Impact of oxidative and osmotic stresses on Candida albicans biofilm formation.

Candida albicans possesses an ability to grow under different host-driven stress conditions by developing robust protective mechanisms. In this investigation the focus was on the impact of osmotic (2M NaCl) and oxidative (5 mM H2O2) stress conditions during C. albicans biofilm formation. Oxidative stress enhanced extracellular DNA secretion into the biofilm matrix, increased the chitin level, and reduced virulence factors, namely phospholipase and proteinase activity, while osmotic stress mainly increased extracellular proteinase and decreased phospholipase activity. Fourier transform infrared and nuclear magnetic resonance spectroscopy analysis of mannan isolated from the C. albicans biofilm cell wall revealed a decrease in mannan content and reduced ß-linked mannose moieties under stress conditions. The results demonstrate that C. albicans adapts to oxidative and osmotic stress conditions by inducing biofilm formation with a rich exopolymeric matrix, modulating virulence factors as well as the cell wall composition for its survival in different host niches.

Efficacy of ferulic acid encapsulated chitosan nanoparticles against Candida albicans biofilm.

Candida albicans, an opportunistic fungal pathogen is a major causative agent of superficial to systemic life-threating biofilm infections on indwelling medical devices. These biofilms acts as double edge swords owing to their resistance towards antibiotics and immunological barriers. To overcome this threat ferulic acid encapsulated chitosan nanoparticles (FA-CSNPs) were formulated to assess its efficacy as an antibiofilm agent against C. albicans. These FA-CSNPs were synthesized using ionotropic gelation method and observed through field emission scanning electron microscopy (FESEM) and fluorescent microscopy. Assessment of successful encapsulation and stability of ferulic acid into chitosan nanoparticles was made using Fourier transform infrared spectrum (FTIR), (1)H NMR and thermal analyses. Synthesized FA-CSNPs, were found to be cytocompatible, when tested using Human Embryonic Kidney (HEK-293) cell lines. XTT assay revealed that FA-CSNPs reduced the cell metabolic activity of C. albicans upto 22.5% as compared to native ferulic acid (63%) and unloaded CSNPs (88%) after 24 h incubation. Disruption of C. albicans biofilm architecture was visualized by FESEM. Results highlighted the potential of FA-CSNPs to be used as an effective alternative to the conventional antifungal therapeutics.

Modulation of Candida albicans Biofilm by Different Carbon Sources.

In the present investigation, the role of carbon sources (glucose, lactate, sucrose, and arabinose) on Candida albicans biofilm development and virulence factors was studied on polystyrene microtiter plates. Besides this, structural changes in cell wall component ß-glucan in presence of different carbon sources have also been highlighted. Biofilm formation was analyzed by XTT (2,3-bis[2-Methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay. Glucose-grown cells exhibited the highest metabolic activity during adhesion among all carbon sources tested (p < 0.05). However, cells exposed to sucrose exhibited highest biofilm formation and matrix polysaccharides secretion after 48 h. The results also correlated with the biofilm height and roughness measurements by atomic force microscopy. Exposure to lactate induced hyphal structures with the highest proteinase activity while arabinose-grown cells formed pseudohyphal structures possessing the highest phospholipase activity. Structural changes in ß-glucan characterized by Fourier transform infrared (FTIR) spectroscopy displayed characteristic band of ß-glucan at 892 cm(-1) in all carbon sources tested. The ß(1→6) to ß(1→3) glucan ratio calculated as per the band area of the peak was less in lactate (1.15) as compared to glucose (1.73), sucrose (1.62), and arabinose (2.85). These results signify that carbon sources influence C. albicans biofilm development and modulate virulence factors and structural organization of cell wall component ß-glucan.

Candida biofilm disrupting ability of di-rhamnolipid (RL-2) produced from Pseudomonas aeruginosa DSVP20.

