Mumio (Shilajit) as a potential chemotherapeutic for the urinary bladder cancer treatment.

Mumio (Shilajit) is a traditional medicinal drug known and used for hundreds of years. Bladder cancer is one of the most common cancer types and better treatments are needed. This study analysed the in vitro effect of Mumio on urinary bladder cancer cells (T24 and 5637) in comparison to normal uroepithelial cells (SV-HUC1). Cytotoxicity of Mumio was analysed in these cell lines via MTT and real-time cell growth assays as well via the assessment of the cytoskeleton, apoptosis, and cell cycle. Mumio affected the viability of both cell types in a time and concentration dependent manner. We observed a selectivity of Mumio against cancer cells. Cell cycle and apoptosis analysis showed that Mumio inhibited G0/G1 or S phase cell cycle, which in turn induced apoptosis. Our results showed that Mumio was significantly more cytotoxic to urinary bladder cancer cells than to normal cells. These results are promising and indicate Mumio as a great candidate for urinary bladder cancer treatment and further investigations should be performed.

A simple "turn-on" fluorescence sensor for salicylaldehyde skeleton based on switch of PET-AIE effect.

The selective detection of salicylaldehyde skeleton is of great significance in phytochemistry and biological research but rarely reported. In this research, a simple and highly selective "turn-on" fluorescence sensor (CDB-Am) for salicylaldehyde skeleton was developed based on switch of photoinduced electron transfer (PET) and aggregation-induced emission (AIE). CDB-Am bearing amino-cyanodistyrene structure responded to salicylaldehyde in the range of 3.1 to 40 µM with a detection limit of 0.94 µM. The sensing process of formation of Schiff-base adduct CDB-SA was confirmed by 1H NMR, MS, and FT-IR spectra, revealing that a recovered AIE property accounted for the turn-on fluorescence response of CDB-Am and the intramolecular hydrogen bonding played a crucial role in the disruption of PET process. This sensing ability was successfully applied for both fluorescence qualitative test of salicylaldehyde skeleton on TLC analysis and quantitative detection of salicylaldehyde skeleton with good accuracy in the root bark of Periploca sepium, suggesting the extensive applications in phytochemistry and traditional Chinese herbal medicine. Furthermore, CDB-Am exhibited the first excellent fluorescence imaging ability in detecting salicylaldehyde skeleton in a living system. This work supplied a new strategy of preparing a novel "turn-on" fluorescence probe for detecting salicylaldehyde skeleton in complex environments and living bodies.

Flinders sensitive line rats are resistant to infarction following transient occlusion of the middle cerebral artery.

BACKGROUND: Depression is a common complication of stroke and increases the risk of mortality and disability. Pre-stroke depression is a possible risk factor for stroke and has also been linked to adverse outcomes. The underlying mechanisms linking depression and stroke remain unclear. Preclinical models may provide novel insights, but models reflecting both conditions are lacking. METHODS: In this study, we investigated the effects of a 45-min transient middle cerebral artery occlusion (MCAo) on infarct size in male adult Flinders Sensitive Line rats, a genetic animal model of depression, and their control strains Flinders Resistant Line and Sprague-Dawley rats. Infarct size was assessed by tetrazolium chloride (TTC) and microtubule-associated protein 2 (MAP2) staining after 48 h of reperfusion. Angiograms of the vascular structure of naïve animals were produced with a µ-CT scanner. RESULTS: Both Flinders strains had significantly smaller infarcts following MCAo compared to Sprague-Dawley rats. This effect does not appear to be due to changes in cerebrovascular architecture, as indicated by an initial exploration of vascular organization using angiograms, or body temperature regulation. CONCLUSIONS: Our study suggests that the rat strain does not influence infarct volumes following MCAo.

INT reduction is a valid proxy for eukaryotic plankton respiration despite the inherent toxicity of INT and differences in cell wall structure.

