Mechanism of the oxidative stress-mediated increase in lipid accumulation by the bacterium,R. opacus PD630: Experimental analysis and genome-scale metabolic modeling

Appropriate species of oleaginous bacteria, with their high growth rates and lipid accumulation capabilities, can be good contenders for industrial triacylglycerol (TAG) production, compared to microalgae. Further, oxidative stress (OS) can be used to significantly increase TAG yields in oleaginous microbes, but the mechanism is unexplored. In a first, this study explored the mechanism behind OS-mediated increase in TAG accumulation by the bacterium, Rhodococccus opacus PD630, through experimental analysis and metabolic modelling. Two mechanisms that could increase acetyl-CoA (TAG-precursor) levels were hypothesized based on literature information. One was OS-mediated inactivation of the aconitase (TCA cycle), and another was the inactivation of the triosephosphate isomerase (TPI; glycolysis). The results negated the involvement of aconitase in increased acetyl-CoA levels. Analysis of the metabolic model showed that inactivation of TPI, re-routed the flux through the pentose phosphate pathway (PPP), supplying both NADPH and acetyl-CoA for TAG synthesis. Additionally, inactivation of TPI increased TAG flux by 143%, whereas, inactivating both TPI and aconitase, increased it by 152%. We present experimental evidence for OS-mediated decrease in TPI activity and increase in activity of glucose-6-phosphate dehydrogenase (PPP enzyme). The findings indicate that increased flux through PPP can be explored to improve TAG accumulation on a large-scale.     

A comparative ecotoxicity analysis of α- and γ-phase aluminium oxide nanoparticles towards a freshwater bacterial isolate Bacillus licheniformis

Crystalline structure of nanoparticles may influence their physicochemical behaviour as well as their toxicological impact on biota. The differences in orientation of the atoms result in the variations in chemical stability. Thus, toxicological impacts of different crystalline phases of aluminium oxide nanoparticles are expected to vary. The present study brings out a comparative toxicity analysis of γ-phase and α-phase aluminium oxide nanoparticles of comparable hydrodynamic size range towards a freshwater bacterial isolate Bacillus licheniformis at low exposure concentrations (5, 1, 0.5 and 0.05 µg/mL). Upon 2-h exposure, the α-aluminium oxide particles showed lower toxicity than the γ-phase aluminium oxide. The lower level of oxidative stress generation and cell membrane damage in case of the α-phase aluminium oxide nanoparticles substantiated the toxicity results. The involvement of protein, lipopolysaccharides in nanoparticle–cell surface interaction, was noted in both the cases. To conclude, the crystallinity of aluminium oxide nanoparticles played an important role in the interaction and the toxicity response.     

All-trans retinoic acid (ATRA) downregulates MMP-9 by modulating its regulatory molecules

The vitamin A derivative all-trans retinoic acid (ATRA) is considered as a potent chemotherapeutic drug for its capability of regulating cell growth and differentiation. We aimed to study the effect of ATRA on MMP-9 in MDA-MB-231, human breast cancer cells and the probable molecular mechanisms through which ATRA exerts its effect. Our experimental findings demonstrate that ATRA enters into the nucleus and regulates various signaling pathways viz. Integrin, FAK, ERK, PI-3K, NFκB and also EGFR and downregulates pro-MMP-9 activity as well as its expression. As a result MDA-MB-231 cell migration on fibronectin medium gets retarded in presence of ATRA. ATRA upregulates TIMP-1 expression. Our study may help to understand the role of ATRA as a regulator of MMP-9 and the possible signaling pathways which are involved in this ATRA mediated downregulation of MMP-9.Key words: MMP-9, ATRA, integrin, EGFR, NFκB     

Immunofluorescent test for simultaneous detection of Edwardsiella ictaluri and Flavobacterium columnare

Enteric septicemia of catfish (ESC) and columnaris disease are 2 bacterial diseases significantly affecting the aquaculture industry, and thus rapid diagnosis of disease is imperative for making judicious management decisions. A rapid indirect fluorescent antibody (IFA) test with antibody conjugated fluorochromes having 2 different spectral properties (Alexa Fluor 488-emitting green fluorescence, and Alexa Fluor 594-emitting red fluorescence) was compared with bacteriological culture (accepted standard) for simultaneous detection of Edwardsiella ictaluri (EI) and Flavobacterium columnare (FC) in 3 groups of experimentally infected channel catfish (Ictalurus punctatus Rafinesque), and a fourth group that acquired an aquarium-infection with F. columnare. A total of 303 samples (derived from kidney, brain and nares) from 101 fish were concurrently examined by both tests. Fish in the 3 experimentally infected groups (I to III) were culture positive for the bacteria with which they were infected, and fish in Group IV, (the spontaneously infected fish) revealed F. columnare only. The IFA test compared favorably in sensitivity (EI= 80.7 %; FC = 87.2%) and specificity (EI = 83.9%; FC = 88.9%) with the standard bacteriological culture. The positive predictive value (EI = 96.2% Group I, 90.8% Group II, 93.7% Groups I and II combined; FC = 95.2% Group II, 95.3% Groups II, III and IV combined) was high, while the negative predictive value (EI = 66.7% Group I, 31.3% Group II, 59.5% Groups I and II combined; FC = 73.7% Group II, 72.7% Groups II, III and IV combined) was relatively low. The IFA test will serve as an efficient tool for rapid simultaneous detection of E. ictaluri and F. columnare in outbreaks of disease.     

