Evaluation of Antimicrobial and Antifungal efficacy of Chitosan as endodontic irrigant against Enterococcus Faecalis and Candida Albicans Biofilm formed on tooth substrate.

BACKGROUND: Bacterial biofilms formed on the root canal wall are often difficult to remove. This study aimed to evaluate the cytotoxic effect and antibacterial efficacy of chitosan when used as root canal irrigant against E. Faecalis and Candida albicans biofilm formed on tooth substrate. MATERIAL AND METHODS: The present study evaluated antibacterial effect of 0.25% Chitosan, 0.5% Chitosan, 2% chlorhexidine and 3% sodium hypochlorite against Enterococcus faecalis and Candida Albicans. Agar-well diffusion methods, minimal inhibitory concentration tests and biofilm susceptibility assays were used to determine antibacterial activity. Teeth specimens were sectioned to obtain a standardized tooth length of 12mm. Specimens were inoculated with 10 mL of the freshly prepared E. Faecalis suspension and Candida albicans for 4 weeks. The specimens were then instrumented with ProTaper rotary files F3 size. After irrigation with test solution, three sterile paper points were placed into one canal, left for 60 s and transferred to a test tube containing 1 mL of reduced transport fluid. The number of CFU in 1 mL was determined. RESULTS: 3-week biofilm qualitative assay showed complete inhibition of bacterial growth with 3% Sodium hypochlorite, 2% Chlorhexidine and Chitosan except saline, which showed presence of bacterial growth. Significant reduction of colony forming units (CFU)/mL was observed for the chitosan groups and the antibacterial activity of the chitosan groups was at par with 3% NaOCl and 2% Chlorhexidine. It was observed that the chitosan showed no cytotoxicity at 3mg/ml and 10% cytotoxicity at 6mg/ml. CONCLUSIONS: The use of chitosan as a root canal irrigant might be an alternative considering the various undesirable properties of NaOCl and chlorhexidine. Key words:Biofilm, Candida albicans, Chitosan, Cytotoxicity, Enterococcus faecalis.

Biocatalytic Synthesis of Novel Partial Esters of a Bioactive Dihydroxy 4-Methylcoumarin by Rhizopus oryzae Lipase (ROL).

Highly regioselective acylation has been observed in 7,8-dihydroxy-4-methylcoumarin (DHMC) by the lipase from Rhizopus oryzae suspended in tetrahydrofuran (THF) at 45 °C using six different acid anhydrides as acylating agents. The acylation occurred regioselectively at one of the two hydroxy groups of the coumarin moiety resulting in the formation of 8-acyloxy-7-hydroxy-4-methylcoumarins, which are important bioactive molecules for studying biotansformations in animals, and are otherwise very difficult to obtain by only chemical steps. Six monoacylated, monohydroxy 4-methylcoumarins have been biocatalytically synthesised and identified on the basis of their spectral data and X-ray crystal analysis.

Chemoenzymatic synthesis of 3'-deoxy-3'-(4-substituted-triazol-1-YL)-5-methyluridine.

An efficient protocol has been developed for the synthesis of a small library of 3'-deoxy-3'-(4-substituted-triazol-1-yl)-5-methyluridine using Cu(I)-catalyzed Huisgen-Sharpless-Meldal 1,3-dipolar cycloaddition reaction of 3'-azido-3'-deoxy-5-methyluridine with different alkynes under optimized condition in an overall yields of 76%-92%. Here, the azido precursor compound, i.e., 3'-azido-3'-deoxy-5-methyluridine was chemoenzymatically synthesized from D-xylose in good yield. Some of the alkynes used in cycloaddition reaction were synthesized by the reaction of hydroxycoumarins or naphthols with propargyl bromide in acetone using K2CO3in excellent yields. All synthesized compounds were unambiguously identified on the basis of their spectral (IR, (1)H-, (13)C NMR spectra, and high-resolution mass spectra) data analysis.

