteolysis. In vitro studies to evaluate pharmacological agents are mainly limited to their direct effects on cell lines. To mimic the communication between breast cancer cells and human osteoclasts, a simple and reproducible cellular model was established to evaluate the effects of zoledronate (zoledronic acid, ZOL), a bisphosphonate which exerts antiresorptive properties.Human precursor osteoclasts were cultured on bone-like surfaces in the presence of stimuli (sRANKL, M-CSF) to ensure their activation. Furthermore, immature as well as activated osteoclasts were co-cultured with MDA-MB-231 breast cancer cells. TRAP5b and type I collagen N-terminal telopeptide (NTx) were used as markers. Osteoclasts’ adhesion to bone surface and subsequent bone breakdown were evaluated by studying the expression of cell surface receptors and certain functional matrix macromolecules in the presence of ZOL.ZOL significantly suppresses the precursor osteoclast maturation, even when the activation stimuli (sRANKL and M-SCF) are present. Moreover, it significantly decreases bone osteolysis and activity of MMPs as well as precursor osteoclast maturation by breast cancer cells. Additionally, ZOL inhibits the osteolytic activity of mature osteoclasts and the expression of integrin β3, matrix metalloproteinases and cathepsin K, all implicated in adhesion and bone resorption.ZOL exhibits a beneficial inhibitory effect by restricting activation of osteoclasts, bone particle decomposition and the MMP-related breast cancer osteolysis.The proposed cellular model can be reliably used for enhancing preclinical evaluation of pharmacological agents in metastatic bone disease.► Zoledronate suppresses precursor osteoclast maturation and osteolytic activity. ► Modulates the expression of matrix macromolecules implicated in adhesion and bone osteolysis ► Potential inhibitor of breast cancer-mediated osteolysis

Keywords: Abbreviations; BPs; bisphosphonates; ZOL; zoledronate; RANKL; receptor activator of nuclear factor-κB ligand; NTx; N-terminal telopeptide; CATK; cathepsin K; OPG; osteoprotegerin; PTHrP; parathyroid hormone-related peptide; M-CSF; macrophage colony-stimulating factor; ECM; extracellular matrix; MMP; matrix metalloproteinase; MT-MMP; membrane type-MMP; TIMP; tissue inhibitors of MMPsBreast cancer; Bone metastasis; Cellular model; Metalloproteinase; Matrix macromolecule

Multi-target-directed design, syntheses, and characterization of fluorescent bisphosphonate derivatives as multifunctional enzyme inhibitors in mevalonate pathway by Jinbo Gao; Jinggong Liu; Yongge Qiu; Xiusheng Chu; Yuqin Qiao; Ding Li (pp. 3635-3642).

Mevalonate pathway is an important cellular metabolic pathway present in all higher eukaryotes and many bacteria. Four enzymes in mevalonate pathway, including MVK, PMK, MDD, and FPPS, play important regulatory roles in cholesterol biosynthesis and cell proliferation.The following methods were used: cloning, expression and purification of enzymes in mevalonate pathway, organic syntheses of multifunctional enzyme inhibitors, measurement of their IC₅₀ values for above four enzymes, kinetic studies of enzyme inhibitions, molecular modeling studies, cell viability tests, and fluorescence microscopy.We report our multi-target-directed design, syntheses, and characterization of two blue fluorescent bisphosphonate derivatives compounds15 and16 as multifunctional enzyme inhibitors in mevalonate pathway. These two compounds had good inhibition to all these four enzymes with their IC₅₀ values at nanomolar to micromolar range. Kinetic and molecular modeling studies showed that these two compounds could bind to the active sites of all these four enzymes. The fluorescence microscopy indicated that these two compounds could easily get into cancer cells.Multifunctional enzyme inhibitors are generally more effective than single enzyme inhibitors, with fewer side effects. Our results showed that these multifunctional inhibitors could become lead compounds for further development for the treatment of soft-tissue tumors and hypercholesteremia.► Enzymes MVK, PMK, MDD, and FPPS, play regulatory roles in mevalonate pathway. ► Two fluorescent bisphosphonates were synthesized as multi-enzyme inhibitors. ► These two compounds had potent inhibition to all four enzymes. ► Kinetic and molecular modeling studies of these two compounds were carried out. ► Fluorescence microscopy indicated that the compounds could get into cancer cells.

Keywords: Abbreviations; FPP; farnesyl pyrophosphate; FPPS; farnesyl pyrophosphate synthase; GPP; geranyl diphosphate; HMG-CoA reductase; 3-hydroxy-3-methylglutaryl-CoA reductase; IPP; isopentenyl diphosphate; MDD; mevalonate 5-diphosphate decarboxylase, also known as mevalonate 5-pyrophosphate decarboxylase or MPD; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; MVAPP; mevalonate 5-diphosphate; MVK; mevalonate kinase; N; -BPs; nitrogen-containing bisphosphonates; PMK; phosphomevalonate kinase; PP; pyrophosphate or diphosphate; TsCl; toluene sulfonyl chlorideBisphosphonate; Multifunctional inhibitor; Mevalonate kinase; Phosphomevalonate kinase; Mevalonate 5-diphosphate decarboxylase; Farnesyl pyrophosphate synthase

Synthesis of 3-O-methylgallic acid a powerful antioxidant by electrochemical conversion of syringic acid by Olfa Dridi Gargouri; Boutheina Gargouri; Souhel Kallel Trabelsi; Mohamed Bouaziz; Abdelhedi Ridha Abdelhédi (pp. 3643-3649).

A kinetic study of the electrochemical oxidation of syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) by cyclic voltammetry at treated gold disk was combined with results of electrolyses at Ta/PbO₂ anode in order to convert it into potentially high-added-value product.The electrochemical oxidation of syringic acid was carried out in order to convert this compound to 3-O-methylgallic acid. This latter was identified by mass spectrophotometry using LC-MS/MS apparatus. The 3-O-methylgallic acid synthesis was controlled by cyclic volammetry, Ortho-diphenolicdeterminations and DPPH radical-scavenging activity.The proposed mechanism is based on the hypothesis of a bielectronic discharge of syringic acid molecule under free and adsorbed form involving two intermediate cation mesomers. Hydrolysis of the more stable of this last one leads to the formation of the 3,4-dihydroxy-5-methoxybenzoic acid (3-O-methylgallic acid) as a major product. The latter aromatic compound was synthesized by anodic oxidation of syringic acid at PbO2 electrode. The cyclic voltammogram of the electrolysis bath of syringic acid shows that the anodic peak potential of 3-O-methylgallic acid was lower (E_pa=128mV) than that of SA (E_pa=320mV). And the strongest antiradical activity was detected when the 3-O-methylgallic acid concentration was higher".The electrochemical oxidation using PbO₂ anode is a rapid, simple and efficient method tool for a conversion of SA into 3-O-methylgallic acid, a potent antioxidant derivativeThe electrochemical process consists in a simple transformation of the syringic acid into 3-O-methylgallic acid having a better antioxidant capacity. This result has been justified by cyclic voltametry which shows that anodic peak of 3-O-methylgallic acid is reversible. Furthermore, its potential is lower than that of the irreversible anodic peak of syringic acid to 3-O-methylgallic acid.► Production of high-added-value compound through electrochemical conversion ► Cyclic voltammetry is used for evaluating the antioxidant capacity of phenolic derivatives. ► Ortho-hydroxyl substitution is an important determinant of antioxidant activity.