Biosurfactant produced from Pseudomonas aeruginosa DSVP20 was evaluated for its potential to disrupt Candida albicans biofilm formed on polystyrene (PS) surfaces in this investigation. P. aeruginosa DSVP20 exhibited optimum production of biosurfactant (5.8 g L(-1)) after 96 h of growth with an ability to reduce surface tension of the aqueous solution from 72 to 28 mN m(-1). Analysis of purified biosurfactant with FT-IR, (1)H and (13)C NMR and MALDI-TOF MS revealed it to be di-rhamnolipid (RL-2) in nature. Biofilm disrupting ability of RL-2 (0.16 mg mL(-1)) on Candida cells when checked using XTT reduction assay revealed that about 50 % of the cells remain adhered to 96-well plate after 2 h of treatment, while up to 90 % reduction in pre-formed C. albicans biofilm on PS surface was observed with RL-2 (5.0 mg mL(-1)) in a dose-dependent manner. Microscopic analyses (SEM and CLSM) further confirm the influence of RL-2 on disruption of Candida biofilm extracellular matrix on PS surface which can be exploited as a potential alternative to the available conventional therapies.

Candida albicans biofilm inhibition by synergistic action of terpenes and fluconazole.

The current treatment options for Candida albicans biofilm-device related infections are very scarce due to their intrinsic increased tolerance to antimycotics. The aim of this work was to study synergistic action of terpenes (eugenol, menthol and thymol) with fluconazole (FLA) on C. albicans biofilm inhibition. The minimum inhibitory concentration (MIC) assayed using CLSI M27-A3 broth micro-dilution method showed antifungal activity against C. albicans MTCC 227 at a concentration of 0.12 % (v/v) for both thymol and eugenol as compared to 0.25 % (v/v) for menthol. FLA was taken as positive control. The effect of these terpenes on metabolic activity of preformed C. albicans biofilm cells was evaluated using 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay in 96-well polystyrene microtiter plate. Thymol and eugenol were more effective at lower concentrations of > or = 1.0 % (v/v) than menthol. Synergistic studies using checkerboard micro-dilution assay showed fractional inhibitory concentration index (sigma FIC = 0.31) between thymol/FLA followed by eugenol/FLA (sigma FIC = 0.37) and menthol/FLA (sigma FIC < 0.5) against pre-formed C. albicans biofilms. Thymol with fluconazole showed highest synergy in reduction of biofilm formation than eugenol and menthol which was not observed when their activities were observed independently. Adherence assay showed 30% viability of C. albicans cells after 2 h of treatment with 0.05 % (v/v) thymol/FLA. Effect of thymol/FLA on C. albicans adhesion visualized by SEM micrographs showed disruption in number of candidal cells and alteration in structural design of C. albicans. Thus, the study demonstrated synergistic effect of terpenes with fluconazole on C. albicans biofilm, which could be future medications for biofilm infections.

Production of microbial surfactants from oily sludge-contaminated soil by Bacillus subtilis DSVP23.

The indigenous microbial community utilizing aliphatic, aromatic, and polar components from the oily sludge as sole source of carbon and energy was selected from the soil samples of Ankleshwar, India for biosurfactant production. Evaluation of biosurfactant production was done using screening assays such as surface tension reduction, hemolytic activity, emulsification activity, drop-collapse assay, and cell surface hydrophobicity studies. Maximum biosurfactant (6.9 g/l) production was achieved after 5 days of growth from Bacillus subtilis DSVP23 which was identified by 16S RNA technique (NCBI GenBank accession no. EU679368). Composition of biosurfactant showed it to be lipopeptide in nature with 15.2% protein content and 18.0% lipid content. Functional group analysis was also done by using Fourier transform infrared spectroscopy which showed it to be a protein-bound lipid thereby imparting them special properties. Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric and nuclear magnetic resonance revealed that the major constituents of lipopeptide are leucine and isoleucine. Gas chromatographic analysis data indicated that oily sludge components of chain length C12-C30 and aromatic hydrocarbons were degraded effectively by B. subtilis DSVP23 after 5 days of incubation. These results collectively points toward the importance of B. subtilis DSVP23 as a potential candidate for bioremediation studies.