A rapid, efficient and sensitive plate assay for detection and screening of l-asparaginase-producing microorganisms.

l-Asparaginase-producing microbes are conventionally screened on phenol red l-asparagine-containing plates. However, sometimes the contrast of the zone obtained (between yellow and pink) is not very sharp and distinct. In the present investigation, an improved method for screening of the microorganisms producing extracellular l-asparaginase is reported wherein bromothymol blue (BTB) is incorporated as pH indicator in l-asparagine-containing medium instead of phenol red. Plates containing BTB at acidic pH are yellow and turn dark blue at alkaline pH. Thus, a dense dark blue zone is formed around microbial colonies producing l-asparaginase, differentiating between enzyme producers and non-producers. The present method is more sensitive and accurate than the conventional method for screening of both fungi and bacteria producing extracellular l-asparaginase. Furthermore, BTB gives a transient green colour at neutral pH (7.0) and dark blue colour at higher pH 8.0-9.0, indicating the potency of the microorganism for l-asparaginase production.

Fermentation behavior of an osmotolerant yeast D. hansenii for Xylitol production.

Realizing the importance of xylitol as a high-valued compound that serves as a sugar substitute, a new, one step thin layer chromatographic procedure for quick, reliable, and efficient determination of xylose and xylitol from their mixture was developed. Two hundred and twenty microorganisms from the laboratory stock cultures were screened for their ability to produce xylitol from D-xylose. Amongst these, an indigenous yeast isolate no.139 (SM-139) was selected and identified as Debaryomyces hansenii on the basis of morphological and biochemical characteristics and (26S) D1/D2 r DNA region sequencing. Debaryomyces hansenii produced 9.33 gL(-1) of xylitol in presence of 50.0 gL(-1) of xylose in 84 h at pH 5.5, 30°C, 200 rpm. In order to utilize even higher concentrations of xylose for maximum xylitol production, a xylose enrichment technique was developed. The strain of Debaryomyces hansenii was obtained through xylose enrichment technique in a statistically optimized medium containing 0.3% yeast extract, 0.2% peptone, 0.03% MgSO(4) .7H(2) O along with 1% methanol. The culture was inoculated with 6% inoculum and incubated at 30°C and 250 rpm. A yield of 0.6 gg(-1) was obtained with a xylitol volumetric productivity of 0.65 g/L h(-1) in the presence of 200 gL(-1) of xylose although up to 300 gL(-1) of xylose could be tolerated through batch fermentation. Through this technique, even higher concentrations of xylose as substrate could be potentially utilized for maximum xylitol production.

Moonlighting protein in Starkeyomyces koorchalomoides: characterization of dihydrolipoamide dehydrogenase as a protein acetyltransferase utilizing acetoxycoumarin as the acetyl group donor.

In this report we have identified for the first time a transacetylase (TAase) in a mesophilic fungi Starkeyomyces koorchalomoides catalyzing the transfer of acetyl group from polyphenolic acetate (PA) to a receptor protein glutathione S-transferase (GST). An elegant assay procedure was established for TAase based on its ability to mediate inhibition of GST by 7,8-diacetoxy-4-methylcoumarin (DAMC), a model PA. Utilizing this assay procedure, S. koorchalomoides TAase was purified to homogeneity. TAase was found to have MW of 50 kDa. The purified enzyme exhibited maximum activity at 45 degrees C at pH 6.8. The N-terminal sequence of purified fungal TAase (ANDASTVED) showed identity with corresponding N-terminal sequence of dihydrolipoamide dehydrogenase (LADH), a mitochondrial matrix enzyme and an E3 component of pyruvate dehydrogenase complex (PDHC). TAase was found to have all the properties of LADH and avidly interacted with the anti-LADH antibody. TAase catalyzed acetylation of GST by DAMC was identified by LC-MS/MS and a single lysine residue (Lys-113) was found to be acetylated. Further, recombinant LADH from Streptococcus pneumoniae lacking lipoyl domain was found to exhibit little TAase activity, suggesting the role of lipoyl domain in the TAase activity of LADH. These observations bear evidence for the protein acetyltransferase activity of LADH. Such an activity of LADH can be attributed as a moonlighting function of the enzyme.

