Crystal structures of SCP2-thiolases of Trypanosomatidae, human pathogens causing widespread tropical diseases: the importance for catalysis of the cysteine of the unique HDCF loop

Thiolases are essential CoA-dependent enzymes in lipid metabolism. In the present study we report the crystal structures of trypanosomal and leishmanial SCP2 (sterol carrier protein, type-2)-thiolases. Trypanosomatidae cause various widespread devastating (sub)-tropical diseases, for which adequate treatment is lacking. The structures reveal the unique geometry of the active site of this poorly characterized subfamily of thiolases. The key catalytic residues of the classical thiolases are two cysteine residues, functioning as a nucleophile and an acid/base respectively. The latter cysteine residue is part of a CxG motif. Interestingly, this cysteine residue is not conserved in SCP2-thiolases. The structural comparisons now show that in SCP2-thiolases the catalytic acid/base is provided by the cysteine residue of the HDCF motif, which is unique for this thiolase subfamily. This HDCF cysteine residue is spatially equivalent to the CxG cysteine residue of classical thiolases. The HDCF cysteine residue is activated for acid/base catalysis by two main chain NH-atoms, instead of two water molecules, as present in the CxG active site. The structural results have been complemented with enzyme activity data, confirming the importance of the HDCF cysteine residue for catalysis. The data obtained suggest that these trypanosomatid SCP2-thiolases are biosynthetic thiolases. These findings provide promise for drug discovery as biosynthetic thiolases catalyse the first step of the sterol biosynthesis pathway that is essential in several of these parasites.

Terminalia paniculata bark extract attenuates non-alcoholic fatty liver via down regulation of fatty acid synthase in high fat diet-fed obese rats

Background: This study was performed to understand the possible therapeutic activity of Terminalia paniculata ethanolic extract (TPEE) on non alcoholic fatty liver in rats fed with high fat diet. Methods: Thirty six SD rats were divided into 6 groups (n = 6): Normal control (NC), high fat diet (HFD), remaining four groups were fed on HFD along with different doses of TPEE (100,150 and 200 mg/kg b.wt) or orlistat, for ten weeks. Liver tissue was homogenized and analyzed for lipid profiles, activities of superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) content. Further, the expression levels of FAS and AMPK-1 alpha were also studied in addition to histopathology examination of liver tissue in all the groups. Results: HFD significantly increased hepatic liver total cholesterol (TC), triglycerides (TG), free fatty acids (FFA) and MDA but decreased the activities of SOD and CAT which were subsequently reversed by supplementation with TPEE in a dose-dependent manner. In addition, TPEE administration significantly down regulated hepatic mRNA expression of FAS but up regulated AMPK-1 alpha compared to HFD alone fed group. Furthermore, western blot analysis of FAS has clearly demonstrated decreased expression of FAS in HFD + TPEE (200 mg/kg b. wt) treated group when compared to HFD group at protein level. Conclusions: Our biochemical studies on hepatic lipid profiles and antioxidant enzyme activities supported by histological and expression studies suggest a potential therapeutic role for TPEE in regulating obesity through FAS.

Haloarchaeal gas vesicle nanoparticles displaying Salmonella SopB antigen reduce bacterial burden when administered with live attenuated bacteria

Innovative vaccines against typhoid and other Salmonella diseases that are safe, effective, and inexpensive are urgently needed. In order to address this need, buoyant, self-adjuvating gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1 were bioengineered to display the highly conserved Salmonella enterica antigen SopB, a secreted inosine phosphate effector protein injected by pathogenic bacteria during infection into the host cell. Two highly conserved sopB gene segments near the 3'-coding region, named sopB4 and B5, were each fused to the gvpC gene, and resulting GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and B5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of recombinant GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 Delta pmrG-HM-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-gamma, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Th1 response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5-GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were found to be stable at elevated temperatures for extended periods without refrigeration in Halobacterium cells. The results all together show that bioengineered GVNPs are likely to represent a valuable platform for the development of improved vaccines against Salmonella diseases. (C) 2014 Elsevier Ltd. All rights reserved.