Keywords: Syringic acid; 3-O-methylgallic acid; Anodic oxidation; Cyclic voltammetry; Antioxidant activity

Prediction and experimental validation of a putative non-consensus binding site for transcription factor STAT3 in serum amyloid A gene promoter by Prabha Tiwari; Lokesh P. Tripathi; Teppei Nishikawa-Matsumura; Shandar Ahmad; Soken-Nakazawa J. Song; Tomoyasu Isobe; Kenji Mizuguchi; Kazuyuki Yoshizaki (pp. 3650-3655).

We previously demonstrated that though the human SAA1 gene shows no typical STAT3 response element (STAT3-RE) in its promoter region, STAT3 and the nuclear factor (NF-κB) p65 first form a complex following interleukin IL-1 and IL-6 (IL-1+6) stimulation, after which STAT3 interacts with a region downstream of the NF-κB RE in the SAA1 promoter. In this study, we employed a computational approach based on indirect read outs of protein–DNA contacts to identify a set of candidates for non-consensus STAT3 transcription factor binding sites (TFBSs). The binding of STAT3 to one of the predicted non-consensus TFBSs was experimentally confirmed through a dual luciferase assay and DNA affinity chromatography. The present study defines a novel STAT3 non-consensus TFBS at nt −75/−66 downstream of the NF-κB RE in the SAA1 promoter region that is required for NF-κB p65 and STAT3 to activate SAA1 transcription in human HepG2 liver cells. Our analysis builds upon the current understanding of STAT3 function, suggesting a wider array of mechanisms of STAT3 function in inflammatory response, and provides a useful framework for investigating novel TF-target associations with potential therapeutic implications.► STAT3 induces serum amyloid A1 ( SAA1) gene expression via the IL-6 transduction. ► No recognizable STAT3-RE is characterized near the NF-κB RE in the SAA gene promoters. ► A computational method was used to identify non-consensus STAT3-REs in SAA1 promoter. ► The candidates were evaluated with dual-luciferase reporter and DNA binding assays. ► By recognizing a wider array of REs, STAT3 may regulate a larger number of targets.

Keywords: IL-6; Indirect readout; DNA structure-based energy; SAA1; STAT3; Transcription factor binding site

Involvement of ASK1–p38 pathway in the pathogenesis of diabetes triggered by pancreatic ß cell exhaustion by Kiyoshi Yamaguchi; Kohsuke Takeda; Hisae Kadowaki; Ikumi Ueda; Yoshio Namba; Yasuyoshi Ouchi; Hideki Nishitoh; Hidenori Ichijo (pp. 3656-3663).

Diabetes mellitus is characterized by high blood glucose levels. Pancreatic ß cell death contributes to type 1 and type 2 diabetes. Akita mice, which harbor a human permanent neonatal diabetes-linked mutation (Cys96Tyr) in the insulin gene, are well established as an animal model of diabetes caused by pancreatic ß cell exhaustion. Mutant Insulin 2 protein (Ins2^C96Y) induces endoplasmic reticulum (ER) stress and pancreatic ß cell death in Akita mice, although the molecular mechanism of Ins^C96Y-induced cell death remains unclear.We investigate the mechanisms of Ins2^C96Y-induced pancreatic ß cell death in vitro and in vivo, using p38 inhibitor (SB203580), MIN6 cell (pancreatic ß cell line), Akita mice and apoptosis signal-regulating kinase 1 (ASK1) knockout mice.The expression of Ins^C96Y activated the ASK1–p38 pathway. Deletion of ASK1 mitigated Ins^C96Y-induced pancreatic ß cell death and delayed the onset of diabetes in Akita mice. Moreover, p38 inhibitor suppressed Ins^C96Y-induced MIN6 cell death.These findings suggest that ER stress-induced ASK1–p38 activation, which is triggered by the accumulation of Ins^C96Y, plays an important role in the pathogenesis of diabetes.Pancreatic ß cell death caused by insulin overload appears to be involved in the pathogenesis of type 1 and type 2 diabetes. Inhibition of the ASK1–p38 pathway may be an effective therapy for various types of diabetes.► The expression of Ins^C96Y activates the ASK1–p38 pathway in ß cell. ► Deletion of ASK1 mitigates Ins^C96Y-induced ß cell death. ► Inhibition of p38 suppresses Ins^C96Y-induced ß cell death. ► Deletion of ASK1 delays the onset of diabetes in Akita mice.

Keywords: Abbreviations; Ins2; Insulin 2 protein; ER; endoplasmic reticulum; ASK1; apoptosis signal-regulating kinase 1; UPR; unfolded protein response; PERK; protein kinase RNA-like ER kinase; ATF6; activating transcription factor 6; IRE1; inositol-requiring enzyme 1; ERAD; ER-associated degradation; WRS; Wolcott-Rallison syndrome; MAP; mitogen-activated protein; CHOP; CCAAT-enhancer-binding protein homologous protein; DMEM; Dulbecco's modified Eagle's medium; FBS; fetal bovine serum; MTT; 3′-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; FITC; fluorescein isothiocyanate; TUNEL; terminal deoxynucleotidyl transferase-dUTP nick end labeling; MEF; mouse embryonic fibroblasts; CA6; carbonic anhydrase VIDiabetes; Akita mouse; ER stress; Apoptosis; ASK1; p38

A high-throughput assay for the detection of Tyr-phosphorylated proteins in urine of bladder cancer patients by A. Khadjavi; A. Notarpietro; F. Mannu; A. Pantaleo; E. Ferru; P. Destefanis; D. Fontana; F. Turrini (pp. 3664-3669).

Bladder cancer has the peculiarity of shedding neoplastic cells and their components in urine representing a valuable opportunity to detect diagnostic markers. Using a semi-quantitative method we previously demonstrated that the levels of Tyr-phosphorylated proteins (TPPs) are highly increased in bladder cancer tissues and that soluble TPPs can also be detected in patient's urine samples. Although the preliminary evaluation showed very promising specificity and sensitivity, insufficient accuracy and very low throughput of the method halted the diagnostic evaluation of the new marker. To overcome this problem we developed a quantitative methodology with high sensitivity and accuracy to measure TPPs in urine.The Immobilized Metal Affinity Chromatography (IMAC) was miniaturized in a 96 well format. Luminescence, visible and infrared fluorescence antibody-based detection methods were comparatively evaluated.Due to their low abundance we evidenced that both phosphoprotein enrichment step and very sensitive detection methods are required to detect TPPs in urine samples. To pursue high throughput, reproducibility and cost containment, which are required for bladder cancer screening programs, we coupled the pre-analytical IMAC procedure with high sensitive detection phases (infrared fluorescence or chemiluminescence) in an automated platform.A high throughput method for measuring with high sensitivity TPP levels in urine samples is now available for large clinical trial for the establishment of the diagnostic and predictive power of TPPs as bladder cancer marker.The new assay represents the first quantitative and high throughput method for the measurement of TPPs in urine.•Urinary TPPs represent a new bladder cancer marker.•Development of a quantitative method for TPP evaluation in urine•High throughput method for large bladder cancer marker screening programs

Keywords: Abbreviations; TPPs; Tyr-phosphorylated proteins; IMAC; immobilized metal affinity chromatography; SCC; standard column chromatography; MIP; miniaturized IMAC procedure; AU; arbitrary units; SU; standard units; AUC; area under the ROC curvePhosphotyrosine proteins; Urinary bladder cancer marker; High-throughput assay

Natural products: A continuing source of novel drug leads by Gordon M. Cragg; David J. Newman (pp. 3670-3695).