The reduction of 2-para (iodophenyl)-3(nitrophenyl)-5(phenyl) tetrazolium chloride (INT) is increasingly being used as an indirect method to measure plankton respiration. Its greater sensitivity and shorter incubation time compared to the standard method of measuring the decrease in dissolved oxygen concentration, allows the determination of total and size-fractionated plankton respiration with higher precision and temporal resolution. However, there are still concerns as to the method's applicability due to the toxicity of INT and the potential differential effect of plankton cell wall composition on the diffusion of INT into the cell, and therefore on the rate of INT reduction. Working with cultures of 5 marine plankton (Thalassiosira pseudonana CCMP1080/5, Emiliania huxleyi RCC1217, Pleurochrysis carterae PLY-406, Scrippsiella sp. RCC1720 and Oxyrrhis marina CCMP1133/5) which have different cell wall compositions (silica frustule, presence/absence of calcite and cellulose plates), we demonstrate that INT does not have a toxic effect on oxygen consumption at short incubation times. There was no difference in the oxygen consumption of a culture to which INT had been added and that of a replicate culture without INT, for periods of time ranging from 1 to 7 hours. For four of the cultures (T. pseudonana CCMP1080/5, P. carterae PLY-406, E. huxleyi RCC1217, and O. marina CCMP1133/5) the log of the rates of dissolved oxygen consumption were linearly related to the log of the rates of INT reduction, and there was no significant difference between the regression lines for each culture (ANCOVA test, F = 1.696, df = 3, p = 0.18). Thus, INT reduction is not affected by the structure of the plankton cell wall and a single INT reduction to oxygen consumption conversion equation is appropriate for this range of eukaryotic plankton. These results further support the use of the INT technique as a valid proxy for marine plankton respiration.

A Novel Niosomal Combination of Selenium Coupled with Glucantime against Leishmania tropica.

There is no effective treatment modality available against different forms of leishmaniasis. Therefore, the aim of this study was to improve the penetration and efficacy of selenium and glucantime coupled with niosomes and compared them with their simple forms alone on in vitro susceptibility assays. In this study, the niosomal formulations of selenium and in combination with glucantime were prepared. The size and morphology of the niosomal formulations were characterized and the effectivity of the new formulation was also evaluated using in vitro MTT assay, intra-macrophage model, and gene expression profile. From the results obtained, no cytotoxicity effect was observed for niosomal and simple forms of drugs, as alone or in combination. Niosomal formulations of the drugs significantly showed more inhibitory effects (P ≤ 0.001) than the simple drugs when the selectivity index was considered. The gene expression levels of Interleukin (IL-10) significantly decreased, while the level of IL-12 and metacaspase significantly increased (P ≤ 0.001). The results of the present study showed that selenium plus glucantime niosome possess a potent anti-leishmanial effect and enhanced their lethal activity as evidenced by the in vitro experiments.

Enhanced cell growth on 3D graphene scaffolds implanted with nitrogen ions.

One of the key challenges in engineering tissues for cell-based therapies is developing biocompatible scaffold materials to direct cell behavior. In this paper, the cytocompatibilities of a flexible three-dimensional graphene scaffold (3D-G) and the same scaffold implanted with nitrogen ions (N+/3D-G) are compared using an in vitro assay based on 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The N+/3D-G samples were prepared from low-temperature hydrothermally synthesized flexible 3D-G by ion implantation and were found to display improved adhesion and proliferation of rat osteoblast and mouse fibroblast cells. In particular, the N+/3D-G sample with a nitrogen content of ∼10% showed the highest levels of cell viability and proliferation. The flexible N+/3D-G has potential applications as a biocompatible scaffold material that provides improved surface area and hydrophilic groups for cell growth and proliferation.

3'-epi-12ß-hydroxyfroside, a new cardenolide, induces cytoprotective autophagy via blocking the Hsp90/Akt/mTOR axis in lung cancer cells.

