Design and characterisation of piperazine-benzofuran integrated dinitrobenzenesulfonamide as Mycobacterium tuberculosis H37Rv strain inhibitors

Molecular hybridisation of four bioactive fragments piperazine, substituted-benzofuran, amino acids, and 2,4-dinitrobenzenesulfonamide as single molecular architecture was designed. A series of new hybrids were synthesised and subjected to evaluation for their inhibitory activity against Mycobacterium tuberculosis (Mtb) H37Rv. 4d–f and 4o found to exhibit MIC as 1.56 µg/mL, equally active as ethambutol whereas 4a, 4c, 4j displayed MIC 0.78 µg/mL were superior to ethambutol. Tested compounds demonstrated an excellent safety profile with very low toxicity, good selectivity index, and antioxidant properties. All the newly synthesised compounds were thoroughly characterised by analytical methods. The result was further supported by molecular modelling studies on the crystal structure of Mycobacterium tuberculosis enoyl reductase.     

Inhibition of angiogenesis by S-adenosylmethionine

Metastasis is a leading cause of mortality and morbidity in cancer. One of the steps in metastasis process is the formation of new blood vessels. Aberrant DNA methylation patterns are common in cancer cells. In recent studies, S-adenosylmethionine (SAM), which is a DNA methylating agent, has been found to have inhibitory effects on some carcinoma cells in vivo and in vitro. In the present study, we have used SAM to investigate whether it is effective against angiogenesis in vitro. Our results have shown that SAM can reduce the formation and organization of capillary-like structures of endothelial cells in tumoral environment. Besides, we have found SAM can block endothelial cell proliferation and the migration of cells towards growth factors-rich media. In conclusion, our study suggests that SAM may be used against angiogenesis as a natural bio-product.     

An efficient protocol for particle bombardment-mediated transformation of <Emphasis Type="Italic">Centella asiatica</Emphasis> callus

In this study, we report an optimization of particle bombardment transformation system for Centella asiatica callus. A total of eight parameters affecting the genetic transformation system were optimized using the synthetic green fluorescent protein (sGFP) as a reporter driven by the CaMV 35S promoter. The results indicated that DNA delivery conditions of 9-cm target distance, 1,100 psi helium pressure, 1.0 μm gold particles size, 27 mmHg chamber vacuum pressure, 2 times number of bombardment, spermidine as precipitation agent, 60 h post-bombardment incubation time and 2 μg plasmid DNA concentration were optimal for C. asiatica callus transformation. The expression of sGFP was monitored using fluorescence microscope and further confirmed using RT-PCR. This optimized genetic transformation system is applicable for rapid transient gene analysis and transgenic C. asiatica production.     

Constructions of expression vectors of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and transient expression of transgenes in immature oil palm embryos

Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a polyhydroxyalkanoate (PHA) bioplastic group with thermoplastic properties is thus high in quality and can be degradable. PHBV can be produced by bacteria, but the process is not economically competitive with polymers produced from petrochemicals. To overcome this problem, research on transgenic plants has been carried out as one of the solutions to produce PHBV in economically sound alternative manner. Four different genes encoded with the enzymes necessary to catalyze PHBV are bktB, phaB, phaC and tdcB. All the genes came with modified CaMV 35S promoters (except for the tdcB gene, which was promoted by the native CaMV 35S promoter), nos terminator sequences and plastid sequences in order to target the genes into the plastids. Subcloning resulted in the generation of two different orientations of the tdcB, pLMIN (left) and pRMIN (right), both 17.557 and 19.967 kb in sizes. Both plasmids were transformed in immature embryos (IE) of oil palm via Agrobacterium tumefaciens. Assays of GUS were performed on one-week-old calli and 90% of the calli turned completely blue. This preliminary test showed positive results of integration. Six-months-old calli were harvested and RNA of the calli were isolated. RT-PCR was used to confirm the transient expression of PHBV transgenes in the calli. The bands were 258, 260, 315 and 200 bp in size for bktB, phaB, phaC and tdcB transgenes respectively. The data obtained showed that the bktB, phaB, phaC and tdcB genes were successfully integrated and expressed in the oil palm genome.     

Calculation of chromophore excited state energy shifts in response to molecular dynamics of pigment-protein complexes

The absorption and energy transfer properties of photosynthetic pigments are strongly influenced by their local environment or “site.” Local electrostatic fields vary in time with protein and chromophore molecular movement and thus transiently influence the excited state transition properties of individual chromophores. Site-specific information is experimentally inaccessible in many light-harvesting pigment–proteins due to multiple chromophores with overlapping spectra. Full quantum mechanical calculations of each chromophores excited state properties are too computationally demanding to efficiently calculate the changing excitation energies along a molecular dynamics trajectory in a pigment–protein complex. A simplified calculation of electrostatic interactions with each chromophores ground to excited state transition, the so-called charge density coupling (CDC) for site energy, CDC, has previously been developed to address this problem. We compared CDC to more rigorous quantum chemical calculations to determine its accuracy in computing excited state energy shifts and their fluctuations within a molecular dynamics simulation of the bacteriochlorophyll containing light-harvesting Fenna–Mathews–Olson (FMO) protein. In most cases CDC calculations differed from quantum mechanical (QM) calculations in predicting both excited state energy and its fluctuations. The discrepancies arose from the inability of CDC to account for the differing effects of charge on ground and excited state electron orbitals. Results of our study show that QM calculations are indispensible for site energy computations and the quantification of contributions from different parts of the system to the overall site energy shift. We suggest an extension of QM/MM methodology of site energy shift calculations capable of accounting for long-range electrostatic potential contributions from the whole system, including solvent and ions.     