Biological activity of the functional epitope of ciguatoxin fragment AB on the neuroblastoma sodium channel in tissue culture.

It is well established that the targeted receptor for ciguatoxin (CTX) in mammalian tissues is the sodium channel, affecting the influx of sodium into cells and altering the action potential and function of the cell. Since the syntheses of fragments of CTX has become available, our focus has been on the receptor functions of the west sphere AB and east sphere JKLM fragments using the neuroblastoma cell assay, guinea pig atrium assay, and the membrane immunobead assay (MIA). The data presented here suggest that the west sphere AB of the ciguatoxin molecule is the active portion and is responsible for the activation of the sodium channels.

Studies on the development of potential biomarkers for rapid assessment of copper toxicity to freshwater fish using Esomus danricus as model.

Living in an environment that has been altered considerably by anthropogenic activities, fish are often exposed to a multitude of stressors including heavy metals. Copper ions are quite toxic to fish when concentrations are increased in environmental exposures often resulting in physiological, histological, biochemical and enzymatic alterations in fish, which have a great potential to serve as biomarkers. Esomus danricus was chosen as model in the present study and the metabolic rate, gill morphology, total glycogen, total protein, superoxide dismutase and catalase were critically evaluated. The 96h LC50 value was found to be 5.5mg/L (Cu as 1.402 mg/L). Fish groups were separately exposed to lethal (5.5 mg/L) and sub lethal concentrations (0.55 mg/L) of copper sulphate over a period of 96h to examine the subtle effects caused at various functional levels. Controls were also maintained simultaneously. Significant decrease in the metabolic rate (p<0.001) of the fish was observed in both the concentrations studied. Studies employing Automated Video Tracking System revealed gross changes in the architecture of gill morphology like loss, fusion, clubbing of secondary gill lamellae, and detachment of gill rakers following softening of gill shaft in fish under lethal exposures indicating reduced respiratory surface area. Biochemical profiles like total glycogen and total protein in gills and muscle of fish exposed to 5.5 mg/L showed appreciable decrease (p<0.05 to 0.001) from control. Significant inhibition of superoxide dismutase (60.83%), catalase (71.57%) from control was observed in fish exposed to 5.5 mg/L at the end of 96h exposure only. Interestingly, in fish exposed to 0.55 mg/L enzyme activity is not affected except for catalase. Toxic responses evaluated at various functional levels are more pronounced in fish exposed to 5.5 mg/L and these can serve as potential biomarkers for rapid assessment of acute copper toxicity in environmental biomonitoring.

Cardiac electrophysiological actions of NS-21 and its active metabolite, RCC-36, compared with terodiline.

Terodiline, an anticholinergic drug with a Ca2+ blocking action, is thought to be associated with torsade de pointes, a serious ventricular tachycardia. NS-21 is a newly developed drug for the treatment of urinary frequency and urinary incontinence and it has pharmacological properties similar to those of terodiline. It remains unknown, however, whether NS-21 and its active metabolite, RCC-36, have any proarrhythmic activity. The electrophysiological properties of NS-21 and RCC-36 were examined in guinea pig ventricular myocytes and were compared with those of terodiline using the whole-cell patch-clamp technique. NS-21, RCC-36 and terodiline inhibited L-type Ca2+ currents in a concentration-dependent manner with IC50 values of 27.0, 27.0 and 33.5 microM, respectively. At a concentration of 10 microM, terodiline inhibited both the time-dependent current and the tail current of the delayed rectifier K+ current, with the latter being significantly inhibited at voltages more positive than +10 mV. In contrast, NS-21 and RCC-36 had almost no effect on either of these currents. Terodiline also inhibited the inward rectifier K+ current significantly at voltages more negative than -100 mV, whereas NS-21 and RCC-36 had little effect. If the proarrhythmic activity of terodiline resulted primarily from the combined inhibition of K+ and Ca2+ currents, one might expect that NS-21 and RCC-36, which inhibit L-type Ca2+ currents without affecting either the delayed rectifier K+ current or the inward rectifier K+ current, would not share the proarrhythmic activities of terodiline.