Rationale: Cardenolides have potential as anticancer drugs. 3'-epi-12ß-hydroxyfroside (HyFS) is a new cardenolide structure isolated by our research group, but its molecular mechanisms remain poorly understood. This study investigates the relationship between its antitumor activities and autophagy in lung cancer cells. Methods: Cell growth and proliferation were detected by MTT, lactate dehydrogenase (LDH) release, 5-ethynyl-20-deoxyuridine (EDU) and colony formation assays. Cell apoptosis was detected by flow cytometry. Autophagic and signal proteins were detected by Western blotting. Markers of autophagy and autophagy flux were also detected by immunofluorescence, transmission electron microscopy and acridine orange staining. Real time RT-PCR was used to analyze the gene expression of Hsp90. Hsp90 ubiquitination was detected by coimmunoprecipitation. The antitumore activities of HyFS were observed in nude mice. Results: HyFS treatment inhibited cell proliferation and induced autophagy in A549 and H460 lung cancer cells, but stronger inhibition of cell proliferation and induction of cell apoptosis were shown when HyFS-mediated autophagy was blocked. The Hsp90/Akt/mTOR axis was found to be involved in the activation of HyFS-mediated autophagy. Evidence of direct interaction between Hsp90 and Akt was observed. HyFS treatment resulted in decreased levels of heat shock protein 90 (Hsp90) and phosphorylated Akt, overexpression of Hsp90 increased activation of autophagy, and inhibition of Hsp90 expression decreased autophagy. In addition, ubiquitin-mediated degradation of Hsp90 and subsequent dephosphorylation of its client protein Akt were also found in HyFS-treated lung cancer cells. Moreover, combination treatment with HyFS and chloroquine showed remarkably increased tumor inhibition in both A549- and H460-bearing mice. Conclusion: Our results demonstrate that HyFS induced cytoprotective autophagy through ubiquitin-mediated degradation of Hsp90, which further blocked the Akt/mTOR pathway in lung cancer cells. Thus, a combination of a HyFS-like cardenolide and an autophagic inhibitor is a potential alternative approach for the treatment of lung cancer.

Anti-Candida activity of antidepressants sertraline and fluoxetine: effect upon pre-formed biofilms.

As an opportunistic fungal pathogen Candida spp. has the ability to form biofilms. The most prescribed drugs for Candida infections, azoles, have shown to be less effective when biofilms are present. In addition, increasing treatment costs and the fact that most prescribed antifungal drugs have only fungistatic activity justify the search for new treatment strategies. One promising approach is third generation antidepressants, selective serotonin re-uptake inhibitors (SSRIs), because of their proven antifungal activity against several Candida spp. Thus, the aim of this work was to determine the ability of two commonly used SSRIs, fluoxetine and sertraline, to impair both biofilm metabolic viability and biofilm biomass. The in vitro effect of fluoxetine and sertraline was individually tested against biofilm metabolic viability and biofilm biomass using the MTT assay and the Crystal Violet assay, respectively. For both drugs, a dose-dependent reduction on both biofilm metabolism and biomass was present. At high concentrations, fluoxetine was able to reduce biofilm metabolism by 96% (C. krusei) and biofilm biomass by 82% (C. glabrata), when compared to the control. At similar conditions, sertraline achieved a reduction of 88% on biofilm biomass (C. glabrata) and 90% on biofilm metabolism (C. parapsilosis). Moreover, fluoxetine showed interesting anti-biofilm activity at previously reported planktonic MIC values and even at sub-MIC values. These results reinforce the potential interest of SSRIs as anti-biofilm agents to be study to counteract resistance phenomena on candidosis.

P2X7 receptor mediates NLRP3-dependent IL-1ß secretion and parasite proliferation in Toxoplasma gondii-infected human small intestinal epithelial cells.