Mixed-ligand complexes of ruthenium(II) containing new photoactive or electroactive ligands: synthesis, spectral characterization and DNA interactions

Mixed-ligand ruthenium(II) complexes of three photoactive ligands, viz., (E)-1-[2-(4-methyl-2-pyridyl)-4-pyridyl]-2-(1-naphthyl)-1-ethene (mppne), (E)-1-(9-anthryl)-2-[2-(4-methyl-2-pyridyl)-4-pyridyl]-1-ethene (mppae) and (E)-1-[2-(4-methyl-2-pyridyl)-4-pyridyl]-2-(1-pyrenyl)-1-ethene (mpppe), in which a 2,2′-bipyridyl unit is linked via an ethylinic linkage to either a naphthalene, an anthracene or a pyrene chromophore and three electroactive ligands, viz., 4-(4-pyridyl)-1,2-benzenediol (catpy), 5,6-dihydroxy-1,10-phenanthroline (catphen) and 1,2-benzenediol (cat), were synthesized in good to moderate yields. Complexes [Ru(bpy)₂(mppne)]²⁺ (bpy is 2, 2′–bipyridyl), [Ru(bpy)₂(mppae)]²⁺, [Ru(bpy)₂(mpppe)]²⁺, [Ru(bpy)₂(sq-py)]⁺, [Ru(bpy)₂(sq-phen)]⁺ and [Ru(phen)₂(bsq)]⁺ (phen is 1,10-phenanthroline) were fully characterized by elemental analysis, IR, ¹H NMR, fast-atom bombardment or electron-impact mass, UV–vis and cyclic voltammetric methods. In the latter three complexes, the ligands catpy, catphen and cat are actually bound to the metal center as the corresponding semiquinone species, viz., 4-(4-pyridyl)-1,2-benzenedioleto(+I) (sq-py), 1,10-phenanthroline-5,6-dioleto(+I) (sq-phen) and 1,2-benzenedioleto(+I) (bsq), thus making the overall charge of the complexes formally equal to + 1 in each case. These three complexes are electron paramagnetic resonance active and exhibit an intense absorption band between 941 and 958 nm owing to metal-to-ligand charge transfer (MLCT, d _Ru→π*_sq) transitions. The other three ruthenium(II) complexes containing three photoactive ligands, mppne, mppae and mpppe, exhibit MLCT (d _Ru→π*_bpy ) bands in the 454–461-nm region and are diamagnetic. These can be characterized by the ¹H NMR method. [Ru(bpy)₂(mppne)]²⁺, [Ru(bpy)₂(mppae)]²⁺ and [Ru(bpy)₂(mpppe)]²⁺ exhibit redox waves corresponding to the Ru^III/Ru^II couple along with the expected ligand (bpy and substituted bpy) based ones in their cyclic and differential pulse voltammograms (CH₃CN, 0.1 M tetrabutylammonium hexafluorophosphate)—corresponding voltammograms of [Ru(bpy)₂(sq-py)]⁺, [Ru(bpy)₂(sq-phen)]⁺ and [Ru(phen)₂(bsq)]⁺ are mainly characterized by waves corresponding to the quinone/semiquinone (q/sq) and semiquinone/1,2-diol (sq/cat) redox processes. The results of absorption and fluorescence titration as well as thermal denaturation studies reveal that [Ru(bpy)₂(mppne)]²⁺ and [Ru(bpy)₂(mppae)]²⁺ are moderate-to-strong binders of calf thymus DNA with binding constants ranging from 10⁵ to 10⁶ M⁻¹. Under the identical conditions of drug and light dose, the DNA (supercoiled pBR 322) photocleavage activities of these two complexes follow the order:[Ru(bpy)₂(mppne)]²⁺>[Ru(bpy)₂(mppae)]²⁺, although the emission quantum yields follow the reverse order. The other ruthenium(II) complexes containing the semiquinone-based ligands are found to be nonluminescent and inefficient photocleavage agents of DNA. However, experiments shows that [Ru(bpy)₂(sq)]⁺-based complexes oxidize the sugar unit and could be used as mild oxidants for the sugar moiety of DNA. Possible explanations for these observations are presented.Electronic Supplementary Material Supplementary material is available for this article at .     