Critical cysteines in Akt1 regulate its activity and proteasomal degradation: implications for neurodegenerative diseases

Impaired Akt1 signaling is observed in neurodegenerative diseases, including Parkinson's disease (PD). In PD models oxidative modification of Akt1 leads to its dephosphorylation and consequent loss of its kinase activity. To explore the underlying mechanism we exposed Neuro2A cells to cadmium, a pan inhibitor of protein thiol disulfide oxidoreductases, including glutaredoxin 1 (Grx1), or downregulated Grx1, which led to dephosphorylation of Akt1, loss of its kinase activity, and also decreased Akt1 protein levels. Mutation of cysteines to serines at 296 and 310 in Akt1 did not affect its basal kinase activity but abolished cadmium- and Grx1 downregulation-induced reduction in Akt1 kinase activity, indicating their critical role in redox modulation of Akt1 function and turnover. Cadmium-induced decrease in phosphorylated Akt1 correlated with increased association of wild-type (WT) Akt1 with PP2A, which was absent in the C296-310S Akt1 mutant and was also abolished by N-acetylcysteine treatment. Further, increased proteasomal degradation of Akt1 by cadmium was not seen in the C296-310S Akt1 mutant, indicating that oxidation of cysteine residues facilitates degradation of WT Akt1. Moreover, preventing oxidative modification of Akt1 cysteines 296 and 310 by mutating them to serines increased the cell survival effects of Akt1. Thus, in neurodegenerative states such as PD, maintaining the thiol status of cysteines 296 and 310 in Akt1 would be critical for Akt1 kinase activity and for preventing its degradation by proteasomes. Preventing downregulation of Akt signaling not only has long-range consequences for cell survival but could also affect the multiple roles that Ala plays, including in the Akt-mTOR signaling cascade. (C) 2014 Elsevier Inc. All rights reserved.

Photoacoustic tomography of the human finger: towards the assessment of inflammatory joint diseases

Inflammatory arthritis is often manifested in finger joints. The growth of new or withdrawal of old blood vessels can be a sensitive marker for these diseases. Photoacoustic (PA) imaging has great potential in this respect since it allows the sensitive and highly resolved visualization of blood. We systematically investigated PA imaging of finger vasculature in healthy volunteers using a newly developed PA tomographic system. We present the PA results which show excellent detail of the vasculature. Vessels with diameters ranging between 100 mu m and 1.5 mm are visible along with details of the skin, including the epidermis and the subpapillary plexus. The focus of all the studies is at the proximal and distal interphalangeal joints, and in the context of ultimately visualizing the inflamed synovial membrane in patients. This work is important in laying the foundation for detailed research into PA imaging of the phalangeal vasculature in patients suffering from rheumatoid arthritis.

Haloarchaeal gas vesicle nanoparticles displaying Salmonella antigens as a novel approach to vaccine development

A safe, effective, and inexpensive vaccine against typhoid and other Salmonella diseases is urgently needed. In order to address this need, we are developing a novel vaccine platform employing buoyant, self-adjuvanting gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1, bioengineered to display highly conserved Salmonella enterica antigens. As the initial antigen for testing, we selected SopB, a secreted inosine phosphate effector protein injected by pathogenic S. enterica bacteria during infection into the host cells. Two highly conserved sopB gene segments near the 3'- region, named sopB4 and sopB5, were each fused to the grIpC gene, and resulting SopB-GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and SopB5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of SopB-GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ApmrG-H111-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-y, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Thl response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were also found to be stable at elevated temperatures for extended periods without refrigeration. The results show that bioengineered GVNPs are likely to represent a valuable platform for antigen delivery and development of improved vaccines against Salmonella and other diseases.

