Biocompatible carbon quantum dots as versatile imaging nanotrackers of fungal pathogen - Candida albicans.

Aim: The development of carbon quantum dots (C-QDs) as nanotrackers to understand drug-pathogen interactions, virulence and multidrug resistance. Methods: Microwave synthesis of C-QDs was performed using citric acid and polyethylene glycol. Further, in vitro toxicity was evaluated and imaging applications were demonstrated in Candida albicans isolates. Results: Well-dispersed, ultra small C-QDs exhibited no cyto/microbial/reactive oxygen species-mediated toxicity and internalized effectively in Candida yeast and hyphal cells. C-QDs were employed for confocal imaging of drug-sensitive and -resistant cells, and a study of the yeast-to-hyphal transition using atomic force microscopy in Candida was conducted for the first time. Conclusion: These biocompatible C-QDs have promising potential as next-generation nanotrackers for in vitro and in vivo targeted cellular and live imaging, after functionalization with biomolecules and drugs.

Scientists have used radiolabeled drugs and radioactive tracking agents for the imaging and study of drug resistance in microbial pathogens. But, these radiolabeled drugs or radiotrackers pose health hazards and environmental risks. However, such limitations can be overcome by designing nontoxic, environment-friendly, nanotechnology-based fluorescent imaging agents. This study demonstrates the development and application of cost-effective, nontoxic carbon-based quantum dots for imaging of drug-sensitive and -resistant microbial strains and transition to different morphological forms (yeast-to-hyphae transition) in fungal pathogens. The results demonstrated the suitability of carbon quantum dots as next-generation nano-based bioimaging/tracking agents for cellular imaging. The availability of such nontoxic fluorescent tracking agents is likely to offer promising solutions in therapeutics and diagnostics by providing insight into various mechanisms and functional links related to drug resistance, virulence and pathogenicity.

Spectroscopic investigations on fungal aspartic protease as target of gallic acid.

Proteases are a major virulence factor in pathogenic fungi and can serve as a potential therapeutic target. The interaction of gallic acid (GA) with Aspartic fungal protease (PepA) was investigated using biophysical and in silico approaches. UV-Vis and fluorescence spectroscopy showed complex formation and static quenching of PepA by GA with Ka of 7.4 × 105 M-1 and stoichiometric binding site (n) of 1.67. CD-spectroscopy showed marked changes in helical content and synchronous fluorescence spectra measurements indicated significant changes in the microenvironment around tryptophan residues in the GA-PepA complex. Outcomes of Isothermal Titration Calorimetry (ITC) measurement and molecular modelling studies validated the spectroscopic results. The binding of GA to Human Serum albumin (HSA) was moderate (Ka = 1.9 × 103 M-1) and did not cause structural disruption of HSA. To conclude, gallic acid is strongly bound to fungal protease leading to structural disruption and inhibition whereas HSA structure was largely conserved. Gallic acid thus appears to be a potential therapeutic agent against fungal proteases.

Quercetin and resveratrol ameliorate nickel-mediated hypercontraction in isolated Wistar rat aorta.

PURPOSE: The ameliorative potential of quercetin and resveratrol on isolated endothelium-intact aortic rings incubated with nickel was examined. METHOD: The effect of varying concentrations of quercetin and resveratrol was investigated on isolated Wistar rat aortic rings using an organ bath system over vasoconstrictor phenylephrine (PE) at 1 µM. To delineate the mechanism of action, isolated aortic rings were pre-incubated with pharmacological modulators, such as verapamil 1 µM, apocynin 100 µM, indomethacin 100 µM or N-G-nitro-L-arginine methyl ester (L-NAME) 100 µM, separately, before incubation with 100 µM quercetin and 30 µM resveratrol. To assess the ameliorative and prophylactic potentials of quercetin and resveratrol, aortic rings were also incubated with quercetin or resveratrol for 40 min, followed by incubation with nickel for 40 min. RESULTS: At 100 µM, quercetin caused 29% inhibition of contraction, while resveratrol at 30 µM caused 55% inhibition of contraction in aortic rings compared with control. Aortic rings incubated with contractile modulators, such as verapamil, apocynin, indomethacin or N-G-nitro-L-arginine methyl ester (L-NAME), along with quercetin or resveratrol at their concentrations producing maximum relaxant effect, showed that both of these natural compounds exert their relaxant effect by inhibiting the generation of reactive oxygen species (ROS) from endothelial and smooth muscle cells, blocking voltage-gated calcium channels, and increasing the release of nitric oxide (NO). The mediation of hypercontraction by nickel is due to the increased ROS and the influx of calcium through voltage-dependent calcium channels. These natural compounds are shown to counter the nickel-induced effects, appearing as effective ameliorators. CONCLUSION: In this study, we found that quercetin and resveratrol act as ameliorators of nickel-mediated hypercontraction by decreasing ROS and enhancing NO release from endothelial cells.