Na+ and high-voltage-activated Ca2+ channel blocking actions of NS-7, a novel neuroprotective agent, in NG108-15 cells.

The actions of a novel neuroprotective compound, 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride (NS-7), on voltage-gated Na+, Ca2+ and K+ channels were investigated in a mouse neuroblastoma and rat glioma hybrid cell line, NG108-15, using a whole-cell voltage clamp technique. NG108-15 cells have a tetrodotoxin-sensitive Na+ channel, three types of Ca2+ channel (L, N and T) and voltage-gated K+ channels, all of which were inhibited by NS-7 in a concentration-dependent manner. However, there was a considerable difference in its potency: the IC50 values for the tetrodotoxin-sensitive Na+ channel, L-type Ca2+ channel and N-type Ca2+ channel were similar (7.8, 4.5 and 7.3 microM, respectively), lower than the IC50 value for the T-type Ca2+ channel (17.1 microM), and much lower than the IC50 value for the voltage-gated K+ channel (160.5 microM). NS-7 altered neither the shape nor the reversal potential of the current-voltage curves for Na+, L-type or N-type Ca2+ channels, although the currents were reduced at every potential tested. These results indicate that NS-7 is a Na+ and high-voltage-activated (L- and N-type) Ca2+ channel blocker, and its channel-blocking properties may contribute to its neuroprotective action.

Long-term potentiation of glycinergic inhibitory synaptic transmission.

1. Tetanizing protocols were used to test whether glycinergic inhibition undergoes long-term plasticity in vivo. For this purpose we studied the inhibition evoked disynaptically in the teleost Mauthner (M) cell by stimulation of the posterior branch of the contralateral VIIIth nerve. The advantage of this experimental design is that the inhibition, which is mediated by identified second-order commissural interneurons, is not contaminated by parallel excitation. 2. The VIIIth-nerve-evoked inhibitory postsynaptic potentials (IPSPs), which are generated at the level of the soma, are depolarizing in Cl(-)-loaded M cells. After VIIIth nerve tetanization, these IPSPs exhibited potentiation lasting > 30 min in 23 of 31 cells. The maximum enhancement measured 5-10 min after the onset of the tetanization averaged 100 +/- 19% (mean +/- SE). In contrast, the non-"tetanized" collateral IPSP induced by antidromic stimulation of the M axon did not increase significantly suggesting synaptic specificity of the potentiation. 3. Single-electrode voltage-clamp studies of Cl(-)-loaded M cells indicated that this plasticity is due to an increased synaptic conductance that occurs without obvious modifications of the kinetics or voltage dependence of the inhibitory postsynaptic currents. 4. The synaptic conductance and its changes during potentiation were quantified by measuring the inhibitory shunt of the antidromic spike while recording with potassium-acetate-filled electrodes. For this purpose the ratio, r', of the inhibitory to resting membrane conductances, was calculated using the expression (V/V')--1, where V and V' are the amplitudes of the control and the test antidromic spikes, respectively. This ratio was called fractional conductance. Measured at the peak of the expected VIIIth-nerve-evoked IPSP, r' increased by 114 +/- 18% (n = 46). Again the collateral inhibitory conductance was not modified. 5. Because there are two synapses in the inhibitory pathway, it became important to determine whether modifications of the second-order inhibitory junctions contribute to the overall potentiation. Several experimental procedures were used for this purpose. 6. The input-output relationship at the inhibitory synapses was determined by comparing the size of the presynaptic volley and r'. The former was recorded intra- or extracellularly as a monophasic positive potential, the so-called extrinsic hyperpolarizing potential, which increases in parallel with the strength of VIIIth nerve stimulation. In 12 experiments where the presynaptic volley was unaffected by the tetanization, suggesting lack of involvement of the first relay, r' nevertheless increased in amplitude by 79 +/- 14%.(ABSTRACT TRUNCATED AT 400 WORDS)