Nature has been a source of medicinal products for millennia, with many useful drugs developed from plant sources. Following discovery of the penicillins, drug discovery from microbial sources occurred and diving techniques in the 1970s opened the seas. Combinatorial chemistry (late 1980s), shifted the focus of drug discovery efforts from Nature to the laboratory bench.This review traces natural products drug discovery, outlining important drugs from natural sources that revolutionized treatment of serious diseases. It is clear Nature will continue to be a major source of new structural leads, and effective drug development depends on multidisciplinary collaborations.The explosion of genetic information led not only to novel screens, but the genetic techniques permitted the implementation of combinatorial biosynthetic technology and genome mining. The knowledge gained has allowed unknown molecules to be identified. These novel bioactive structures can be optimized by using combinatorial chemistry generating new drug candidates for many diseases.The advent of genetic techniques that permitted the isolation / expression of biosynthetic cassettes from microbes may well be the new frontier for natural products lead discovery. It is now apparent that biodiversity may be much greater in those organisms. The numbers of potential species involved in the microbial world are many orders of magnitude greater than those of plants and multi-celled animals. Coupling these numbers to the number of currently unexpressed biosynthetic clusters now identified (>10 per species) the potential of microbial diversity remains essentially untapped.► We have given a history of natural products as drugs. ► We have discussed the reasons for the decreased interest in NPs in the Pharmaceutical industry. ► We have shown that the use of genomic techniques has allowed the recognition of new microbial sources of structures ► We demonstrate that the new frontier will be the interplay of genomics, chemistry and controlled biosynthesis. ► We demonstrate that biodiversity is in the microbial realm.

Keywords: Microbial diversity; synthesis; genomics; natural product drugs

Small molecule agonists of integrin CD11b/CD18 do not induce global conformational changes and are significantly better than activating antibodies in reducing vascular injury by Mohd Hafeez Faridi; Mehmet M. Altintas; Camilo Gomez; Juan Camilo Duque; Roberto I. Vazquez-Padron; Vineet Gupta (pp. 3696-3710).

CD11b/CD18 is a key adhesion receptor that mediates leukocyte adhesion, migration and immune functions. We recently identified novel compounds, leukadherins, that allosterically enhance CD11b/CD18-dependent cell adhesion and reduce inflammation in vivo, suggesting integrin activation to be a novel mechanism of action for the development of anti-inflammatory therapeutics. Since a number of well-characterized anti-CD11b/CD18 activating antibodies are currently available, we wondered if such biological agonists could also become therapeutic leads following this mechanism of action.We compared the two types of agonists using in vitro cell adhesion and wound-healing assays and using animal model systems. We also studied effects of the two types of agonists on outside-in signaling in treated cells.Both types of agonists similarly enhanced integrin-mediated cell adhesion and decreased cell migration. However, unlike leukadherins, the activating antibodies produced significant CD11b/CD18 macro clustering and induced phosphorylation of key proteins involved in outside-in signaling. Studies using conformation reporter antibodies showed that leukadherins did not induce global conformational changes in CD11b/CD18 explaining the reason behind their lack of ligand-mimetic outside-in signaling. In vivo, leukadherins reduced vascular injury in a dose-dependent fashion, but, surprisingly, the anti-CD11b activating antibody ED7 was ineffective.Our results suggest that small molecule allosteric agonists of CD11b/CD18 have clear advantages over the biologic activating antibodies and provide a mechanistic basis for the difference.CD11b/CD18 activation represents a novel strategy for reducing inflammatory injury. Our study establishes small molecule leukadherins as preferred agonists over activating antibodies for future development as novel anti-inflammatory therapeutics.Display Omitted► CD11b/CD18 regulates leukocyte adhesion, migration and inflammatory functions. ► Increasing adhesion by activating CD11b/CD18 is a novel therapeutic approach. ► We compared two types of CD11b/CD18 agonists using in vitro and in vivo assays. ► Small molecules, leukadherins, induced no outside-in signaling. ► Leukadherins were superior to activating mAbs in reducing inflammation.

Keywords: Cell adhesion; Leukocyte; CD11b/CD18; Drug discovery; Anti-adhesion therapy; Inflammation

Crystal structure of a Bombyx mori sigma-class glutathione transferase exhibiting prostaglandin E synthase activity by Kohji Yamamoto; Akifumi Higashiura; Mamoru Suzuki; Kosuke Aritake; Yoshihiro Urade; Nobuko Uodome; Atsushi Nakagawa (pp. 3711-3718).

Glutathione transferases (GSTs) are members of a major family of detoxification enzymes. Here, we report the crystal structure of a sigma-class GST of Bombyx mori, bmGSTS1, to gain insight into the mechanism catalysis.The structure of bmGSTS1 and its complex with glutathione were determined at resolutions of 1.9Å and 1.7Å by synchrotron radiation and the molecular replacement method.The three-dimensional structure of bmGSTS1 shows that it exists as a dimer and is similar in structure to other GSTs with respect to its secondary and tertiary structures. Although striking similarities to the structure of prostaglandin D synthase were also detected, we were surprised to find that bmGSTS1 can convert prostaglandin H₂ into its E₂ form. Comparison of bmGSTS1 with its glutathione complex showed that bound glutathione was localized to the glutathione-binding site (G-site). Site-directed mutagenesis of bmGSTS1 mutants indicated that amino acid residues Tyr8, Leu14, Trp39, Lys43, Gln50, Met51, Gln63, and Ser64 in the G-site contribute to catalytic activity.We determined the tertiary structure of bmGSTS1 exhibiting prostaglandin E synthase activity.These results are, to our knowledge, the first report of a prostaglandin synthase activity in insects.•We determined the crystal structure of sigma-class GST from Bombyx mori (bmGSTS1).•Secondary and tertiary structures are highly conserved in bmGSTS1.•bmGSTS1 can convert prostaglandin H₂ into its E₂ form.•Comparison of bmGSTS1 with its glutathione complex showed that G-site is important for glutathione binding.•These results are, to our knowledge, the first report of a prostaglandin synthase activity in insects.