BACKGROUND: Toxoplasma gondii can invade and replicate in all nucleated cells in a wide range of host species, and infection induces IL-1ß production. IL-1ß plays central roles in the stimulation of the innate immune system and inflammation. However, little is known of the innate immune responses in human fetal small intestinal epithelial cells (FHs 74 Int cells) after T. gondii infection. METHODS: FHs 74 Int cells were infected with the T. gondii GFP-RH strain. Then, IL-1ß production and its mechanisms of action were evaluated using ELISA, MTT cell viability assays, Western blotting, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR), and gene-specific small interfering RNA (siRNA) transfection. RESULTS: Infection of FHs 74 Int cells by T. gondii triggered significant time- and dose-dependent IL-1ß production. Although T. gondii activated NLRP1, NLRP3, NLRC4 and AIM2 inflammasomes in FHs 74 Int cells, NLRP3 levels were consistently and significantly time-dependently increased, while the other inflammasomes were not. Transfection with siRNA targeting NLRP3, cleaved caspase-1 (Casp-1) or ASC significantly reduced T. gondii-induced IL-1ß production, whereas T. gondii proliferation was markedly increased. Toxoplasma gondii infection activated P2X7 receptor (P2X7R) levels in FHs 74 Int cells in a time-dependent manner; however, transfection with siRNA targeting P2X7R significantly reduced T. gondii-induced IL-1ß secretion and substantially increased T. gondii proliferation, which is mediated by decreased protein expression levels of NLRP3, cleaved Casp-1 and ASC. Collectively, NLRP3-dependent IL-1ß secretion is mediated by P2X7R in small intestinal epithelial cells in response to T. gondii infection, thereby controlling parasite proliferation. CONCLUSIONS: This study revealed that the P2X7R/NLRP3 pathway plays important roles in IL-1ß secretion and inhibition of T. gondii proliferation in small intestinal epithelial cells. These results not only contribute to our understanding of the mucosal immune mechanisms of T. gondii infection but also offer new insight into the identification of innate resistance in the gut epithelium.

Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition.

Metabolic viability based high throughput assays like MTT and MTS are widely used in assessing the cell viability. However, alteration in both mitochondrial content and metabolism can influence the metabolic viability of cells and radiation is a potential mitochondrial biogenesis inducer. Therefore, we tested if MTT assay is a true measure of radiation induced cell death in widely used cell lines. Radiation induced cellular growth inhibition was performed by enumerating cell numbers and metabolic viability using MTT assay at 24 and 48 hours (hrs) after exposure. The extent of radiation induced reduction in cell number was found to be larger than the decrease in MTT reduction in all the cell lines tested. We demonstrated that radiation induces PGC-1α and TFAM to stimulate mitochondrial biogenesis leading to increased levels of SDH-A and enhanced metabolic viability. Radiation induced disturbance in calcium (Ca2+) homeostasis also plays a crucial role by making the mitochondria hyperactive. These findings suggest that radiation induces mitochondrial biogenesis and hyperactivation leading to increased metabolic viability and MTT reduction. Therefore, conclusions drawn on radiation induced growth inhibition based on metabolic viability assays are likely to be erroneous as it may not correlate with growth inhibition and/or loss of clonogenic survival.

A Primary Study of the Antioxidant, Hypoglycemic, Hypolipidemic, and Antitumor Activities of Ethanol Extract of Brown Slimecap Mushroom, Chroogomphus rutilus (Agaricomycetes).

In vivo and in vitro treatments were carried out to investigate the effects of a 95% ethanol extract of Chroogomphus rutilus (CRE) on antioxidant, hypoglycemic, hypolipidemic, and antitumor properties. CRE showed potent radical scavenging activity against DPPH in vitro. It could increase antioxidant enzymatic activities (superoxide dismutase and glutathione peroxidase) and could reduce malondialdehyde content in vivo in mice in which aging was induced by D-galactose. CRE had hypoglycemic activity and could significantly inhibit α-glucosidase activity in vitro and decrease blood glucose concentration in vivo. CRE could decrease the serum total cholesterol, triglyceride, and low-density lipoprotein cholesterol levels and increase the high-density lipoprotein cholesterol level in diabetic mice. The MTT assay showed that CRE also had a certain inhibitory effect on the tumor cells. These results suggest that CRE may be beneficial for human health and could be useful for applications in medicine, the food industry, and agriculture.