Interaction of fungal lipase with potential phytotherapeutics.

Interaction of thymol, carvacrol and linalool with fungal lipase and Human Serum Albumin (HSA) have been investigated employing UV-Vis spectroscopy Fluorescence and Circular dichroism spectroscopy (CD) along with docking studies. Thymol, carvacrol and linalool displayed approximately 50% inhibition at 1.5 mmol/litre concentrations using para-nitrophenyl palmitate (pNPP). UV-Vis spectroscopy give evidence of the formation of lipase-linalool, lipase-carvacrol and lipase-thymol complex at the ground state. Three molecules also showed complex formation with HSA at the ground state. Fluorescence spectroscopy shows strong binding of lipase to thymol (Ka of 2.6 x 109 M-1) as compared to carvacrol (4.66 x 107 M-1) and linalool (5.3 x 103 M-1). Number of binding sites showing stoichiometry of association process on lipase is found to be 2.52 (thymol) compared to 2.04 (carvacrol) and 1.12 (linalool). Secondary structure analysis by CD spectroscopy results, following 24 hours incubation at 25°C, with thymol, carvacrol and linalool revealed decrease in negative ellipticity for lipase indicating loss in helical structure as compared with the native protein. The lowering in negative ellipticity was in the order of thymol > carvacrol > linalool. Fluorescence spectra following binding of all three molecules with HSA caused blue shift which suggests the compaction of the HSA structure. Association constant of thymol and HSA is 9.6 x 108 M-1 which along with 'n' value of 2.41 suggests strong association and stable complex formation, association constant for carvacrol and linalool was in range of 107 and 103 respectively. Docking results give further insight into strong binding of thymol, carvacrol and linalool with lipase having free energy of binding as -7.1 kcal/mol, -5.0 kcal/mol and -5.2 kcal/mol respectively. To conclude, fungal lipases can be attractive target for controlling their growth and pathogenicity. Employing UV-Vis, Fluorescence and Circular dichroism spectroscopy we have shown that thymol, carvacrol and linalool strongly bind and disrupt structure of fungal lipase, these three phytochemicals also bind well with HSA. Based on disruption of lipase structure and its binding nature with HSA, we concluded thymol as a best anti-lipase molecule among three molecules tested. Results of Fluorescence and CD spectroscopy taken together suggests that thymol and carvacrol are profound disrupter of lipase structure.

Target-based drug repurposing against Candida albicans-A computational modeling, docking, and molecular dynamic simulations study.

The emergence of multidrug-resistant strains of Candida albicans has become a global threat mostly due to co-infection with immune-compromised patients leading to invasive candidiasis. The life-threatening form of the disease can be managed quickly and effectively by drug repurposing. Thus, the study used in silico approaches to evaluate Food and Drug Administration (FDA) approved drugs against three drug targets-TRR1, TOM40, and YHB1. The tertiary structures of three drug targets were modeled, refined, and evaluated for their structural integrity based on PROCHECK, ERRAT, and PROSA. High-throughput virtual screening of FDA-approved drugs (8815), interaction analysis, and energy profiles had revealed that DB01102 (Arbutamine), DB01611 (Hydroxychloroquine), and DB09319 (Carindacillin) exhibited better binding affinity with TRR1, TOM40, and YHB1, respectively. Notably, the molecular dynamic simulation explored that Gln45, Thr119, and Asp288 of TRR1; Thr107 and Ser121 of TOM40; Arg193, Glu213, and Ser228 of YHB1 are crucial residues for stable drug-target interaction. Additionally, it also prioritized Arbutamine-TRR1 as the best drug-target complex based on MM-PBSA (-52.72 kcal/mol), RMSD (2.43 Å), and radius of gyration (-21.49 Å) analysis. In-depth, PCA results supported the findings of molecular dynamic simulations. Interestingly, the conserved region (>70%) among the TRR1 sequences from pathogenic Candida species indicated the effectiveness of Arbutamine against multiple species of Candida as well. Thus, the study dispenses new insight and enriches the understanding of developing an advanced technique to consider potential antifungals against C. albicans. Nonetheless, a detailed experimental validation is needed to investigate the efficacy of Arbutamin against life-threatening candidiasis.