Keywords: Abbreviations; GSH; glutathione; GST; glutathione transferase; GSTS; sigma-class GST; PG; prostaglandin; PGDS; prostaglandin D synthase; PGES; prostaglandin E synthase; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresisCrystal structure; Glutathione; Lepidoptera; Prostaglandin; Prostaglandin synthase

TMEM126A is a mitochondrial located mRNA (MLR) protein of the mitochondrial inner membrane by Sylvain Hanein; Mathilde Garcia; Lucas Fares-Taie; Valérie Serre; Yves De Keyzer; Thierry Delaveau; Isabelle Perrault; Nathalie Delphin; Sylvie Gerber; Alain Schmitt; Jean-Marc Masse; Arnold Munnich; Josseline Kaplan; Frédéric Devaux; Jean-Michel Rozet (pp. 3719-3733).

Hereditary optic neuropathies (HONs) are a heterogeneous group of disorders that affect retinal ganglion cells (RGCs) and axons that form the optic nerve. Leber's Hereditary Optic Neuropathy and the autosomal dominant optic atrophy related to OPA1 mutations are the most common forms. Nonsyndromic autosomal recessive optic neuropathies are rare and their existence has been long debated. We recently identified the first gene responsible for these conditions, TMEM126A. This gene is highly expressed in retinal cellular compartments enriched in mitochondria and supposed to encode a mitochondrial transmembrane protein of unknown function.A specific polyclonal antibody targeting the TMEM126A protein has been generated. Quantitative fluorescent in situ hybridization, cellular fractionation, mitochondrial membrane association study, mitochondrial sub compartmentalization analysis by both proteolysis assays and transmission electron microscopy, and expression analysis of truncated TMEM126A constructs by immunofluorescence confocal microscopy were carried out. TMEM126A mRNAs are strongly enriched in the vicinity of mitochondria and encode an inner mitochondrial membrane associated cristae protein. Moreover, the second transmembrane domain of TMEM126A is required for its mitochondrial localization. TMEM126A is a mitochondrial located mRNA (MLR) that may be translated in the mitochondrial surface and the protein is subsequently imported to the inner membrane. These data constitute the first step toward a better understanding of the mechanism of action of TMEM126A in RGCs and support the importance of mitochondrial dysfunction in the pathogenesis of HON.Local translation of nuclearly encoded mitochondrial mRNAs might be a mechanism for rapid onsite supply of mitochondrial membrane proteins.• TMEM126A mRNAs are found close to mitochondria suggesting a local translation.•TMEM126A is anchored to the mitochondrial inner membrane close to cristae.•TMEM126A has 4 transmembrane domains; both soluble N- and C-ends face the matrix.•The second transmembrane domain of TMEM126A is involved in mitochondrial targeting.•Co-translation import may be a way to satisfy a rapid onsite supply of TMEM126A.

Keywords: Abbreviations; HON; hereditary optic neuropathy; RGC; retinal ganglion cell; mtDNA; mitochondrial DNA; LHON; Leber's Hereditary Optic Neuropathy; OPA; optic atrophy; TMEM126A; transmembrane protein 126A; IMS; intermembrane space; OM; outer membrane; IM; inner membrane; SAPS; Statistical Analysis of Protein Sequences; TMEM126B; transmembrane protein 126B; DUF; domain of unknown function; MLR; mitochondrial-located mRNA; AA; amino acid; FISH; fluorescent in situ hybridization; UQCRC1; ubiquinol-cytochrome c reductase core protein I; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; RNA12S; mitochondrially encoded 12S RNA; mt-rRNA; mitochondrial ribosomal RNA; DPBS; Phosphate Buffered Saline; MB; mitochondrial buffer; MAPK1; mitogen-activated protein kinase 1; MAPK3; mitogen-activated protein kinase 3; MT-CO2; mt-DNA encoded cytochrome c oxidase II; NDUFA9; NADH dehydrogenase ubiquinone 1 alpha subcomplex 9 39; kDa; VDAC1; voltage-dependent anion channel 1; CYCS; cytochrome c somatic; NDUFB6; NADH dehydrogenase ubiquinone 1 beta subcomplex 6 17; kDa; PDHA1; pyruvate dehydrogenase lipoamide alpha 1; BCL2; B-cell CLL/lymphoma 2; ATP5B; ATP synthase H; + transporting, mitochondrial F1 complex beta polypeptide; SDHA; succinate dehydrogenase complex subunit A flavoprotein; TEM; transmission electron microscopy; qFISH; quantitative fluorescent in situ hybridization; MTS; mitochondrial targeting leader sequence; TM; transmembrane; Puf3p; Pumilio-Fem-3-binding factor (FBF) (Puf) RNA binding protein; UTR; untranslated region; PUM1; pumilio protein 1; PUM2; pumilio protein 2; miRNA; micro RNA; CDS; coding sequence; G3BP1; GTPase activating protein (SH3 domain) binding protein 1; YB-1; Y box binding protein 1TMEM126A; Optic neuropathy; Mitochondrial inner membrane; Cristae; Mitochondria-localized mRNA; MLR

Biochemical characterization of C4 protein of Cotton Leaf Curl Kokhran Virus- Dabawali by Debojit Guha; C.G. Poornima Priyadarshini; Arunima Purakayastha; R. Thippeswamy; M. Lakshmikanth; H.S. Savithri (pp. 3734-3744).

Cotton leaf curl Kokhran Virus-Dabawali (CLCuKV-Dab) is a monopartite begomovirus encoding two proteins V1 and V2 in the virion sense and four proteins C1, C2, C3 and C4 in the complementary sense. The C4 protein of monopartite begomoviruses has been implicated to play a role in symptom determination and virus movement. The present work aims at the biochemical characterization of this protein.The C4 protein of CLCuKV-Dab was purified in fusion with GST and tested for the ability to hydrolyze ATP and other phosphate containing compounds. ATPase activity was assayed by using radiolabeled γ-[32P]-ATP and separating the product of reaction by thin layer chromatography. The hydrolysis of other compounds was monitored by the formation of a blue colored phosphomolybdate complex which was estimated by measuring the absorbance at 655nm.The purified GST-C4 protein exhibited metal ion dependent ATPase and inorganic pyrophosphatase activities. Deletion of a sequence resembling the catalytic motif present in phosphotyrosine phosphatases resulted in 70% reduction in both the activities. Mutational analysis suggested arginine 13 to be catalytically important for the ATPase and cysteine 8 for the pyrophosphatase activity of GST-C4. Interaction of V2 with GST-C4 resulted in an increase in both the enzymatic activities of GST-C4.The residues important for the enzymatic activities of GST-C4 are present in a motif different from the classical Walker motifs and the non-classical ATP binding motifs reported so far.The C4 protein of CLCuKV-Dab, a putative natively unfolded protein, exhibits enzymatic activities.•C4 of Cotton leaf curl Kokhran virus- Dabawali affinity purified in fusion with GST.•GST-C4 exhibited metal ion dependent ATPase and inorganic pyrophosphatase activities.•Catalytic motif resembling phosphotyrosine phosphatases found at the N terminus of C4.•Arginine 13 was found to be important for ATPase activity by mutational analysis.•Cysteine 8 was found to be important for pyrophosphatase activity by mutational analysis.