Cytotoxicity of Etch-and-Rinse, Self-Etch, and Universal Dental Adhesive Systems in Fibroblast Cell Line 3T3.

The aim of this study was to evaluate in fibroblast cultures the direct cytotoxic effects of etch-and-rinse, self-etch, and universal adhesive systems. The sterile glass cover slips (n = 3) were then immersed in culture medium to obtain the eluates for the experimental groups: (1) Adper™ Single Bond 2; (2) Ambar; (3) Adper™ Scotchbond™ Multi-Purpose; (4) Scotchbond™ Universal; (5) Ambar Universal; and (6) OptiBond All-In-One. As a negative control, sterile glass cover slips were immersed in culture medium only. After 24 h, the eluate obtained was applied on fibroblast culture. Cell viability and cell morphology were evaluated by MTT assay and SEM, respectively. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (α = 0.05). All adhesive systems except universal reduced cell viability in 3T3 cells to between 26.04% and 56.57%, and Scotchbond Universal and Ambar Universal reduced cell viability to 2.13% and 3.57%, respectively, when compared to the negative control. Cytoplasmic membrane shrinkage and cell-free areas with residual membrane fragments from dead cells were observed. In conclusion, improvements in universal adhesive system formulations and their mechanisms of action are not accompanied by increased toxicity compared with those in other systems, warranting commitment to the use of these dentin-pulp complexes.

First report on a novel Nigrospora sphaerica isolated from Catharanthus roseus plant with anticarcinogenic properties.

This paper reports on the vinca alkaloid produced by a novel Nigrospora sphaerica isolated from Catharanthus roseus. Through liquid chromatography-mass spectrometry (LCMS), only the crude mycelia extract of this fungus was positive for determination of vinblastine. This vinca alkaloid was then purified by using high-performance liquid chromatography (HPLC) and tested for cytotoxicity activity using MTT assays. The breast cell line cancer (MDA-MB 231) was treated with a purified vinblastine which was intracellulary produced by N. sphaerica. The purified vinblastine from extracted leaf of C. roseus was used as a standard comparison. A positive result with a value of half maximal inhibitory concentration (IC50 ) of > 32 µg ml-1 was observed compared with standard (IC50 ) of 350 µg ml-1 only. It showed that a vinblastine produced by N. sphaerica has a high cytotoxicity activity even though the concentration of vinblastine produced by this endophytic fungus was only 0.868 µg ml-1 .

An in vitro study of antileishmanial effect of Portulaca oleracea extract.