Statistical optimization for succinic acid production from E. coli in a cost-effective medium.

Response surface methodology (RSM) was employed for optimization of medium components and cultural parameters in cost effective cane molasses based medium for attaining high yield of succinic acid. The important factors obtained by "one-variable-at-a-time-approach" (cane molasses, corn steep liquor, sodium carbonate, and inoculum density) were further optimized by RSM. The optimum values of the parameters obtained through RSM (cane molasses 12.5%, corn steep liquor 7.5%, and sodium carbonate 25 mM) led to almost double yield of succinic acid (15.2 g/l in 36 h) as against "one-variable-at-a-time-approach" (7.1 g/l in 36 h) in 500-ml anaerobic bottles containing 300-ml cane molasses based medium. Subsequently, in 10-l bioreactor succinic acid production from Escherichia coli was further improved to 26.2 g/l in 30 h under conditions optimized through RSM. This fermentation-derived succinic acid will definitely help in replacing existing environmentally hazardous and cost-intensive chemical methods for the production of succinic acid.

Novel method of using dynamic electrical impedance signals for noninvasive diagnosis of knee osteoarthritis.

Osteoarthritis (OA) of knee is the most commonly occurring non-fatal irreversible disease, mainly in the elderly population and particularly in female. Various invasive and non-invasive methods are reported for the diagnosis of this articular cartilage pathology. Well known techniques such as X-ray, computed tomography, magnetic resonance imaging, arthroscopy and arthrography are having their disadvantages, and diagnosis of OA in early stages with simple effective noninvasive method is still a biomedical engineering problem. Analyzing knee joint noninvasive signals around knee might give simple solution for diagnosis of knee OA. We used electrical impedance data from knees to compare normal and osteoarthritic subjects during the most common dynamic conditions of the knee, i.e. walking and knee swing. It was found that there is substantial difference in the properties of the walking cycle (WC) and knee swing cycle (KS) signals. In experiments on 90 pathological (combined for KS and WC signals) and 72 normal signals (combined), suitable features were drawn. Then signals were used to classify as normal or pathological. Artificial multilayer feed forward neural network was trained using back propagation algorithm for the classification. On a training data set of 54 signals for KS signals, the classification efficiency for a test set of 54 was 70.37% and 85.19% with and without normalization respectively wrt base impedance. Similarly, the training set of 27 WC signals and test set of 27 signals resulted in 77.78% and 66.67% classification efficiency. The results indicate that dynamic electrical impedance signals have potential to be used as a novel method for noninvasive diagnosis of knee OA.

Rapid screening procedures for identification of succinic acid producers.

Succinic acid, an intermediate of tricarboxylic acid cycle, is produced and accumulated by anaerobic microorganisms. The long-standing interest in the production of this organic acid is because it is a key compound in producing more than 30 commercially important products. The detection of succinic acid is generally carried out by gas chromatography (GC), enzymatic assays, ion-exclusion chromatography (IEC) or by high performance liquid chromatography (HPLC). However, these methods are time consuming, require sophisticated instrumentation and are expensive. In the present investigation we are reporting two rapid, cost effective screening methods for the detection of this important organic acid. These methods can be utilized to screen a large number of microbes producing succinic acid in a very short span of time.

Chemical variability in the essential oil components of Achillea millefolium agg. from different Himalayan habitats (India).

Essential oil diversity was studied in wild Achillea millefolium from two different high altitude Himalayan habitats (1600 m, 2850 m) and their cultivated populations under uniform environmental conditions at lower altitudes of Jammu (300 m). The populations proved to represent two different ecotypes: the 1,8-cineole type and the borneol type with appreciable differences in the contents of oils and mono- and sesquiterpenes. Populations from all these habitats showed considerable overlap in various constituents and the major components were characterized as beta-pinene (10.6 % - 17.7 %), 1,8-cineole (3.0 % - 15.1 %), borneol (0.2 % - 12.1 %), and beta-caryophyllene (8.5 % - 16.2 %). No variation in morphology and chromosome number was observed under comparable environmental conditions from different habitats. Preliminary investigation indicates the existence of different ecotypes from the Himalayan habitats.