Keywords: Geminivirus; Natively unfolded; ATPase; Phosphotyrosine phosphatase; Inorganic pyrophosphatase; Protein–protein interaction

Kineococcus radiotolerans Dps forms a heteronuclear Mn–Fe ferroxidase center that may explain the Mn-dependent protection against oxidative stress by Matteo Ardini; Annarita Fiorillo; Maria Fittipaldi; Simonetta Stefanini; Dante Gatteschi; Andrea Ilari; Emilia Chiancone (pp. 3745-3755).

The ferroxidase center of DNA-binding protein from starved cells (Dps) is a major player in the iron oxidation/detoxification process that leads to a decreased reactive oxygen species production. The possible Mn(II) participation in this process has been studied in Dps from Kineococcus radiotolerans, a radiation-resistant bacterium with a high cytosolic Mn/Fe ratio and a high capacity to survive ionizing and stress conditions.The X-ray structure of recombinant K. radiotolerans Dps loaded with Mn(II) has been solved at 2.0Å resolution. Mn(II) binding to K. radiotolerans Dps and its effect on Fe(II) oxidation have been characterized in spectroscopic measurements.In K. radiotolerans Dps, the Fe–Fe ferroxidase center can have a Mn–Fe composition. Mn(II) binds only at the high affinity, so-called A site, whereas Fe(II) binds also at the low affinity, so-called B site. The Mn–Fe and Fe–Fe centers behave distinctly upon iron oxidation by O₂. A site-bound Mn(II) or Fe(II) plays a catalytic role, while B site-bound Fe(II) behaves like a substrate and can be replaced by another Fe(II) after oxidation. When H₂O₂^{is the Fe(II) oxidant, single electrons are transferred to aromatic residues near the ferroxidase center and give rise to intra-protein radicals thereby limiting OH} release in solution. The presence of the Mn–Fe center results in significant differences in the development of such intra-protein radicals.Mn(II) bound at the Dps ferroxidase center A site undergoes redox cycling provided the B site contains Fe.The results provide a likely molecular mechanism for the protective role of Mn(II) under oxidative stress conditions as it participates in redox cycling in the hetero-binuclear ferroxidase center.► Mn(II) binds to the A site of the binuclear K. radiotolerans^{Dps ferroxidase center. ► A heterometallic ferroxidase center is formed when Fe(II) binds to the B site. ► During the catalyzed Fe(II) oxidation Mn(II) participates in redox cycling. ► Both the Mn/Fe and the canonic Fe/Fe ferroxidase centers limit OH} release in solution. ► Mn(II) participation in redox cycling of KrDps may explain the Mn-dependent protection against oxidative stress.

Keywords: Kineococcus radiotolerans; DNA-binding protein from starved cell structure; Hetero-nuclear manganese–iron center; Manganese(II) redox cycling; Oxidative stress protection

MicroRNA-33a functions as a bone metastasis suppressor in lung cancer by targeting parathyroid hormone related protein by Po-Lin Kuo; Szi-Hui Liao; Jen-Yu Hung; Ming-Shyan Huang; Ya-Ling Hsu (pp. 3756-3766).

Bone is a common site of metastasis for lung cancer, and is associated with significant morbidity and a dismal prognosis. MicroRNAs (miRNAs) are increasingly implicated in regulating the progression of malignancies.The efficacy of miR-33a or anti-miR-33a plasmid was assessed by Real-time PCR. Luciferase assays were using One-Glo Luciferase Assay System. Measurement of secreted factors was determined by ELISA kit.We have found that miR-33a, which is downregulated in lung cancer cells, directly targets PTHrP (parathyroid hormone-related protein), a potent stimulator of osteoclastic bone resorption, leading to decreased osteolytic bone metastasis. We also found that miR-33a levels are inversely correlated with PTHrP expression between human normal bronchial cell line and lung cancer cell lines. The reintroduction of miR-33a reduces the stimulatory effect of A549 on the production of osteoclastogenesis activator RANKL (receptor activator of nuclear factor kappa-B ligand) and M-CSF (macrophage colony-stimulating factor) on osteoblasts, while the expression of PTHrP is decreased in A549 cells. miR-33a overexpression also reduces the inhibitory activity of A549 on the production of OPG (osteoprotegerin), an osteoclastogenesis inhibitor. In addition, miR-33a-mediated PTHrP downregulation results in decreased IL-8 secretion in A549, which contributes to decreased lung cancer-mediated osteoclast differentiation and bone resorption.These findings have led us to conclude that miR-33a may be a potent tumor suppressor, which inhibits direct and indirect osteoclastogenesis through repression of PTHrP.miR-33a may even predict a poor prognosis for lung cancer patients.•miR-33a is downregulated in lung cancer cell lines.•miR-33a regulates the expression of PTHrP.•Overexpression of miR-33a decreases the induction of A549 on the production of RANKL and M-CSF in osteoblasts.•Ectopic expression of miR-33a decreases the stimulatory effect of A549 on osteoclastogenesis.•Knockdown of miR-33a triggers the inductive effect of BEAS-2B on the production of RANKL and M-CSF in osteoblasts.

Keywords: Abbreviations; PTHrP; parathyroid hormone-related protein; RANKL; receptor activator of nuclear factor kappa-B ligand; M-CSF; macrophage colony-stimulating factor; OPG; osteoprotegerin; Real-time qRT-PCR; Real time quantitative reverse transcription PCRLung cancer; mir-33a; PTHrP; OPG; Osteoclastogenesis

Design, structural and functional characterization of a Temporin-1b analog active against Gram-negative bacteria by Concetta Avitabile; Fortuna Netti; Giuseppina Orefice; Maddalena Palmieri; Nunzia Nocerino; Gaetano Malgieri; Luca D. D'Andrea; Rosanna Capparelli; Roberto Fattorusso; Alessandra Romanelli (pp. 3767-3775).

Temporins are small antimicrobial peptides secreted by the Rana temporaria showing mainly activity against Gram-positive bacteria. However, different members of the temporin family, such as Temporin B, act in synergy also against Gram-negative bacteria. With the aim to develop a peptide with a wide spectrum of antimicrobial activity we designed and analyzed a series of Temporin B analogs.Peptides were initially obtained by Ala scanning on Temporin B sequence; antimicrobial activity tests allowed to identify the TB_G6A sequence, which was further optimized by increasing the peptide positive charge (TB_KKG6A). Interactions of this active peptide with the LPS of E. coli were investigated by CD, fluorescence and NMR.TB_KKG6A is active against Gram-positive and Gram-negative bacteria at low concentrations. The peptide strongly interacts with the LPS of Gram-negative bacteria and folds upon interaction into a kinked helix.Our results show that it is possible to widen the activity spectrum of an antimicrobial peptide by subtle changes of the primary structure. TB_KKG6A, having a simple composition, a broad spectrum of antimicrobial activity and a very low hemolytic activity, is a promising candidate for the design of novel antimicrobial peptides.The activity of antimicrobial peptides is strongly related to the ability of the peptide to interact and break the bacterial membrane. Our studies on TB_KKG6A indicate that efficient interactions with LPS can be achieved when the peptide is not perfectly amphipathic, since this feature seems to help the toroidal pore formation process.Display Omitted► A new Temporin B analog, TB_KKG6A, was designed. ► TB_KKG6A is active against Gram-positive and Gram-negative bacteria. ► The peptide strongly interacts with the LPS of E. coli. ► The peptide folds into a kinked helix upon interaction with LPS. ► The peptide shows very low hemolytic activity.

