Enhancing Production of Bio-Isoprene Using Hybrid MVA Pathway and Isoprene Synthase in E. coli

The depleting petroleum reserve, increasingly severe energy crisis, and global climate change are reigniting enthusiasm for seeking sustainable technologies to replace petroleum as a source of fuel and chemicals. In this paper, the efficiency of the MVA pathway on isoprene production has been improved as follows: firstly, in order to increase MVA production, the source of the “upper pathway” which contains HMG-CoA synthase, acetyl-CoA acetyltransferase and HMG-CoA reductase to covert acetyl-CoA into MVA has been changed from Saccharomyces cerevisiae to Enterococcus faecalis; secondly, to further enhance the production of MVA and isoprene, a alanine 110 of the mvaS gene has been mutated to a glycine. The final genetic strain YJM25 containing the optimized MVA pathway and isoprene synthase from Populus alba can accumulate isoprene up to 6.3 g/L after 40 h of fed-batch cultivation

cDNA Immunization of Mice with Human Thyroglobulin Generates Both Humoral and T Cell Responses: A Novel Model of Thyroid Autoimmunity

Thyroglobulin (Tg) represents one of the largest known self-antigens involved in autoimmunity. Numerous studies have implicated it in triggering and perpetuating the autoimmune response in autoimmune thyroid diseases (AITD). Indeed, traditional models of autoimmune thyroid disease, experimental autoimmune thyroiditis (EAT), are generated by immunizing mice with thyroglobulin protein in conjunction with an adjuvant, or by high repeated doses of Tg alone, without adjuvant. These extant models are limited in their experimental flexibility, i.e. the ability to make modifications to the Tg used in immunizations. In this study, we have immunized mice with a plasmid cDNA encoding the full-length human Tg (hTG) protein, in order to generate a model of Hashimoto's thyroiditis which is closer to the human disease and does not require adjuvants to breakdown tolerance. Human thyroglobulin cDNA was injected and subsequently electroporated into skeletal muscle using a square wave generator. Following hTg cDNA immunizations, the mice developed both B and T cell responses to Tg, albeit with no evidence of lymphocytic infiltration of the thyroid. This novel model will afford investigators the means to test various hypotheses which were unavailable with the previous EAT models, specifically the effects of hTg sequence variations on the induction of thyroiditis

Triose Phosphate Isomerase Deficiency Is Caused by Altered Dimerization–Not Catalytic Inactivity–of the Mutant Enzymes

Triosephosphate isomerase (TPI) deficiency is an autosomal recessive disorder caused by various mutations in the gene encoding the key glycolytic enzyme TPI. A drastic decrease in TPI activity and an increased level of its substrate, dihydroxyacetone phosphate, have been measured in unpurified cell extracts of affected individuals. These observations allowed concluding that the different mutations in the TPI alleles result in catalytically inactive enzymes. However, despite a high occurrence of TPI null alleles within several human populations, the frequency of this disorder is exceptionally rare. In order to address this apparent discrepancy, we generated a yeast model allowing us to perform comparative in vivo analyses of the enzymatic and functional properties of the different enzyme variants. We discovered that the majority of these variants exhibit no reduced catalytic activity per se. Instead, we observed, the dimerization behavior of TPI is influenced by the particular mutations investigated, and by the use of a potential alternative translation initiation site in the TPI gene. Additionally, we demonstrated that the overexpression of the most frequent TPI variant, Glu104Asp, which displays altered dimerization features, results in diminished endogenous TPI levels in mammalian cells. Thus, our results reveal that enzyme deregulation attributable to aberrant dimerization of TPI, rather than direct catalytic inactivation of the enzyme, underlies the pathogenesis of TPI deficiency. Finally, we discovered that yeast cells expressing a TPI variant exhibiting reduced catalytic activity are more resistant against oxidative stress caused by the thiol-oxidizing reagent diamide. This observed advantage might serve to explain the high allelic frequency of TPI null alleles detected among human populations

Synthesis and bioactivity of nobilamide B

An alternative and facile solution/solid-phase approach is reported for the total synthesis of neuroactive peptide, nobilamide B. Z-Dhb was formed in solution via EDC/CuCl induced elimination. The solid-phase synthesis employed HBTU/Oxyma Pure™ coupling using Barlos resin. Synthetic nobilamide B was also found to be neuroactive in primary cultures of dorsal root ganglion (DRG) neurons. © the Partner Organisations 2014