BACKGROUND & OBJECTIVES: Leishmaniasis is caused by protozoa of Leishmania genus and is considered as a zoonotic disease. It is a major public health problem worldwide, with high endemicity in developing countries like Iran. Various chemical drugs are used for leishmaniasis treatment, but their side-effects and the emergence of drug resistance have led to look for new effective compounds. The aim of this study was to introduce purslane (Portulaca oleracea) as a traditional and medicinal herb which might act as a valuable source for designing new pharmaceutical drug/lead against Leishmania sp. METHODS: This study was conducted in the laboratory of Seddigheh Tahereh Infectious Disease Research Center, Isfahan, Iran during the spring of 2015. The essence from the purslane plant was prepared through water distillation and the alcoholic extract was prepared through maceration method. The essence was dried, and diluted with DMSO (5%). Leishmania major promastigotes were cultured in 25 ΁ 2΀C temperature in the stationary phase of RPMI-1640 medium, enriched with 10% fetal calf serum and penicillin-streptomycin to yield higher quantity. The biological activity of herb essence was evaluated on L. major promastigotes and compared to glucantime reference drug using methylthiazole tetrazolium (MTT) colorometric assay. The optical density absorbance was measured with Eliza reader set, and the IC50 value was calculated at different time intervals. All tests were repeated thrice. Results were analyzed by using Tukey test and t-test. RESULTS: The IC50 values after 48 h, for glucantime against standard parasite promastigotes and clinical strains were equal to 12 and 19 mg/ml, respectively, whereas for purslane herb leaves and stems essence; it was equal to 360 and 680 mg/ml, respectively. Although, the glucantime pharmaceutical drug was more efficient compared to the investigated herb essence, the essense had significant effect on L. major promastigotes with increasing density (p <0.05). The ingredients of the herb leaves and stem essence were-Phytol, squalene, palmitic acid, ethyl- linoleate, ferulic acid, linolenic acid, scopoletin, linoleic acid, rhein, apigenin, and bergapten. INTERPRETATION & CONCLUSION: The study showed that essence of purslane has considerable antileishmanial effects and can stop the growth of parasites in the laboratory compared to glucantime. More experiments are necessary to investigate its effect on Leishmania parasite in animal model.

Does biomass production correlate with metabolic activity in Staphylococcus aureus?

We assessed agreement between the crystal violet binding assay and the XTT assay in the classification of biofilm production in 492 Staphylococcus aureus strains from bacteremic patients. We found that the overall correlation between the procedures was 46.5%. Biomass production and metabolic activity must be assessed simultaneously.

Antibacterial and antibiofilm activities of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) against periodontopathic bacteria.

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are two major omega-3 polyunsaturated fatty acids (n-3 PUFAs) with antimicrobial properties. In this study, we evaluated the potential antibacterial and antibiofilm activities of DHA and EPA against two periodontal pathogens, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). MTT assay showed that DHA and EPA still exhibited no cytotoxicity to human oral tissue cells when the concentration came to 100 µM and 200 µM, respectively. Against P. gingivalis, DHA and EPA showed the same minimum inhibitory concentration (MIC) of 12.5 µM, and a respective minimum bactericidal concentration (MBC) of 12.5 µM and 25 µM. However, the MIC and MBC values of DHA or EPA against F. nucleatum were both greater than 100 µM. For early-stage bacteria, DHA or EPA displayed complete inhibition on the planktonic growth and biofilm formation of P. gingivalis from the lowest concentration of 12.5 µM. And the planktonic growth of F. nucleatum was slightly but not completely inhibited by DHA or EPA even at the concentration of 100 µM, however, the biofilm formation of F. nucleatum at 24 h was significantly restrained by 100 µM EPA. For exponential-phase bacteria, 100 µM DHA or EPA completely killed P. gingivalis and significantly decreased the viable counts of F. nucleatum. Meanwhile, the morphology of P. gingivalis was apparently damaged, and the virulence factor gene expression of P. gingivalis and F. nucleatum was strongly downregulated. Besides, the viability and the thickness of mature P. gingivalis biofilm, together with the viability of mature F. nucleatum biofilm were both significantly decreased in the presence of 100 µM DHA or EPA. In conclusion, DHA and EPA possessed antibacterial activities against planktonic and biofilm forms of periodontal pathogens, which suggested that DHA and EPA might be potentially supplementary therapeutic agents for prevention and treatment of periodontal diseases.

Antibacterial, cyto and genotoxic activities of A22 compound ((S-3, 4 -dichlorobenzyl) isothiourea hydrochloride).