Keywords: Temporin; Structure; LPS; Antimicrobial activity; Binding

Inhibition of TNF-α, and NF-κB and JNK pathways accounts for the prophylactic action of the natural phenolic, allylpyrocatechol against indomethacin gastropathy by Sudhir K. Yadav; Biplab Adhikary; Sandip K. Bandyopadhyay; Subrata Chattopadhyay (pp. 3776-3786).

The gastro-intestinal disorders, induced by the NSAIDs including indomethacin (IND) remain unresolved medical problems. Herein, we disclose allylpyrocatechol (APC) as a potential agent against IND-gastropathy and rationalize its action mechanistically.Mice were pre-treated with APC for 1h followed by IND (18mgkg−¹) administration, and the ulcer-prevention capacity of APC was evaluated on the 3rd day by histology. Its effect on the inflammatory (MPO, cytokines, adhesion molecules), ulcer-healing (COX, prostaglandins, growth factors and their receptors) and signaling parameters (NF-κB and MAPKs) were assessed by immunoblots/mRNA, and ELISA at the time points of their maximal changes due to IND administration.IND induced oxidative stress, triggering mucosal TNF-α that activated NF-κB and JNK MAPK signaling in mice. These increased the pro-inflammatory biochemical parameters, but reduced the healing factors. APC reversed all the adverse effects to prevent gastric ulceration. APC (5mgkg−¹), trolox (50mgkg−¹) and NAC (250mgkg−¹) showed similar protection that was better than that by misoprostol (5μgkg−¹) and omeprazole (3mgkg−¹).The anti-ulcer effect of APC can be primarily attributed to its antioxidant action that helped in controlling various inflammatory parameters and augmenting angiogenesis.Given that APC is an effective, non-toxic antioxidant with appreciable natural abundance, further evaluation of its pharmacokinetics and dynamics would help in promoting it as a new anti-inflammatory agent.•Rational formulation of allylpyrocatechol (APC) as a potential anti-ulcer agent.•Identification of TNF-α as the potential target of APC.•Establishment of molecular mechanism of action of APC.•Suggestion of different mechanisms of action of misoprostol and omeprazole.

Keywords: Gastric ulcer; NSAID; Indomethacin; Inflammatory modulator; Angiogenesis

Antibacterial performance of nanoscaled visible-light responsive platinum-containing titania photocatalyst in vitro and in vivo by Yao-Hsuan Tseng; Der-Shan Sun; Wen-Shiang Wu; Hao Chan; Ming-Syuan Syue; Han-Chen Ho; Hsin-Hou Chang (pp. 3787-3795).

Traditional antibacterial photocatalysts are primarily induced by ultraviolet light to elicit antibacterial reactive oxygen species. New generation visible-light responsive photocatalysts were discovered, offering greater opportunity to use photocatalysts as disinfectants in our living environment. Recently, we found that visible-light responsive platinum-containing titania (TiO₂–Pt) exerted high performance antibacterial property against soil-borne pathogens even in soil highly contaminated water. However, its physical and photocatalytic properties, and the application in vivo have not been well-characterized.Transmission electron microscopy, energy dispersive spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction, ultraviolet–visible absorption spectrum and the removal rate of nitrogen oxides were therefore analyzed. The antibacterial performance under in vitro and in vivo conditions was evaluated.The apparent quantum efficiency for visible light illuminated TiO₂–Pt is relatively higher than several other titania photocatalysts. The killing effect achieved approximately 2 log reductions of pathogenic bacteria in vitro. Illumination of injected TiO₂–Pt successfully ameliorated the subcutaneous infection in mice.This is the first demonstration of in vivo antibacterial use of TiO₂–Pt nanoparticles. When compared to nanoparticles of some other visible-light responsive photocatalysts, TiO₂–Pt nanoparticles induced less adverse effects such as exacerbated platelet clearance and hepatic cytotoxicity in vivo.These findings suggest that the TiO₂–Pt may have potential application on the development of an antibacterial material in both in vitro and in vivo settings.The TiO₂–Pt nanoparticle is a visible-light responsive photocatalyst, which has superior apparent quantum efficiency and antibacterial activity, as compared with several commercially available and laboratory prepared photocatalysts. Antibacterial experiments in vitro indicated that the high performance of TiO₂–Pt on the attenuation of pathogenic bacteria is contributed by two major effects: the killing and the reduction of resistance against phagocytes of the bacteria. A mouse model further demonstrated that skin penetrative visible light can trigger TiO₂–Pt-mediated photocatalysis and thus ameliorate the subcutaneous Staphylococcus aureus infection in vivo. These findings suggest that TiO₂–Pt may have potential application on the development of a high performance photocatalyst to be used in both in vitro and in vivo settings.Display Omitted•Superior apparent quantum efficiency of TiO₂–Pt than several titania photocatalysts•First demonstration of in vivo antibacterial use of TiO₂–Pt nanoparticles (NPs)•Less adverse effect of TiO₂–Pt to induce platelet and hepatic cytotoxicity in vivo

Keywords: TiO; ₂; Platinum-containing TiO; ₂; Visible light responsive photocatalyst

Antiadipogenic effect of carnosic acid, a natural compound present in Rosmarinus officinalis, is exerted through the C/EBPs and PPARγ pathways at the onset of the differentiation program by Mauro Gaya; Victoria Repetto; Judith Toneatto; Claudia Anesini; Graciela Piwien-Pilipuk; Silvia Moreno (pp. 3796-3806).