C5'- and C3'-sugar radicals produced via photo-excitation of one-electron oxidized adenine in 2'-deoxyadenosine and its derivatives

We report that photo-excitation of one-electron-oxidized adenine [A(-H)•] in dAdo and its 2′-deoxyribonucleotides leads to formation of deoxyribose sugar radicals in remarkably high yields. Illumination of A(-H)• in dAdo, 3′-dAMP and 5′-dAMP in aqueous glasses at 143 K leads to 80-100% conversion to sugar radicals at C5′ and C3′. The position of the phosphate in 5′- and 3′-dAMP is observed to deactivate radical formation at the site of substitution. In addition, the pH has a crucial influence on the site of sugar radical formation; e.g. at pH ~5, photo-excitation of A(-H)• in dAdo at 143 K produces mainly C5′• whereas only C3′• is observed at high pH ~12. ¹³C substitution at C5′ in dAdo yields ¹³C anisotropic couplings of (28, 28, 84) G whose isotropic component 46.7 G identifies formation of the near planar C5′•. A β-¹³C 16 G isotropic coupling from C3′• is also found. These results are found to be in accord with theoretically calculated ¹³C couplings at C5′ [DFT, B3LYP, 6-31(G) level] for C5′• and C3′•. Calculations using time-dependent density functional theory [TD-DFT B3LYP, 6-31G(d)] confirm that transitions in the near UV and visible induce hole transfer from the base radical to the sugar group leading to sugar radical formation.     

Ginseng: Cardiotonic in adult rat cardiomyocytes, cardiotoxic in neonatal rat cardiomyocytes

Ginsengs are widely used to improve cardiac health and circulation. Loosely termed as ginsengs, Asian (Panax), Siberian and Ashwagandha (Indian Ginseng) Indian ginsengs are prepared from different plants. We tested the popular belief of cardiotonic effects of ginsengs using both neonatal and adult rat cardiomyocytes, comparing extracts from the three ginsengs. Addition of 10% v/v of extract (100 microl of extract/ml of culture medium) of each of the ginsengs resulted in a rapid (<10 s) cessation of beating in neonatal cardiomyocytes due to calcium overload, while sequential dilutions revealed that treatment with a low dose (0.01% v/v, 0.1 microl/ml of the medium) resulted in constant, regular beats (transients), and a slight elevation of diastolic calcium without overload. Addition of extracts to sparking, calcium-tolerant adult cardiomyocytes resulted in initiation of calcium transients, and adult cells were able to tolerate exposure to high concentrations of extract. Cardiotonic effects in adult cells (cardiotoxicity in neonatal cells) were most profound with Asian ginseng (2.6 times that of Siberian ginseng, 1.6 times that of Indian ginseng) probably due to the active ingredients (ginsenosides in Asian, eleutherosides in Siberian and withanolides in Indian) being structurally different. We conclude that fully developed cardiomyocytes are able to accommodate higher doses of ginseng than neonatal cells, and that the effects of ginseng on newly formed, developing myocytes, could be extremely deleterious to the fetus. However, for adults, ginseng might well be a 'tonic' in its ability to increase beating and intramyocytic calcium levels.     

Dose-finding study with nicotine as a proconvulsant agent in PTZ-induced seizure model in mice

The present study was conducted to investigate the possible interaction between low doses of nicotine and pentylenetetrazole (PTZ) in vivo and also to evaluate the influence of nicotine on the antiseizure efficacy of topiramate and sodium valproate in the PTZ-induced seizure model in mice. Graded dose–response study with nicotine showed the CD50 value for nicotine at 6.76 mg/kg. i.p. Subtheshold dose of nicotine (4 mg/kg, i.p.) pretreatment significantly decreased the CD50 value for PTZ from 47.86 mg/kg, i.p. (of PTZ per se) to 31.62 mg/kg, i.p. Sodium valproate but not topiramate, significantly inhibited PTZ-induced seizures in mice with an ED50 value of 177.83 mg/kg, i.p. Nonconvulsive dose of nicotine (1 mg/kg, i.p.) significantly antagonized the protective efficacy of sodium valproate against PTZ-induced seizures and increased the ED50 value to 338.84 mg/kg, i.p. PTZ-induced seizures significantly increased the mouse brain levels of MDA and reduced the level of GSH while sodium valproate reversed such changes. Nicotine pretreatment reversed the anti-lipid peroxidative action of sodium valproate in the PTZ-induced seizure model in mice. The study highlighted the convulsant as well as proconvulsant role of nicotine and established dose discrimination for nicotine as a proconvulsant agent and an anti-antiseizure agent. The study bears significant clinical relevance particularly amongst epileptic smokers who may show failure of efficacy of antiepileptic agents and present with breakthrough seizure attacks on exposure to nicotine.