Tunable blinking kinetics of cy5 for precise DNA quantification and single-nucleotide difference detection

Fluorescence correlation spectroscopy (FCS) can resolve the intrinsic fast-blinking kinetics (FBKs) of fluorescent molecules that occur on the order of microseconds. These FBKs can be heavily influenced by the microenvironments in which the fluorescent molecules are contained. In this work, FCS is used to monitor the dynamics of fluorescence emission from Cy5 labeled on DNA probes. We found that the FBKs of Cy5 can be tuned by having more or less unpaired guanines (upG) and thymines (upT) around the Cy5 dye. The observed FBKs of Cy5 are found to predominantly originate from the isomerization and back-isomerization processes of Cy5, and Cy5-nucleobase interactions are shown to slow down these processes. These findings lead to a more precise quantification of DNA hybridization using FCS analysis, in which the FBKs play a major role rather than the diffusion kinetics. We further show that the alterations of the FBKs of Cy5 on probe hybridization can be used to differentiate DNA targets with single-nucleotide differences. This discrimination relies on the design of a probe-target-probe DNA three-way-junction, whose basepairing configuration can be altered as a consequence of a single-nucleotide substitution on the target. Reconfiguration of the three-way-junction alters the Cy5-upG or Cy5-upT interactions, therefore resulting in a measurable change in Cy5 FBKs. Detection of single-nucleotide variations within a sequence selected from the Kras gene is carried out to validate the concept of this new method.     

Arabidopsis ANTR1 is a thylakoid Na+-dependent phosphate transporter: functional characterization in Escherichia coli

In this study, the putative anion transporter 1 (ANTR1) from Arabidopsis thaliana was shown to be localized to the chloroplast thylakoid membrane by Western blotting with two different peptide-specific antibodies. ANTR1 is homologous to the type I of mammalian Na+-dependent inorganic phosphate (Pi) transporters. The function of ANTR1 as a Na+-dependent Pi transporter was demonstrated by heterologous expression and uptake of radioactive Pi into Escherichia coli cells. The expression of ANTR1 conferred increased growth rates to the transformed cells and stimulated Pi uptake in a pH- and Na+-dependent manner as compared with the control cells. Among various tested effectors, Pi was the preferred substrate. Although it competed with the uptake of Pi, glutamate was not transported by ANTR1 into E. coli. In relation to its function as a Pi transporter, several physiological roles for ANTR1 in the thylakoid membrane are proposed, such as export of Pi produced during nucleotide metabolism in the thylakoid lumen back to the chloroplast stroma and balance of the trans-thylakoid H+ electrochemical gradient storage.     

Design and Optimization of Mefloquine Hydrochloride Microparticles for Bitter Taste Masking

The objective of the present investigation was to reduce the bitterness with improved dissolution, in acidic medium (pH 1.2), of mefloquine hydrochloride (MFL). Microparticles were prepared by coacervation method using Eudragit E (EE) as polymer and sodium hydroxide as precipitant. A 3² full factorial design was used for optimization wherein the drug concentration (A) and polymer concentration (B) were selected as independent variables and the bitterness score, particle size and dissolution at various pH were selected as the dependent variables. The desirability function approach has been employed in order to find the best compromise between the different experimental responses. The model is further cross validated for bias. The optimized microparticles were characterized by FT-IR, DSC, XRPD and SEM. Bitterness score was evaluated by human gustatory sensation test. Multiple linear regression analysis revealed that the reduced bitterness of MFL can be obtained by controlling the dissolution of microparticles at pH 6.8 and increasing the EE concentration. The increase in polymer concentration leads to reduction in dissolution of microparticles at pH > 5 due to its insolubility. However the dissolution studies at pH 1.2 demonstrated enhanced dissolution of MFL from microparticles might be due to the high porosity of the microparticles, hydrophilic nature of the EE, and improved wettability, provided by the dissolved EE. The bitterness score of microparticles was decreased to zero compared to 3+ of pure ARM. In conclusion the bitterness of MFL was reduced with improved dissolution at acidic pH.     

Exploration of in vitro time point quantitative evaluation of newly synthesized benzimidazole and benzothiazole derivatives as potential antibacterial agents

Present communication deals with the in vitro time point quantitative antibacterial evaluation of newly synthesized 1,2-disubstituted benzimidazoles (3a–p) and 2-substituted benzothiazoles (5a–h) against Gram-positive bacteria Staphylococcus aureus, Bacillus cereus, and Gram-negative bacteria Vibrio cholerae, Shigella dysenteriae and Escherichia coli. These compounds were synthesized under mild reaction conditions using Al₂O₃–Fe₂O₃ nanocrystals as heterogeneous catalyst. Bio-evaluation studies revealed that, compounds 3a, 5a and 5d exhibited moderate to good antibacterial activity against all the tested bacterial stains. The compounds 3a, 3f and 5a have shown enhanced inhibitory activity compared with standard antibacterial drug ciprofloxacin against V. cholerae, B. cereus, and S. dysenteriae, respectively. Additionally, the compounds 3a, 3e, 3f, 3h and 5b displayed complete bactericidal activity within 24 h, whereas ciprofloxacin took 48 h to kill those bacteria completely.     