Obesity is a serious health problem all over the world, and inhibition of adipogenesis constitutes one of the therapeutic strategies for its treatment. Carnosic acid (CA), the main bioactive compound of Rosmarinus officinalis extract, inhibits 3T3-L1 preadipocytes differentiation. However, very little is known about the molecular mechanism responsible for its antiadipogenic effect.We evaluated the effect of CA on the differentiation of 3T3-L1 preadipocytes analyzing the process of mitotic clonal expansion, the level of adipogenic markers, and the subcellular distribution of C/EBPβ.CA treatment only during the first day of 3T3-L1 differentiation process was enough to inhibit adipogenesis. This inhibition was accompanied by a blockade of mitotic clonal expansion. CA did not interfere with C/EBPβ and C/EBPδ mRNA levels but blocked PPARγ, and FABP4 expression. C/EBPβ has different forms known as LIP and LAP. CA induced an increase in the level of LIP within 24h of differentiation, leading to an increment in LIP/LAP ratio. Importantly, overexpression of LAP restored the capacity of 3T3-L1 preadipocytes to differentiate in the presence of CA. Finally, CA promoted subnuclear de-localization of C/EBPβ.CA exerts its anti-adipogenic effect in a multifactorial manner by interfering mitotic clonal expansion, altering the ratio of the different C/EBPβ forms, inducing the loss of C/EBPβ proper subnuclear distribution, and blocking the expression of C/EBPα and PPARγ.Understanding the molecular mechanism by which CA blocks adipogenesis is relevant because CA could be new a food additive beneficial for the prevention and/or treatment of obesity.•The antiadipogenic mechanism of carnosic acid is multifactorial.•Carnosic acid inhibits mitotic clonal expansion of 3T3-L1 cells.•The expression of C/EBPα, PPARγ and FABP2 are inhibited by carnosic acid.•Carnosic acid alters the ratio of C/EBPβ forms and its subnuclear distribution.•Carnosic acid is a potential agent for the treatment/prevention of obesity.

Keywords: Abbreviations; CA; carnosic acid; RE; Rosmarinus officinalis; extract; COH; carnosol; RA; rosmarinic acid; C/EBP; CCAAT/Enhancer Binding Protein; PPARγ; peroxisome proliferator-activated receptor γ; IBMX; isobutyl-3-methylxanthine; MTS; 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; FBS; fetal bovine serum; DAPI; 6-diamino-2-phenylindole; IIF; indirect immunofluorescenceAdipogenesis; Obesity; Carnosic acid; Rosemary; C/EBPs; PPARγ

Superb resolution and contrast of transmission electron microscopy images of unstained biological samples on graphene-coated grids by Jaekyun Jeon; Michael S. Lodge; Ben D. Dawson; Masa Ishigami; Frank Shewmaker; Bo Chen (pp. 3807-3815).

In standard transmission electron microscopy (TEM), biological samples are supported on carbon films of nanometer thickness. Due to the similar electron scattering of protein samples and graphite supports, high quality images with structural details are obtained primarily by staining with heavy metals.Single-layered graphene is used to support the protein self-assemblies of different molecular weights for qualitative and quantitative characterizations.We show unprecedented high resolution and contrast images of unstained samples on graphene on a low-end TEM. We show for the first time that the resolution and contrast of TEM images of unstained biological samples with high packing density in their native states supported on graphene can be comparable or superior to uranyl acetate-stained TEM images.Our results demonstrate a novel technique for TEM structural characterization to circumvent the potential artifacts caused by staining agents without sacrificing image resolution or contrast, and eliminate the need for toxic metals. Moreover, this technique better preserves sample integrity for quantitative characterization by dark-field imaging with reduced beam damage.This technique can be an effective alternative for bright-field qualitative characterization of biological samples with high packing density and those not amenable to the standard negative staining technique, in addition to providing high quality dark-field unstained images at reduced radiation damage to determine quantitative structural information of biological samples.Display Omitted•Unstained protein aggregation samples supported on graphene on a conventional TEM•Deposited in their native buffer conditions with image resolution and contrast comparable to negatively stained samples•Ideal for samples sensitive to buffer condition and avoid potential artifacts associated with staining•Reduced beam damage to samples and eliminates hazardous staining agents

Keywords: Prion; Electron microscopy; Molecular structure; Mass per length

Secreted glyceraldehye-3-phosphate dehydrogenase is a multifunctional autocrine transferrin receptor for cellular iron acquisition by Navdeep Sheokand; Santosh Kumar; Himanshu Malhotra; Vikas Tillu; Chaaya Iyengar Raje; Manoj Raje (pp. 3816-3827).

The long held view is that mammalian cells obtain transferrin (Tf) bound iron utilizing specialized membrane anchored receptors. Here we report that, during increased iron demand, cells secrete the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which enhances cellular uptake of Tf and iron.These observations could be mimicked by utilizing purified GAPDH injected into mice as well as when supplemented in culture medium of model cell lines and primary cell types that play a key role in iron metabolism. Transferrin and iron delivery was evaluated by biochemical, biophysical and imaging based assays.This mode of iron uptake is a saturable, energy dependent pathway, utilizing raft as well as non-raft domains of the cell membrane and also involves the membrane protein CD87 (uPAR). Tf internalized by this mode is also catabolized.Our research demonstrates that, even in cell types that express the known surface receptor based mechanism for transferrin uptake, more transferrin is delivered by this route which represents a hidden dimension of iron homeostasis.Iron is an essential trace metal for practically all living organisms however its acquisition presents major challenges. The current paradigm is that living organisms have developed well orchestrated and evolved mechanisms involving iron carrier molecules and their specific receptors to regulate its absorption, transport, storage and mobilization. Our research uncovers a hidden and primitive pathway of bulk iron trafficking involving a secreted receptor that is a multifunctional glycolytic enzyme that has implications in pathological conditions such as infectious diseases and cancer.Display Omitted•Enhanced secretion of GAPDH upon iron depletion increases Tf–Fe delivery into cells.•Tf trafficking by this novel route is more efficient than via classical membrane bound receptors.•A long suspected but uncharacterized primitive pathway of cellular iron acquisition uncovered.•Implications for diseases involving high glycolysis and iron demand such as infectious diseases and cancer.

Keywords: Abbreviations; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; sGAPDH; secreted GAPDH; Tf; transferrin; TfR; transferrin receptor; DFO; desferrioxamine; CTX; cholera toxin; SFM; serum free mediumGlyceraldehyde-3-phosphate dehydrogenase; Transferrin receptor; Iron; Trafficking; Multifunctional protein; Urokinase plasminogen activator receptor

Effect of Lyso-phosphatidylcholine and Schnurri-3 on Osteogenic Transdifferentiation of Vascular Smooth Muscle Cells to Calcifying Vascular Cells in 3D Culture by Fernando Castro-Chavez; Kasey C. Vickers; Jae Sam Lee; Ching-Hsuan Tung; Joel D. Morrisett (pp. 3828-3834).