Investigation of Serum Trace Element,Malondialdehyde and Immune Status in Drug Abuser Patients Undergoing Detoxification

Drug abuser patients (n = 104), age ranging from 19 to 42 years, were randomly recruited to investigate the serum levels of trace elements (Cu, Zn, Fe, and Mg), malondialdehyde (MDA), and immunoglobulin (IgG, IgA, and IgM) before and after clinical intervention. Control group also included 104 healthy individuals. Blood samples were analyzed for determining trace elements, MDA, and immunoglobulin using atomic absorption spectroscopy, Ultraviolet-Visible (UV-VIS) spectroscopy, and turbidimetry method, respectively. For serum level of Zn and Fe, the differences between the groups (before intervention, after intervention, and control) were not significant (p > 0.05). However, significant differences were found in serum copper levels between control group, drug abuser patients, and before and after intervention (p < 0.05). The concentration of Mg was found to be significantly higher (p = 0.007) in drug abuser patients than the controls, and after intervention, the level was restored to control value. A displacement of elemental homeostasis was observed in drug abuser patients compared to control, and it was improved after intervention. An increase in serum concentration of MDA was found in drug abuser patients compared to control subjects (p > 0.05) but was not statistically significant. After intervention, the concentration was restored to control value (p > 0.05). The serum concentrations of IgA and IgM were found to be significantly higher (p < 0.05) in drug abuser patients before intervention than the controls, and the level tended to be restored to control level after clinical intervention. Serum IgG level was found to be lower in drug abuser patients compared to controls and further declined significantly (p < 0.05) after intervention. These findings may suggest a possible imbalance in the levels of micronutrients, antioxidants, and immunoglobulin in drug abuser patients, which tend to be restored to control values after detoxification.     

Engineering a prokaryotic Cys-loop receptor with a third functional domain

Prokaryotic members of the Cys-loop receptor ligand-gated ion channel superfamily were recently identified. Previously, Cys-loop receptors were only known from multicellular organisms (metazoans). Contrary to the metazoan Cys-loop receptors, the prokaryotic ones consist of an extracellular (ECD) and a transmembrane domain (TMD), lacking the large intracellular domain (ICD) present in metazoa (between transmembrane segments M3 and M4). Using a chimera approach, we added the 115-amino acid ICD from mammalian serotonin type 3A receptors (5-HT(3A)) to the prokaryotic proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC). We created 12 GLIC-5-HT(3A)-ICD chimeras by replacing a variable number of amino acids in the short GLIC M3M4 linker with the entire 5-HT(3A)-ICD. Two-electrode voltage clamp recordings after expression in Xenopus laevis oocytes showed that only two chimeras were functional and produced currents upon acidification. The pH(50) was comparable with wild-type GLIC. 5-HT(3A) receptor expression can be inhibited by the chaperone protein RIC-3. We have shown previously that the 5-HT(3A)-ICD is required for the attenuation of 5-HT-induced currents when RIC-3 is co-expressed with 5-HT(3A) receptors in X. laevis oocytes. Expression of both functional 5-HT(3A) chimeras was inhibited by RIC-3 co-expression, indicating appropriate folding of the 5-HT(3A)-ICD in the chimeras. Our results indicate that the ICD can be considered a separate domain that can be removed from or added to the ECD and TMD while maintaining the overall structure and function of the ECD and TMD.     

Ubiquitin promoter�Cterminator cassette promotes genetically stable expression of the taste-modifying protein miraculin in transgenic lettuce

Lettuce is a commercially important leafy vegetable that is cultivated worldwide, and it is also a target crop for plant factories. In this study, lettuce was selected as an alternative platform for recombinant miraculin production because of its fast growth, agronomic value, and wide availability. The taste-modifying protein miraculin is a glycoprotein extracted from the red berries of the West African native shrub Richadella dulcifica. Because of its limited natural availability, many attempts have been made to produce this protein in suitable alternative hosts. We produced transgenic lettuce with miraculin gene driven either by the ubiquitin promoter/terminator cassette from lettuce or a 35S promoter/nos terminator cassette. Miraculin gene expression and miraculin accumulation in both cassettes were compared by quantitative real-time PCR analysis, Western blotting, and enzyme-linked immunosorbent assay. The expression level of the miraculin gene and protein in transgenic lettuce was higher and more genetically stable in the ubiquitin promoter/terminator cassette than in the 35S promoter/nos terminator cassette. These results demonstrated that the ubiquitin promoter/terminator cassette is an efficient platform for the genetically stable expression of the miraculin protein in lettuce and hence this platform is of benefit for recombinant miraculin production on a commercial scale.     

Thermodynamic heuristics with case-based reasoning: combined insights for RNA pseudoknot secondary structure

The secondary structure of RNA pseudoknots has been extensively inferred and scrutinized by computational approaches. Experimental methods for determining RNA structure are time consuming and tedious; therefore, predictive computational approaches are required. Predicting the most accurate and energy-stable pseudoknot RNA secondary structure has been proven to be an NP-hard problem. In this paper, a new RNA folding approach, termed MSeeker, is presented; it includes KnotSeeker (a heuristic method) and Mfold (a thermodynamic algorithm). The global optimization of this thermodynamic heuristic approach was further enhanced by using a case-based reasoning technique as a local optimization method. MSeeker is a proposed algorithm for predicting RNA pseudoknot structure from individual sequences, especially long ones. This research demonstrates that MSeeker improves the sensitivity and specificity of existing RNA pseudoknot structure predictions. The performance and structural results from this proposed method were evaluated against seven other state-of-the-art pseudoknot prediction methods. The MSeeker method had better sensitivity than the DotKnot, FlexStem, HotKnots, pknotsRG, ILM, NUPACK and pknotsRE methods, with 79% of the predicted pseudoknot base-pairs being correct.