A systematic review on distribution and antifungal resistance pattern of Candida species in the Indian population.

The emergence of antifungal drug resistance in Candida species has led to increased morbidity and mortality in immunocompromised patients. Understanding species distribution and antifungal drug resistance patterns is an essential step for novel drug development. A systematic review was performed addressing this challenge in India with keywords inclusive of 'Candida', 'Antifungal Drug Resistance', 'Candidemia', 'Candidiasis' and 'India'. A total of 106 studies (January 1978-March 2020) from 20 Indian states were included. Of over 11,429 isolates, Candida albicans was the major species accounting for 37.95% of total isolates followed by C. tropicalis (29.40%), C. glabrata (11.68%) and C. parapsilosis (8.36%). Rates of antifungal resistance were highest in non-albicans Candida (NAC) species - C. haemuloni (47.16%), C. krusei (28.99%), C. lipolytica (28.89%) and C. glabrata (20.69%). Approximately 10.34% isolates of C. albicans were observed to be drug resistant. Candida species were frequently resistant to certain azoles (ketoconazole-22.2%, miconazole-22.1% and fluconazole-21.8%). In conclusion, the present systematic review illustrates the overall distribution and antifungal resistance pattern of Candida species among the Indian population that could be helpful in the future for the formation of treatment recommendations for the region but also elsewhere. LAY SUMMARY: A total of 106 studies were reviewed to define the prevalence, distribution and antifungal resistance pattern of Candida species in India. The presented data could become the point of reference for all reported findings on Candida species in India.

Mechanistic Insights into the Anticandidal Action of Vanillin Reveal Disruption of Cell Surface Integrity and Mitochondrial Functioning.

BACKGROUND: Considering the emergence of multidrug resistance (MDR) in prevalent human fungal pathogen, Candida albicans, there is a parallel spurt in the development of novel strategies aimed to disrupt MDR. Compounds from natural resources could be exploited as efficient antifungal drugs owing to their structural diversity, cost effectiveness and negligible side effects. OBJECTIVE: The present study elucidates the antifungal mechanisms of Vanillin (Van), a natural food flavoring agent against Candida albicans. METHODS: Antifungal activities were assessed by broth microdilution and spot assays. Membrane and cell wall perturbations were studied by PI uptake, electron microscopy, plasma membrane H+ extrusion activity and estimation of ergosterol and chitin contents. Mitochondrial functioning was studied by growth on non-fermentable carbon sources, rhodamine B labeling and using retrograde signaling mutants. Gene expressions were validated by semi-quantitative RT-PCR. RESULTS: We observed that the antifungal activity of Van was not only limited to clinical isolates of C. albicans but also against non-albicans species of Candida. Mechanistic insights revealed the effect of Van on cell surface integrity as evident from hypersensitivity against membrane perturbing agent SDS, depleted ergosterol levels, transmission electron micrographs and diminished plasma membrane H+ extrusion activity. In addition, spot assays with cell wall perturbing agents, scanning electron micrographs, delayed sedimentation rate and lower chitin content further substantiate cell wall damage by Van. Furthermore, Van treated cells underwent mitochondrial dysfunctioning via impaired retrograde signaling leading to abrogated iron homeostasis and DNA damage. All the perturbed phenotypes were also validated by RT-PCR depicting differential regulation of genes (NPC2, KRE62, FTR2 and CSM3) in response to Van. CONCLUSION: Together, our results suggested that Van is promising antifungal agent that may be advocated for further investigation in therapeutic strategies to treat Candida infections.