In vitro cell culture is a widely used technique for investigating a range of processes such as stem cell behavior, regenerative medicine, tissue engineering, and drug discovery. Conventional cell culture is performed in Petri dishes or flasks where cells typically attach to a flat glass or plastic surface as a cell monolayer. However, 2D cell monolayers do not provide a satisfactory representation of in vivo conditions. A 3D culture could be a much better system for representing the conditions that prevail in vivo.To simulate 3D conditions, vascular smooth muscle cells (VSMCs) were loaded with gold–polyvmer–iron oxide hydrogel, enabling levitation of the cells by using spatially varying magnetic fields. These magnetically levitated 3D cultures appeared as freely suspended, clustered cells which proliferated 3–4 times faster than cells in conventional 2D cultures. When the levitated cells were treated with 10nM lysophosphatidylcholine (LPC), for 3days, cell clusters exhibited translucent extensions/rods 60–80μm wide and 200–250μm long. When 0.5μg/μl Schnurri-3 was added to the culture containing LPC, these extensions were smaller or absent. When excited with 590–650nm light, these extensions emitted intrinsic fluorescence at >667nm. When the 3D cultures were treated with a fluorescent probe specific for calcium hydroxyapatite (FITC-HABP-19), the cell extensions/rods emitted intensely at 518nm, the λ_max for FITC emission. Pellets of cells treated with LPC were more enriched in calcium, phosphate, and glycosaminoglycans than cells treated with LPC and Schnurri-3.In 3D cultures, VSMCs grow more rapidly and form larger calcification clusters than cells in 2D cultures. Transdifferentiation of VSMC into calcifying vascular cells is enhanced by LPC and attenuated by Schnurri-3.The formation of calcified structures in 3D VSMC cultures suggests that similar structures may be formed in vivo.•LPC enhances vascular SMC transdifferentiation into calcifying vascular cells.•Schnurri-3 attenuates differentiation of vascular SMC into calcifying vascular cells.•Magnetic levitation can be used to create 3D cultures of vascular SMC.•Vascular SMC grows much faster in levitated 3D cultures than in 2D cultures.

Keywords: Abbreviations; CHA; calcium hydroxyapatite; LPC; lysophosphatidylcholine; VSMC; vascular smooth muscle cell; CVC; calcifying vascular cell; HABP-19; hydroxyapatite binding peptide containing 19 amino acids; CEA; carotid endarterectomy; Ca/P; calcium/phosphorus; FBS; fetal bovine serum; Shn3; Schnurri-3; PO; phosphate; GAG; glycosaminoglycan; FITC; fluoroscein isothiocyanate; Gla γE; gamma-carboxy glutamic acid; βA; beta alanine; MLM; magnetic levitationOsteocalcin; Magnetic levitation; Calcifying cell; Atherosclerosis; Arteriosclerosis; Gamma-carboxyglutamic acid

Oroxylin A sensitizes non-small cell lung cancer cells to anoikis via glucose-deprivation-like mechanisms: c-Src and hexokinase II by Libin Wei; Qinsheng Dai; Yuxin Zhou; Meijuan Zou; Zhiyu Li; Na Lu; Qinglong Guo (pp. 3835-3845).

Cellular metabolism, particularly glycolysis, is altered during the metastatic process and is highly associated with tumor progression and apoptosis resistance. Oroxylin A, a natural plant flavonoid, exhibits chemopreventive and therapeutic anti-inflammatory and anticancer potential. However, the anticancer effects of oroxylin A on non-small cell lung carcinoma (NSCLC) remain poorly understood.In vitro studies were performed using 2D and 3D conditions. The effects on anoikis-sensitization and glycolysis-inhibition of oroxylin A in human non-small cell lung cancer A549 cells were examined. In vivo murine lung metastasis experiments were utilized to assess the anti-metastatic capacity of oroxylin A.ROS-mediated activation of c-Src following detachment caused anoikis resistance in A549 cells. Oroxylin A sensitized A549 cells to anoikis by inactivating the c-Src/AKT/HK II pathway in addition to inducing the dissociation of HK II from mitochondria. Prior to sensitizing A549 cells to anoikis, oroxylin A decreased the ATP level and inhibited glycolysis. Furthermore, oroxylin A inhibited lung metastasis of A549 cells in vivo in nude mice.Oroxylin A sensitized anoikis, which underlies distinct glucose-deprivation-like mechanisms that involved c-Src and HK II.The findings in this study indicated that oroxylin A could potentially be utilized in the development of improved metastatic cancer treatments.•ROS-mediated activation of c-Src following detachment causes anoikis resistance.•Oroxylin A inhibits the metastasis of A549 cells by sensitizing anoikis.•Oroxylin A inactivates the c-Src/AKT/HK II pathway.•Oroxylin A induces dissociation of HK II from mitochondria.•Glucose-deprivation is a novel anticancer mechanism of oroxylin A in NSCLC.

Keywords: Abbreviations; polyHEMA; poly-2-hydroxyethylmethacrylate; 3-BrPA; 3-bromopyruvate; PP2; 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine; NAC; Nacetylcysteine; MTT; 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide; DAPI; 4′, 6-diamidino-2-phenylindole; DCFH-DA; 2′,7′-dichlorfluorescein-diacetate; PARP; poly-ADP-ribose polymerase; AIF; apoptosis inducing factor; ROS; reactive oxygen specials; HK II; Hexokinase II; PGM; phosphoglycerate mutase; PFK; 6-phosphofructo-2-kinase; SCO2; cytochrome c oxidase 2; G-6-P; glucose-6-phosphate; MMP; mitochondrial membrane potential; VDAC; voltage-dependent anion channel; OMM; outer mitochondrial membrane; 18FDG-PET; positron emission tomographyOroxylin A; Anoikis; Glycolysis; c-Src; Hexokinase II

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7 by Valentina Bosello-Travain; Marcus Conrad; Giorgio Cozza; Alessandro Negro; Silvia Quartesan; Monica Rossetto; Antonella Roveri; Stefano Toppo; Fulvio Ursini; Mattia Zaccarin; Matilde Maiorino (pp. 3846-3857).

Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (C_P) it lacks the resolving Cys (C_R), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.Oxidation of the C_P is fast ( k+₁>10³M−¹s−¹), however the rate of reduction by GSH is slow ( k′+₂=12.6M−¹s−¹) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized C_P can be reduced at a fast rate by human protein disulfide isomerase ( HsPDI) ( k+₁>10³M−¹s−¹), but not by Trx. By surface plasmon resonance analysis, a K_D=5.2μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical C_R in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.► The ER contains GPx7, a Cys-glutathione peroxidase missing a resolving Cys. ► The catalytic Cys is oxidized at a fast rate by hydroperoxides. ► GSH binds to the active site, but the release of the reduced enzyme is slow. ► GPx7 is competent for a fast oxidation of PDI. ► Kinetics suggest that physiological GSH regulates the oxidation of PDI by GPx7.

Keywords: Abbreviations; CysGPxs; subfamily of glutathione peroxidases containing a Cys redox center; 1CysGPx; a glutathione peroxidase containing the C; _P; only; 2CysGPxs; glutathione peroxidases containing both the C; _P; and the C; _R; C; _P; peroxidatic Cys; C; _R; resolving Cys; GPx7; glutathione peroxidase 7; ER; endoplasmic reticulum; GSH; reduced glutathione; GSSG; oxidized glutathione; GPxs; glutathione peroxidases; PDI; protein disulfide isomerase; PCOOH; phosphatidylcholine hydroperoxide; Prdxs; peroxiredoxins; ROOH; hydroperoxide; Sec; Selenocysteine; SecGPxs; subfamily of glutathione peroxidases containing a Sec redox center; Syn; synuclein; Trx; thioredoxin; TrxR; thioredoxin reductase; U; _P; peroxidatic SelenocysteineKinetics; Glutathione peroxidase; Peroxide; Redox switch; Redoxin

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BBA - General Subjects (v.1830, #6)