The A22 is a chemical compound that acts as a reversible inhibitor of a bacterial cell wall protein MreB leading the rods to the coccoid form. Thus, by changing the bacterial form, many properties can be affected, as the acquisition of nutrients, cell division, the clamping surfaces, motility and pathogenesis. Infections caused by strains of Pseudomonas aeruginosa have great clinical importance because these microorganisms can include more than one resistance mechanism acting together, limiting treatment options. Thus, it is important to investigate the action of A22 against P. aeruginosa, once there are urgent needs for new antimicrobial compounds for increase the arsenal therapeutic to treat diseases caused by this microrganism. Therefore, this study investigated for the first time the antimicrobial activity of A22 against seve standards strains of Gram negative microorganisms and twenty-eight clinical isolates of P. aeruginosa. This study performed an additional investigation to analyze the cyto and genotoxic potential effects from A22 on human peripheral blood mononuclear cells (PBMCs). The antibacterial activity of A22 was studied by broth microdilution method and time-kill assay. The cytotoxicity was evaluated by MTT assay at 24, 48 and 72 h of exposure to A22 and the genotoxicity was evaluated by the Comet assay. The susceptibility tests showed A22 has a relevant antibacterial activity against P. aeruginosa, including multidrug-resistant (MDR) clinical isolates. The A22 treatment not showed genotoxic effects against PBMCs in almost all concentrations tested at 24 and 48 h of exposure. Only for concentration of 32 µg/mL (highest tested) the damage index was significantly higher in all moments. The MTT assay demonstrated that A22 was able to maintain cell viability in all exposure times. In summary, the A22 demonstrated important anti-Pseudomonas activity and showed no cyto and genotoxic significant effect. These results need to be considered in future in vitro and in vivo studies in order to introduce the A22 as a possible therapeutic option.

Biofilm inhibitory effect of chlorhexidine conjugated gold nanoparticles against Klebsiella pneumoniae.

Klebsiella pneumoniae (K. pneumoniae) is one of the major pathogen associated with nosocomial infections, especially catheter associated urinary tract infections which involved biofilm formation. This study was designed to evaluate the antibiofilm efficacy of gold nanoparticle conjugated with chlorhexidine (Au-CHX) against K. pneumoniae isolates. Au-CHX was synthesized and analyzed for stability by using UV-Visible spectrophotometry, atomic force microscopy (AFM), fourier transform infrared spectroscopy (FT-IR) and electrospray ionization mass spectroscopy (ESI-MS). Biofilm inhibition and eradication was performed by crystal violet, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and further confirmed by florescence and AFM microscopy. Au-CHX showed the maxima surface plasmon resonance (SPR) band at 535 nm, spherical morphology and polydispersity with size in the range of 20-100 nm. The micro molar concentrations (i.e. 25 and 100 µM) of Au-CHX completely inhibited the biofilm formation and metabolic activity within biofilms of K. pneumoniae reference and three tested clinical isolates, respectively. Time dependant biofilm inhibition assay showed that Au-CHX inhibited the early stage of biofilm formation. While at 75 and 100 µM concentrations, it also eradicated the established biofilms of K. pneumoniae isolates as compared to 2 mM chlorhexidine. Reduced florescence signals and surface roughness during microscopic analysis further confirms the antibiofilm activity of Au-CHX against K. pneumoniae ATCC13882 and clinical isolates. Thus it is concluded that chlorhexidine coated gold nanoparticle not only inhibits the biofilm formation of K. pneumoniae ATCC and clinical isolates but also eradicated the preformed biofilm.

Imipramine alters the sterol profile in Leishmania amazonensis and increases its sensitivity to miconazole.