Genome-wide screening and in silico gene knockout to predict potential candidates for drug designing against Candida albicans.

C. albicans infections are increasingly becoming a threat to public health with emergence of drug resistant strains. It emphasizes the need to look for alternate drug targets through genome-wide screening. In the present study, whole proteome of C. albicans SC5314 was analyzed in single click target mining workflow of TiDv2. A protein-protein interaction network (PPI) for the resulting putative targets was generated based on String database. Ninety four proteins with higher connectivity (degree ≥ 10) in the network are noted as hub genes. Among them, 24 are observed to be known targets while 70 are novel ones. Further, chokepoint analysis revealed FAS2, FOL1 and ERG5 as chokepoint enzymes in their respective pathways. Subsequently, the chokepoints were selected as prior interest for in silico gene knockout via MATLAB and COBRA Toolbox. In silico gene knockout pointed that FAS2 inhibition reduced the growth rate of pathogen from 0.2879 mmol.gDW-1.h-1 to zero. Furthermore, enzyme inhibition assay of FAS2 with cerulenin strengthen the computational outcome with MIC 1.25 µg/mL. Hence, the study establishes FAS2 as a promising target to design therapeutics against C. albicans.

Cobalt-Induced Hypercontraction is Mediated by Generationof Reactive Oxygen Species and Influx of Calcium in Isolated RatAorta.

To investigate the mechanism of cobalt-mediated phenylephrine (PE)-induced contraction in endothelium-intact isolated Wistar rat aortic rings. Effect of dose-dependent concentrations of cobalt on PE-induced contraction was investigated in isolated Wistar rat aortic rings using an organ bath system. Aortic rings were pre-incubated with verapamil (1 µM and 20 µM), gadolinium, apocynin, indomethacin or N-G-nitro-L-arginine methyl ester (L-NAME) separately before incubation with cobalt. Endothelium-intact aortic rings were incubated with 800 nM, 1 µM, 10 µM, 50 µM cobalt; we observed 20%, 22%, 32% and 27% increased contractions respectively, while no effect was seen in tension recording on cobalt exposure. Incubation of endothelium-intact aortic rings with 100 µM apocynin and 100 µM L-NAME suggested the role of NADPH oxidase in generation of reactive oxygen species (ROS) and decrease in bioavailability of nitric oxide (NO) from eNOS on exposure to cobalt. Aortic rings pre-incubated with 1 µM and 20 µM verapamil suggested role of both L-type and T-type calcium channels in influx of extracellular calcium in smooth muscle cells. We observed no role of store-operated calcium channels (SOCC) in calcium influx due to cobalt exposure and cyclooxygenase in generation of prostanoids in isolated aortic rings. Cobalt caused rise of PE-induced contractions as a result of the endothelial generation of ROS, by decreasing bioavailability of NO. Generation of ROS may be responsible for causing the influx of extracellular calcium through L-type and T-type Ca2+ channels in smooth muscle cells.

Octyl gallate triggers dysfunctional mitochondria leading to ROS driven membrane damage and metabolic inflexibility along with attenuated virulence in Candida albicans.

Recently the high incidence of worldwide Candida infections has substantially increased. The growing problem about toxicity of antifungal drugs and multidrug resistance aggravates the need for the development of new effective strategies. Natural compounds in this context represent promising alternatives having potential to be exploited for improving human health. The present study was therefore designed to evaluate the antifungal effect of a naturally occurring phenolic, octyl gallate (OG), on Candida albicans and to investigate the underlying mechanisms involved. We demonstrated that OG at 25 µg/ml could effectively inhibit C. albicans. Mechanistic insights revealed that OG affects mitochondrial functioning as Candida cells exposed to OG did not grow on non-fermentable carbon sources. Dysfunctional mitochondria triggered generation of reactive oxygen species (ROS), which led to membrane damage mediated by lipid peroxidation. We explored that OG inhibited glucose-induced reduction in external pH and causes decrement in ergosterol levels by 45%. Furthermore, OG impedes the metabolic flexibility of C. albicans by inhibiting the glyoxylate enzyme isocitrate lyase, which was also confirmed by docking analysis. Additionally, OG affected virulence traits such as morphological transition and cell adherence. Furthermore, we depicted that OG not only prevented biofilm formation but eliminates the preformed biofilms. In vivo studies with Caenorhabditis elegans nematode model confirmed that OG could enhance the survival of C. elegans after infection with Candida. Toxicity assay using red blood cells showed only 27.5% haemolytic activity. Taken together, OG is a potent inhibitor of C. albicans that warrants further structural optimization and pharmacological investigations.