Synthesis and bioactivity of nobilamide B

An alternative and facile solution/solid-phase approach is reported for the total synthesis of neuroactive peptide, nobilamide B. Z-Dhb was formed in solution via EDC/CuCl induced elimination. The solid-phase synthesis employed HBTU/Oxyma Pure™ coupling using Barlos resin. Synthetic nobilamide B was also found to be neuroactive in primary cultures of dorsal root ganglion (DRG) neurons. © the Partner Organisations 2014

Toward antituberculosis drugs: in silico screening of synthetic compounds against Mycobacterium tuberculosis L,D-transpeptidase 2

Junie B Billones,1,2 Maria Constancia O Carrillo,1 Voltaire G Organo,1 Stephani Joy Y Macalino,1 Jamie Bernadette A Sy,1 Inno A Emnacen,1 Nina Abigail B Clavio,1 Gisela P Concepcion31Office of the Vice President for Academic Affairs – Emerging Interdisciplinary Research Program: “Computer-aided Discovery of Compounds for the treatment of Tuberculosis in the Philippines,” Department of Physical Sciences and Mathematics, College of Arts and Sciences, 2Institute of Pharmaceutical Sciences, National Institutes of Health, University of the Philippines Manila, Manila, 3Marine Science Institute, University of the Philippines Diliman, Diliman, Quezon City, PhilippinesAbstract: Mycobacterium tuberculosis (Mtb) the main causative agent of tuberculosis, is the main reason why this disease continues to be a global public health threat. It is therefore imperative to find a novel antitubercular drug target that is unique to the structural machinery or is essential to the growth and survival of the bacterium. One such target is the enzyme L,D-transpeptidase 2, also known as LdtMt2, a protein primarily responsible for the catalysis of 3→3 cross-linkages that make up the mycolyl–arabinogalactan–peptidoglycan complex of Mtb. In this study, structure-based pharmacophore screening, molecular docking, and in silico toxicity evaluations were employed in screening compounds from a database of synthetic compounds. Out of the 4.5 million database compounds, 18 structures were identified as high-scoring, high-binding hits with very satisfactory absorption, distribution, metabolism, excretion, and toxicity properties. Two out of the 18 compounds were further subjected to in vitro bioactivity assays, with one exhibiting a good inhibitory activity against the Mtb H37Ra strain.Keywords: antituberculosis drug discovery, virtual screening, dockin

In silico discovery and in vitro activity of inhibitors against Mycobacterium tuberculosis 7,8-diaminopelargonic acid synthase (Mtb BioA)

Junie B Billones,1,2 Maria Constancia O Carrillo,1 Voltaire G Organo,1 Jamie Bernadette A Sy,1 Nina Abigail B Clavio,1 Stephani Joy Y Macalino,1 Inno A Emnacen,1 Alexandra P Lee,1 Paul Kenny L Ko,1 Gisela P Concepcion3 1OVPAA-EIDR Program, “Computer-Aided Discovery of Compounds for the Treatment of Tuberculosis in the Philippines”, Department of Physical Sciences and Mathematics, College of Arts and Sciences, University of the Philippines Manila, Manila, Philippines; 2Institute of Pharmaceutical Sciences, National Institutes of Health, University of the Philippines Manila, Manila, Philippines; 3Marine Science Institute, College of Science, University of the Philippines Diliman, Quezon City, Philippines Abstract: Computer-aided drug discovery and development approaches such as virtual screening, molecular docking, and in silico drug property calculations have been utilized in this effort to discover new lead compounds against tuberculosis. The enzyme 7,8-diaminopelargonic acid aminotransferase (BioA) in Mycobacterium tuberculosis (Mtb), primarily involved in the lipid biosynthesis pathway, was chosen as the drug target due to the fact that humans are not capable of synthesizing biotin endogenously. The computational screening of 4.5 million compounds from the Enamine REAL database has ultimately yielded 45 high-scoring, high-affinity compounds with desirable in silico absorption, distribution, metabolism, excretion, and toxicity properties. Seventeen of the 45 compounds were subjected to bioactivity validation using the resazurin microtiter assay. Among the 4 actives, compound 7 ((Z)-N-(2-isopropoxyphenyl)-2-oxo-2-((3-(trifluoromethyl)cyclohexyl)amino)acetimidic acid) displayed inhibitory activity up to 83% at 10 µg/mL concentration against the growth of the Mtb H37Ra strain. Keywords: CADDD, ADMET, TOPKAT, BioA inhibitor, structure-based pharmacophore, pharmacophore, molecular docking, resazurin microtiter assa