Essential oil composition of aerial parts of Angelica glauca growing wild in North-West Himalaya (India).

Fresh aerial parts of Angelica glauca, growing wild in Kashmir valley in higher Himalaya (Jammu and Kashmir, India), collected at flowering stage from different locations, on hydro-distillation provided a refreshing light pale coloured essential oil with characteristic floral woody flavour. The oil was found to be a complex mixture of mono- and sesquiterpenes and 34 compounds accounting for nearly 97.4% of the oil were characterized with the help of capillary GC, GC-MS, and NMR. Major compounds of the oil were characterized as alpha-phellandrene (13.5%), trans-carveol (12.0%), beta-pinene (11.7%), thujene (7.5%), beta-caryophyllene oxide (7.2%), beta-caryophyllene (7.0%), gamma-terpinene (6.7%), nerolidol (6.5%), beta-bisabolene (5.2%) and germacrene D (4.5%). It is the first report to exploit the essential oil from Himalayan A. glauca herb collected at flowering stage.

Statistical optimization of tannase production from Penicillium variable using fruits (chebulic myrobalan) of Terminalia chebula.

Statistically based experimental designs were applied to the optimization of cultural conditions for tannase production, an enzyme of great importance, from Penicillium variable. First, D-optimal design was used to evaluate the effects of variables, including concentrations of substrate (chebulic myrobalan, fruits of the tree Terminalia chebula ), pH, inoculum density, agitation and incubation period, on tannase production. The optimum value of pH and inoculum density thus obtained was 5.0 and 5 x 10(7) spores/50 ml respectively. Among these variables, substrate concentration, agitation and incubation period were identified to have the significant effects. Subsequently, the concentrations of substrate, agitation and incubation period were optimized using central composite design. The optimum values of the parameters thus obtained from the response surface methodology were 5.8 g of the substrate/50 ml of the medium, pH 5.0, 5 x 10(7) spores/50 ml of inoculum density, 150 rev./min agitation rate and 72 h of incubation period. The subsequent verification experiments confirmed the validity of the models. This optimization strategy led to a 2.4-fold increase in the enzyme production from 13.6 units/ml obtained in D-optimal design to 33 units/ml in central composite design.

Synthetic and novel biocatalytic resolution studies on (+/-)-5/6/7-acetoxy-4-aryl-3,4-dihydrocoumarins.

Eleven (+/-)-5/6/7-acetoxy-4-aryl-3,4-dihydrocoumarins have been synthesised in two steps starting from the coupling of cinnamic acid/substituted cinnamic acid with appropriate phenols, followed by acetylation in 50-83% overall yields. All hydroxy- and acetoxycoumarins were unambiguously identified on the basis of their spectral data. Candida antarctica lipase-catalysed deacetylation of these racemic acetoxydihydrocoumarins in dioxane occurred with moderate enantioselectivity. This is one of the rare examples of resolution using phenolic ester moiety as a remote handle for chiral recognition by a lipase.

Isolation, part characterization, immunogenicity, and specificity study of Plasmodium falciparum culture supernatant.

A Plasmodium falciparum malaria blood stage antigen was isolated from in vitro parasite culture supernatant. The chemical composition of the antigen was studied by high-performance thin-layer chromatography, thin-layer chromatography, gas-liquid chromatography, and other chemical methods. Such analysis indicated it to be a glycophospholipid (GPL) and to be composed of xylose, mannose, galactose, and glucose linked to a phospholipid, but no inositol. The extracted and purified antigen's sensitivity and specificity properties were assessed by laser immuno assay and enzyme-linked immunosorbent assay. The results of the sensitivity study showed a very high malaria antibody-binding response compared to other known antigens. The specificity study of GPL antigen with different nonmalarial samples showed no positive response within the limit of significance. This isolated GPL antigen appears to be better than other antigens.