Impact of length of replication competent genome of hepatitis B virus over the differential antigenic secretion.

Hepatitis B virus (HBV) genome consists of circular partially double stranded DNA of 3.2 kb size which gets converted into covalently closed circular DNA (cccDNA) during its life cycle. It then acts as a template for formation of pregenomicRNA (pgRNA) of 3.5 kb. Absence of appropriate animal models prompted a need to establish a better in vitro culture system to uncover the propagation and survival mechanisms of the virus. There is scarcity of data to represent the significance of varying length of replication competent viral genome on the secretion of viral secretory proteins/antigens and in turn on the overall effects on the accomplishment of the viral life cycle. The present study was undertaken to ascertain a suitable replication competent construct in which the viral life cycle of HBV with varying clinical relevance can be studied efficiently. Two constructs (pHBV 1.3 and pHBV 1X) of different sizes were used to transfect hepatoma cells and consequently the secretory antigens were monitored. In vector free approach (pHBV 1X), 3.2 kb viral DNA is directly transfected in the culture system whereas in vector mediated approach more than full length of viral genome is cloned in a vector (pHBV 1.3X) and transfected to obtain a 3.5 kb pgRNA intermediate. HBV secretes two important antigens; HBsAg and HBeAg. HBsAg is a hallmark of infection and is the first to be secreted in the blood stream whereas HBeAg is a secretory protein and remains associated with the viral replication. The construct pHBV 1.3X referring to as more than full length, by virtue of being capable of undergoing transcription without the synthesis of cccDNA intermediate (unlike the clinical situation where an intermediate step of cccDNA synthesis is an essential component to initiate the viral life cycle) appears to be better system for studying viral life cycle in in vitro culture system. The reasons could be assigned to the fact that as low as 100 ng of viral DNA was shown to quantify the replicative phenotypes with this construct. The better efficiency of this construct at prima facie, appears to be mediated through the significantly higher levels of pgRNA transcript during the viral life cycle.

Role of calcium channels and endothelial factors in nickel induced aortic hypercontraction in Wistar rats.

AIM: To investigate the mechanism of nickel augmented phenylephrine (PE)-induced contraction in isolated segments of Wistar rat aorta. MATERIALS AND METHODS: Effect of varying concentrations of nickel on PE-induced contraction were investigated in isolated segments of Wistar rat aorta using an organ bath system. Aortic rings were pre-incubated with verapamil (1 µM and 20 µM), gadolinium, apocynin, indomethacin or N-G-nitro-L-arginine methyl ester (L-NAME) separately before incubation with nickel. RESULTS: Endothelium intact aortic rings incubated with 100 nM, 1 µM or 100 µM of nickel exhibited 80%, 43% and 28% increase in PE-induced contraction, respectively, while no such enhancing responses were observed in endothelium denuded aorta. Incubation of aortic rings with 1 µM and 20 µM verapamil suggested an involvement of influx of calcium through T-type calcium channels in smooth muscle cells, while aortic rings pre-incubated with gadolinium showed no role of store operated calcium channels in the nickel effect on PE-induced contractions. The enhancing effect of nickel on PE-induced contractions was inhibited by apocynin, indomethacin or L-NAME. CONCLUSION: Nickel has caused augmentation of PE-induced contractions as a result of the endothelial generation of reactive oxygen species (ROS) and cyclooxygenase 2 (COX2) dependent endothelium contracting factors (EDCFs), which increases the influx of extracellular calcium through T-type Ca2+ channels in smooth muscle cells.