Effect of Latent Iron Deficiency on 5-Hydroxytryptamine Metabolism in Rat Brain

Eight weeks of latent iron deficiency in weaned rats maintained on an experimental low iron content diet (18-20 mg/kg) did not significantly alter the packed cell volume and hemoglobin concentration; however, the hepatic and brain nonheme iron contents decreased by 66% and 21% (p less than 0.001), respectively. The tryptophan concentration decreased by 31% and 34% in liver and brain, respectively, in rats on experimental diet (p less than 0.01). The brain 5-hydroxytryptamine and 5-hydroxyindoleacetic acid contents were reduced by 21% and 23% (p less than 0.01 and p less than 0.02), respectively. However, in the brain, weight, protein, DNA, and the activities of monoamine oxidase, aldehyde dehydrogenase, and liver tryptophan oxygenase were found to remain unaltered. When rehabilitated with a diet containing 390 mg/kg iron, rats previously maintained on the experimental diet for 2 weeks showed partial recovery in tryptophan levels both in liver and brain. However, brain 5-hydroxytryptamine and 5-hydroxyindoleacetic acid levels remained unaltered. The hepatic iron content improved without any change in brain iron content. The latent iron deficiency produced significant alterations in the metabolism of 5-hydroxytryptamine and brain iron content that could not be recovered 2 weeks after the iron rehabilitation.     

Protein profiles of two rice varieties by 2-D gel electrophoresis under moisture stress

Two rice varieties N22 (drought-tolerant) and Panidhan (drought-susceptible) were studied for the protein profiles under control, moisture stress and abscisic acid (ABA) treatments. Both qualitative and quantitative differences were observed. In control and ABA treatment, the number of polypeptides was more in N22, compared to Panidhan. However, a significant number of polypeptides were induced in Panidhan, compared to N22 under mild and severe stress.     

Pyruvate-fortified cardioplegia suppresses oxidative stress and enhances phosphorylation potential of arrested myocardium

Cardioplegic arrest for bypass surgery imposes global ischemia on the myocardium, which generates oxyradicals and depletes myocardial high-energy phosphates. The glycolytic metabolite pyruvate, but not its reduced congener lactate, increases phosphorylation potential and detoxifies oxyradicals in ischemic and postischemic myocardium. This study tested the hypothesis that pyruvate mitigates oxidative stress and preserves the energy state in cardioplegically arrested myocardium. In situ swine hearts were arrested for 60 min with a 4:1 mixture of blood and crystalloid cardioplegia solution containing 188 mM glucose alone (control) or with additional 23.8 mM lactate or 23.8 mM pyruvate and then reperfused for 3 min with cardioplegia-free blood. Glutathione (GSH), glutathione disulfide (GSSG), and energy metabolites [phosphocreatine (PCr), creatine (Cr), P(i)] were measured in myocardium, which was snap frozen at 45 min arrest and 3 min reperfusion to determine antioxidant GSH redox state (GSH/GSSG) and PCr phosphorylation potential {[PCr]/([Cr][P(i)])}. Coronary sinus 8-isoprostane indexed oxidative stress. Pyruvate cardioplegia lowered 8-isoprostane release approximately 40% during arrest versus control and lactate cardioplegia. Lactate and pyruvate cardioplegia dampened (P < 0.05 vs. control) the surge of 8-isoprostane release following reperfusion. Pyruvate doubled GSH/GSSG versus lactate cardioplegia during arrest, but GSH/GSSG fell in all three groups after reperfusion. Myocardial [PCr]/([Cr][P(i)]) was maintained in all three groups during arrest. Pyruvate cardioplegia doubled [PCr]/([Cr][P(i)]) versus control and lactate cardioplegia after reperfusion. Pyruvate cardioplegia mitigates oxidative stress during cardioplegic arrest and enhances myocardial energy state on reperfusion.     

Bioprospecting microbes for single-cell oil production from starchy wastes

Production of lipid from oleaginous yeast using starch as a carbon source is not a common practice; therefore, the purpose of this investigation was to explore the capability of starch assimilating microbes to produce oil, which was determined in terms of biomass weight, productivity, and lipid yield. Saccharomyces pastorianus, Rhodotorula mucilaginosa, Rhodotorula glutinis, and fungal isolate Ganoderma wiiroense were screened for the key parameters. The optimization was also performed by one-factor-at-a-time approach. Considering the specific yield of lipid and cell dry weight yield, R. glutinis and R. mucilaginosa showed superiority over other strains. G. wiiroense, a new isolate, would also be a promising strain for starch waste utilization in terms of extracellular and intracellular specific yield of lipids. Extracellular specific yield of lipid was highest in R. glutinis culture (0.025?g?g^?1 of biomass) followed by R. mucilaginosa (0.022?g?g^?1 of biomass) and G. wiiroense (0.020?g?g^?1 of biomass). Intracellular lipid was again highest in R. glutinis (0.048?g?g^?1 of biomass). The most prominent fatty acid methyl esters among the lipid as detected by GC-MS were saturated lipids mainly octadecanoic acid, tetradecanoate, and hexadecanoate. Extracellular lipid produced on starch substrate waste would be a cost-effective alternative for energy-intensive extraction process in biodiesel industry.