BACKGROUND: Imipramine, a tricyclic antidepressant widely used clinically, has other pharmacological effects, such as antileishmanial activity. Tricyclic antidepressants interact with lipid bilayers, and some studies have shown that imipramine inhibits methyltransferases. Leishmania spp. produces compounds with an ergostane skeleton instead of a cholesterol skeleton, and the inhibition of enzymes of the sterol biosynthesis pathway is an interesting therapeutic target. Among these enzymes, C-24 methyltransferase has been suggested to play an essential role, as its inhibition kills the parasites. In this context, we investigated whether imipramine alters the biosynthesis of sterols in L. amazonensis and evaluated the efficacy of imipramine alone and in combination with miconazole, a classical inhibitor of another step in this pathway. METHODS: To analyze the interference of imipramine with sterol metabolism, promastigotes of L. amazonensis were cultured with medium alone, 15 or 30 µM imipramine or 4 µM miconazole, and their lipids were extracted with methanol/chloroform/water (1:0.5:0.4 v/v) and analyzed by GC/MS. To assess the antileishmanial activity of the treatments, promastigotes of L. amazonensis were incubated with various concentrations of imipramine up to 100 µM and up to 24 µM miconazole. Promastigotes were also treated with the combination of imipramine and miconazole at concentrations up to 12.5 µM of imipramine and 24 µM of miconazole. Parasite growth was evaluated by the MTT assay. The fractional inhibitory concentration index (FICI) was calculated to determine whether there were synergistic effects. Peritoneal macrophages with and without L. amazonensis infection were treated with miconazole (0 - 16 µM) or imipramine (0 to 50 µM) for 72 hours. For assays of the combined treatment in amastigotes, the concentration of imipramine was fixed at 12.5 µM and various concentrations of miconazole were used up to 16 µM. The infection rate was determined by counting the infected macrophages under a light microscope. FINDINGS: Promastigotes treated with imipramine accumulated cholesta-5,7,22-trien-3ß-ol and cholesta-7-24-dien- 3ß-ol, sterols that normally increase after treatment with classical inhibitors of C-24 methyltransferase. The IC50 of miconazole in promastigotes decreased when it was used in combination with imipramine, resulting in an additive effect, with a FICI value of 0.83. Imipramine also showed activity against intracellular amastigotes and enhanced the activity of miconazole, without apparent toxicity to the host cells. CONCLUSIONS: Imipramine was confirmed to have antileishmanial activity in both forms of the parasite, affecting the sterol biosynthesis of the organisms. Using imipramine in combination with azoles may be advantageous for the treatment of leishmaniasis.

Chemical composition and antifungal activity of Satureja hortensis L. essentiall oil against planktonic and biofilm growth of Candida albicans isolates from buccal lesions of HIV(+) individuals.

ETHNOPHARMACOLOGICAL RELEVANCE: Oral candidiasis is an opportunistic infection of the oral cavity which usually occurs in the immunocompromised individuals. Candida albicans (C. albicans) is the most common species of yeast responsible for oral candidiasis. This study investigated the effects of Satureja hortensis L. essentiall oil (EO) on the planktonic, biofilm formation and mature biofilms of C. albicans isolates from buccal lesions of HIV(+) individuals. MATERIALS AND METHODS: MTT reduction assay, broth micro-dilution method and scanning electron microscopy (SEM) were employed to determine the effect of mentioned EO on the C. albicans planktonic and biofilm forms. GC-GC/MS was used to detect the major active compounds of EO. RESULTS: Thymol (45.9%), gamma-terpinen (16.71%), carvacrol (12.81%) and p-cymene (9.61%) were found as the most abundant constituents. MIC values ranged from 250 to 400 µg/ml and MFC values ranged from 350 to 500 µg/ml. All C. albicans isolates formed biofilm on polystyrene plats but the quantity of biofilm mass (optical density) was different for the isolates ranging from 0.850 to 0.559 nm. The mean of biofilm formation by C. albicans isolates was reduced by 87.1 ± 3.7%, 73.6 ± 5.1%, 69.4 ± 5.3% and 67 ± 4.2% at 4800, 3200, 2400 and 1600 µg/ml, respectively. In sub-MIC concentration, SEM analysis revealed loosening of cells, deformity of three dimensional structures of biofilms and shrinkage in cell membranes of sessile cells. CONCLUSIONS: In conclusion, the substantial anti-fungal activity showed by S. hortensis L. EO suggests exploitation of this oil as potential natural anti-biofilm product to deal with the problem of buccal cavity lesion associated with C. albicans.