Therapeutic Potential of Dietary Phenolic Acids.

Although modern lifestyle has eased the quality of human life, this lifestyle's related patterns have imparted negative effects on health to acquire multiple diseases. Many synthetic drugs are invented during the last millennium but most if not all of them possess several side effects and proved to be costly. Convincing evidences have established the premise that the phytotherapeutic potential of natural compounds and need of search for novel drugs from natural sources are of high priority. Phenolic acids (PAs) are a class of secondary metabolites spread throughout the plant kingdom and generally involved in plethora of cellular processes involved in plant growth and reproduction and also produced as defense mechanism to sustain various environmental stresses. Extensive research on PAs strongly suggests that consumption of these compounds hold promise to offer protection against various ailments in humans. This paper focuses on the naturally derived PAs and summarizes the action mechanisms of these compounds during disease conditions. Based on the available information in the literature, it is suggested that use of PAs as drugs is very promising; however more research and clinical trials are necessary before these bioactive molecules can be made for treatment. Finally this review provides greater awareness of the promise that natural PAs hold for use in the disease prevention and therapy.

Diversity and antifungal resistance patterns of prevalent opportunistic pathogenic yeasts colonizing the oral cavities of asymptomatic human immunodeficiency virus-infected individuals, and their relation to CD4(+) counts.

BACKGROUND: Yeasts are important opportunistic pathogens, in individuals infected with human immunodeficiency virus (HIV). Yeast species inhabiting the oral mucosa of HIV-infected persons can act as source of oral lesions, especially as the individual progresses towards immunocompromised state. Present study was conducted to evaluate the diversity of yeasts in oral cavities of asymptomatic HIV-infected persons and their association with CD4(+) cell counts. MATERIALS AND METHODS: 100 HIV seropositive subjects and 100 healthy controls were screened for oral yeast carriage using standard procedures. RESULTS: Of the 100 HIV-seropositive persons screened, 48 were colonized by different yeasts, either alone or in association with another species. Candida albicans was the most common species (56.90%) while non C. albicans Candida (NCAC) accounted for 39.65%. Among NCAC, Candida tropicalis and Candida krusei were most common. One isolate each of rare opportunistic pathogenic yeasts, Geotrichum candidum and Saccharomyces cereviseae, was recovered. The control group had an oral candidal carriage rate of 23%; C. albicans was the predominant species, followed by Candida glabrata, C. tropicalis and Candida parapsilosis. Antifungal susceptibility testing revealed no resistance in C. albicans, to the commonly used antifungal agents, whereas resistance or dose dependent susceptibility to fluconazole was observed in some of the NCAC species. CONCLUSION: Oral carriage of opportunistic pathogenic yeasts was greater in HIV-seropositive persons heading towards immunocompromised state, as evidenced by their CD4(+) cell count. The predominant yeast isolated in this study (C. albicans), was found to be susceptible to commonly used antifungals.

Increased A3AR-dependent vasoconstriction in diabetic mice is promoted by myeloperoxidase.

Vascular dysfunction importantly contributes to mortality and morbidity in various cardiac and metabolic diseases. Among endogenous molecules regulating vascular tone is adenosine, with the adenosine A3 receptor (A3AR) exerting cardioprotective properties in ischemia and reperfusion. However, overexpression of A3AR is suggested to result in vascular dysfunction and inflammation. The leukocyte enzyme myeloperoxidase (MPO) is an important modulator of vascular function with nitric oxide-consuming and proinflammatory properties. Increased MPO plasma levels are observed in patients with cardiovascular disorders like heart failure, acute coronary syndromes, and arrhythmias. Given that vascular dysfunction and inflammation are also hallmarks of diabetes, the role of MPO in adenosine-dependent vasomotor function was investigated in a murine model of diabetes mellitus. Wild-type (WT) and MPO-deficient (Mpo) mice were treated with Streptozotocin (STZ), which induced an increase of MPO plasma levels in WT mice and led to enhanced aortic superoxide generation as assessed by dihydroethidium staining in STZ-treated WT mice as compared with controls. The vasoconstriction of aortic segments in response to the A3AR agonist Cl-IB-MECA (2-Chloro-N6-(3-iodobenzyl)-N-methyl-5-carbamoyladenosine) as determined by isometric force measurements was augmented in diabetic WT as compared with diabetic Mpo mice. Moreover, A3AR protein expression was enhanced in STZ-treated mice but was attenuated by MPO deficiency. The current data reveal an MPO-mediated increase of vascular A3AR expression under diabetic conditions, which leads to enhanced vasoconstriction in response to A3AR agonists and discloses an additional mechanism of MPO-mediated vascular dysfunction.