A galactose-functionalized dendritic siRNA-nanovector to potentiate hepatitis C inhibition in liver cells

A RNAi based antiviral strategy holds the promise to impede hepatitis C viral (HCV) infection overcoming the problem of emergence of drug resistant variants, usually encountered in the interferon free direct-acting antiviral therapy. Targeted delivery of siRNA helps minimize adverse `off-target' effects and maximize the efficacy of therapeutic response. Herein, we report the delivery of siRNA against the conserved 5'-untranslated region (UTR) of HCV RNA using a liver-targeted dendritic nano-vector functionalized with a galactopyranoside ligand (DG). Physico-chemical characterization revealed finer details of complexation of DG with siRNA, whereas molecular dynamic simulations demonstrated sugar moieties projecting ``out'' in the complex. Preferential delivery of siRNA to the liver was achieved through a highly specific ligand-receptor interaction between dendritic galactose and the asialoglycoprotein receptor. The siRNA-DG complex exhibited perinuclear localization in liver cells and co-localization with viral proteins. The histopathological studies showed the systemic tolerance and biocompatibility of DG. Further, whole body imaging and immunohistochemistry studies confirmed the preferential delivery of the nucleic acid to mice liver. Significant decrease in HCV RNA levels (up to 75%) was achieved in HCV subgenomic replicon and full length HCV-JFH1 infectious cell culture systems. The multidisciplinary approach provides the `proof of concept' for restricted delivery of therapeutic siRNAs using a target oriented dendritic nano-vector.

Asparagine requirement in Plasmodium berghei as a target to prevent malaria transmission and liver infections

The proteins of Plasmodium, the malaria parasite, are strikingly rich in asparagine. Plasmodium depends primarily on host haemoglobin degradation for amino acids and has a rudimentary pathway for amino acid biosynthesis, but retains a gene encoding asparagine synthetase (AS). Here we show that deletion of AS in Plasmodium berghei (Pb) delays the asexual-and liver-stage development with substantial reduction in the formation of ookinetes, oocysts and sporozoites in mosquitoes. In the absence of asparagine synthesis, extracellular asparagine supports suboptimal survival of PbAS knockout (KO) parasites. Depletion of blood asparagine levels by treating PbASKO-infected mice with asparaginase completely prevents the development of liver stages, exflagellation of male gametocytes and the subsequent formation of sexual stages. In vivo supplementation of asparagine in mice restores the exflagellation of PbASKO parasites. Thus, the parasite life cycle has an absolute requirement for asparagine, which we propose could be targeted to prevent malaria transmission and liver infections.

Combined Effect of Cryogel Matrix and Temperature-Reversible Soluble-Insoluble Polymer for the Development of in Vitro Human Liver Tissue

Hepatic cell culture on a three-dimensional (3D) matrix or as a hepatosphere appears to be a promising in vitro biomimetic system for liver tissue engineering applications. In this study, we have combined the concept of a 3D scaffold and a spheroid culture to develop an in vitro model to engineer liver tissue for drug screening. We have evaluated the potential of poly(ethylene glycol)-alginate-gelatin (PAG) cryogel matrix for in vitro culture of human liver cell lines. The synthesized cryogel matrix has a flow rate of 7 mL/min and water uptake capacity of 94% that enables easy nutrient transportation in the in vitro cell culture. Youngs modulus of 2.4 kPa and viscoelastic property determine the soft and elastic nature of synthesized cryogel. Biocompatibility of PAG cryogel was evaluated through MTT assay of HepG2 and Huh-7 cells on matrices. The proliferation and functionality of the liver cells were enhanced by culturing hepatic cells as spheroids (hepatospheres) on the PAG cryogel using temperature-reversible soluble-insoluble polymer, poly(N-isopropylacrylamide) (PNIPAAm). Pore size of the cryogel above 100 mu m modulated spheroid size that can prevent hypoxia condition within the spheroid culture. Both the hepatic cells have shown a significant difference (P < 0.05) in terms of cell number and functionality when cultured with PNIPAAm. After 10 days of culture using 0.05% PNIPAAm, the cell number increased by 11- and 7-fold in case of HepG2 and Huh-7 cells, respectively. Similarly, after 10 days of hepatic spheroids culture on PAG cryogel, the albumin production, urea secretion, and CYP450 activity were significantly higher in case of culture with PNIPAAm. The developed tissue mass on the PAG cryogel in the presence of PNIPAAm possess polarity, which was confirmed using F-actin staining and by presence of intercellular bile canalicular lumen. The developed cryogel matrix supports liver cells proliferation and functionality and therefore can be used for in vitro and in vivo drug testing.