Ocimum sanctum (L.) essential oil and its lead molecules induce apoptosis in Candida albicans.

Manipulation of endogenous responses during programmed cell death (PCD) in fungi can lead to development of effective therapeutic strategies. In the present study, we evaluated the physiology of cell death in Candida albicans in response to Ocimum sanctum essential oil (OSEO) and its two major constituents - methyl chavicol (MET CHAV) and linalool (LIN) at varying inhibitory concentrations. Apoptotic cell death was studied on the basis of externalization of membrane phosphatidylserine (PS) revealed by annexin-V-FITC labeling, morphological alterations revealed by transmission electron microscopy and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Exposure of fungal cells to MIC/4 of OSEO, MET CHAV and LIN resulted in morphological features characteristic of apoptosis, while necrosis was observed at higher concentrations. Necrotic cells displayed reduced TUNEL staining and an inability to exclude propidium iodide. In addition, they lacked a defined nucleus and an intact external morphology. Exposed cells were TUNEL-positive, showed chromatin condensation and margination, nuclear envelope separation, nuclear fragmentation, cytoplasmic shrinkage and plasma membrane blebbing. A dose-dependent decrease in cytochrome c oxidase activity was observed with each compound, but the decrease was not comparable to that elicited by H2O2, eliminating the primary involvement of cytochrome c release in apoptosis thus induced. Previously reported data revealed induction of apoptosis at low concentrations as a result of oxidative insult. Studies aimed at identifying other mitochondrial factors activated during this course to mediate apoptosis will further elucidate the mechanism of antifungal action of these natural products.

Inhibition of As(III) and Hg(II) caused aortic hypercontraction by eugenol, linalool and carvone.

Acute and chronic exposure to arsenic and mercury is known to produce vasoconstriction. There is, however, no clarity concerning the pathways leading to this increased contraction. In this study we elicit and compare maximum contractility of rat aortas under resting conditions in the presence of arsenic and mercury, and delineate pathways mediating this effect. Phenylephrine (PE) induced hypercontraction of 37% and 32% were obtained when isolated aortic segments were exposed to 25 ?M As(III) and 6 nM Hg(II), respectively. Isometric contraction measurements in presence of apocynin, verapamil and sodium nitroprusside indicates that the major causes of increased contraction are reactive oxygen species (ROS) and depletion of nitric oxide (NO). Calcium influx plays a minor role in arsenic and mercury caused hypercontraction. In unexposed aorta, eugenol causes relaxation by inhibiting ROS and elevating NO, linalool by blocking voltage dependent calcium channel (VDCC) and elevating NO, and carvone by blocking calcium influx through VDDC. Since the arsenic and mercury hypercontraction is mediated by increased ROS and depleted NO, we hypothesize that molecules which neutralize ROS or elevate NO will be better ameliorators. In line with this argument, we found eugenol to be the best ameliorator of arsenic and mercury hypercontraction followed by linalool and carvone.

Influences of cinnamic aldehydes on H⁺ extrusion activity and ultrastructure of Candida.

The antifungal effects of cinnamaldehyde, 4-hydroxy-3-methoxycinnamaldehyde (coniferyl aldehyde) and 3,5-dimethoxy-4-hydroxycinnamaldehyde (sinapaldehyde) were investigated against 65 strains of Candida (six standard, 39 fluconazole-sensitive and 20 fluconazole-resistant). MICs of cinnamaldehyde, coniferyl aldehyde and sinapaldehyde ranged from 100 to 500 µg ml(-1), 100 to 300 µg ml(-1) and 100 to 200 µg ml(-1), respectively. All tested isolates showed a marked sensitivity towards these aldehydes in spot and time-kill assays. Sinapaldehyde was found to be the most effective, followed by coniferyl aldehyde and cinnamaldehyde. At their respective MIC(90) values, the three compounds caused mean inhibition levels of glucose-stimulated H(+)-efflux of 36, 34 and 41 % (cinnamaldehyde), 41, 42 and 47 % (coniferyl aldehyde) and 43, 45 and 51 % (sinapaldehyde) for standard-sensitive, clinical-sensitive and clinical-resistant isolates, respectively. Inhibition levels of H(+)-efflux caused by plasma membrane ATPase inhibitors N,N'-dicyclohexylcarbodiimide (100 µM) and diethylstilbestrol (10 µM) were 34, 45 and 44 %, and 57, 39 and 35 %, for standard-sensitive, clinical-sensitive and clinical-resistant isolates, respectively. Intracellular pH (pHi) was found to decrease by 0.34, 0.42 and 0.50 units following incubation with three tested aldehydes from the control pHi of 6.70. Scanning electron microscopy and transmission electron microscopy analysis was performed on a representative strain, C. albicans 10261, showing alterations in morphology, cell wall, plasma membrane damage and lysis. Haemolytic activity of the three compounds varied from 10 to 15 % at their highest MIC compared to an activity level of 20 % shown by fluconazole at 30 µg ml(-1). In conclusion, this study shows significant activity of cinnamic aldehydes against Candida, including azole-resistant strains, suggesting that these molecules can be developed as antifungals.

Anticandidal activity of curcumin and methyl cinnamaldehyde.

Cinnamaldehyde, its derivatives and curcumin are reported to have strong antifungal activity. In this work we report and compare anticandidal activity of curcumin (CUR) and α-methyl cinnamaldehyde (MCD) against 38 strains of Candida (3; standard, fluconazole sensitive, 24; clinical, fluconazole sensitive, 11; clinical, fluconazole resistant). The minimum inhibitory concentrations (MIC90) of CUR ranged from 250 to 650 µg/ml for sensitive strains and from 250 to 500 µg/ml for resistant strains. MIC90 of MCD varied between 100 and 250 µg/ml and 100-200 µg/ml for sensitive and resistant strains, respectively. Higher activity of MCD as compared to CUR was further reinforced by spot assays and growth curve studies. At their respective MIC90 values, in the presence of glucose, average inhibition of H+-efflux caused by CUR and MCD against standard, clinical and resistant isolates was 24%, 31%, 32% and 54%, 52%, 54%, respectively. Inhibition of H+-extrusion leads to intracellular acidification and cell death, average pHi for control, CUR and MCD exposed cells was 6.68, 6.39 and 6.20, respectively. Scanning electron micrographs of treated cells show more extensive damage in case of MCD. Haemolytic activity of CUR and MCD at their highest MIC was 11.45% and 13.00%, respectively as against 20% shown by fluconazole at typical MIC of 30 µg/ml. In conclusion, this study shows significant anticandidal activity of CUR and MCD against both azole-resistant and sensitive clinical isolates, MCD is found to be more effective.

Exposure of Candida to p-anisaldehyde inhibits its growth and ergosterol biosynthesis.

p-Anisaldehyde (4-methoxybenzaldehyde), an extract from Pimpinella anisum seeds, is a very common digestive herb of north India. Antifungal activity of p-anisaldehyde was investigated on 10 fluconazole-resistant and 5 fluconazole-sensitive Candida strains. Minimum inhibitory concentrations (MIC(90)) ranged from 250 µg/ml to 600 µg/ml for both sensitive and resistant strains. Ergosterol content was drastically reduced by p-anisaldehyde-62% in sensitive and 66% in resistant strains-but did not corelate well with MIC(90) values. It appears that p-anisaldehyde exerts its antifungal effect by decreasing NADPH routed through up-regulation of putative aryl-alcohol dehydrogenases. Cellular toxicity of p-anisaldehyde against H9c2 rat cardiac myoblasts was less than 20% at the highest MIC value. These findings encourage further development of p-anisaldehyde.