Anti-Bacterial and Anti-Candidal Activity of Silver Nanoparticles Biosynthesized Using Citrobacter spp. MS5 Culture Supernatant.

The present study described the extracellular synthesis of silver nanoparticles (AgNPs) using environmental bacterial isolate Citrobacter spp. MS5 culture supernatant. To our best knowledge, no previous study reported the biosynthesis of AgNPs using this bacterial isolate. The biosynthesized AgNPs were characterized using different techniques like UV-Vis spectroscopy, fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) equipped with energy dispersive X-ray (EDX). The analysis of UV-Vis spectra revealed absorption maxima at 415 nm due to surface plasmon resonance (SPR) indicated the formation of AgNPs and FTIR spectrum confirmed the participation of proteins molecule in AgNPs synthesis. XRD and EDX spectrum confirmed the metallic and crystalline nature of AgNPs. TEM and SEM showed spherical nanoparticles with a size range of 5-15 nm. The biosynthesized AgNPs showed effective independent as well as enhanced combined antibacterial activity against extended spectrum ß-lactamase (ESBL) producing multidrug resistant Gram-negative bacteria. Further, effective antifungal activity of AgNPs was observed towards pathogenic Candida spp. The present study provides evidence for eco-friendly biosynthesis of well-characterized AgNPs and their potential antibacterial as well as antifungal activity.

Systemic p-ANCA vasculitis with fatal outcome, arising in the setting of methimazole use.

Here we report a fatal case of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) due to methimazole use in a 64-year-old woman. She was initially hospitalized for abdominal pain and possible colitis, and subsequently developed hematuria, renal failure, and hemoptysis. The serologic work-up revealed positive antinuclear antibody (ANA) and perinuclear-antineutrophilic cytoplasm antibodies (p-ANCA), with positive antimyeloperoxidase. Three weeks following admission, the patient was found to be pulseless, and expired. At autopsy, microscopic review included widespread transmural necrotizing vasculitis and crescentic glomerulonephritis in the kidney, and diffuse pulmonary alveolar hemorrhage; focal coronary artery intimal vasculitis and necrotizing pericarditis were also noted. Several drugs have been associated with the development of ANCA-positive diseases, including propylthiouracil, hydralazine, allopurinol, penicillamine, and levamisole in cocaine. Association of ANCA vasculitis with methimazole exposure is less known, and severe presentation with fatal outcome, as seen in our patient, is exceedingly rare. We reviewed clinical and histopathologic features of drug-induced ANCA vasculitis associated with methimazole to raise awareness of this potentially life-threatening complication associated with this agent.

An empirical investigation of the determinants of CO2 emissions: evidence from Pakistan.

The aim of this paper is to examine the relationship between CO2 emissions and its possible determinants and their direction of causality for Pakistan over the period of 1972 to 2017. The survey of literature guides us that the most frequently discussed factors are real GDP per capita, energy consumption, urbanization, trade openness, and financial development. Testing of environmental Kuznets curve (EKC) hypothesis is the most common in environment literature so we also incorporated the real GDP per capita squared term in the model. Autoregressive distributed lag (ARDL) bound testing approach to cointegration with structural break and error correction method (ECM) are applied to the selected time series to investigate the long-run relationship between CO2 emissions and real GDP per capita, real GDP per capita squared term, energy consumption, urbanization, trade openness, and financial development. The empirical evidence confirms the cointegration among the variables and EKC holds for Pakistan support H1 of the study, which though contradictory to the previous studies conducted on Pakistan but all of previous work faces the exclusion bias and their findings were skewed. The findings also suggest that energy consumption and urbanization have a positive effect on CO2 emissions, supporting H2 and H3. However, H4 and H5 rejected as trade openness and financial development found positively significant. Moreover, bidirectional Granger causality was exists only between CO2 emissions and trade openness. The findings suggests that Pakistan need to settle the economic agenda of the nation through the resolution of economic controversies, energy mix need to tilt toward clean and renewable energy, and rural-urban migration need to manage for better air, water, and living.

Immunoprophylactic potential of wheat grass extract on benzene-induced leukemia: An in vivo study on murine model.

OBJECTIVES: Wheat grass (Triticum aestivum) is a gift of nature given to mankind. A number of scientific research on wheatgrass establishes its anticancer and antioxidant potential. Current work was focused to determine antileukemic effect of wheat grass. MATERIALS AND METHODS: The commercial wheatgrass powder was extracted with 95% of methanol. Methanol extract of wheat grass was studied for acute oral toxicity as per revised Organization for Economic Cooperation and Development Guidelines number 423. Leukemia was successfully induced in Wister rats by intravenous injection of benzene. The blood was collected and analyzed for hematological parameters. Phagocytotic activity of the extract was determined. RESULTS: Phytochemical screening revealed the presence of flavonoids, phenolics, carbohydrates, and amino acids. From acute toxicity studies, it was found that the methanol extract of wheatgrass was safe up to a dose level of 2000 mg/kg of body weight. Outcomes of hematological parameters in various experimental groups of murine model demonstrated antileukemic effect of extract. Methanol extract of wheatgrass aroused the process of phagocytosis of killed Candida albicans and also demonstrated a significant chemotactic activity at all tested concentrations. CONCLUSION: In the current work, methanol extract of wheat grass demonstrated antileukemic potential that might be due to the presence of flavonoids and polyphenolics in it. Further isolation, structural characterization of active constituents is necessary to extrapolate the mechanism of action.

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.

Cinnamic aldehydes affect hydrolytic enzyme secretion and morphogenesis in oral Candida isolates.

Effect of cinnamaldehyde (CD), 4-hydroxy-3-methoxy cinnamaldehyde (HMCD) and 3,5-dimethoxy-4-hydroxy cinnamaldehyde (HDMCD) on growth and virulence factors of standard (Candida albicans 90028) and 26 oral isolates of C. albicans has been investigated. Growth was significantly inhibited by all three compounds in both solid and liquid medium, no systematic difference was observed between various isolates. MIC90 ranged from 125 to 450 µg/ml for CD, 100-250 µg/ml for HMCD and 62.5-125 µg/ml for HDMCD. All oral isolates were found to be proteinase and phospholipase secretors, both proteinase and phospholipase secretion was significantly inhibited by all the three tested molecules. No systematic difference in secretion or its inhibition was observed between standard and oral isolates as also between various isolates. Average drop in proteinase and phospholipase secretion caused by ½ MIC of CD was 33% and 28%, HMCD; 46% and 44%, HDMCD; 59% and 54%. The standard strain and all the 26 oral isolates displayed morphogenesis under triggering experimental conditions; no difference was seen between standard and various isolates. In the absence of test compounds hyphae development at 300 min was 83% for standard strain whereas average hyphae development for oral isolates was 85%. Average hyphal transition was suppressed by all tested compounds. At ½ MIC concentration at 300 min average hyphal transition of standard and oral isolates was CD; 49% and 57%, HMCD; 45% and 38%, HDMCD; 5% and 5%. Average haemolytic activity of the three tested compounds varied from 10 to 15% at their highest MIC compared to 20% shown by fluconazole at typical MIC of 30 µg/ml.

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.

Proton pumping ATPase mediated fungicidal activity of two essential oil components.

This work evaluates the antifungal activity of two essential oil components against 28 clinical isolates (17 sensitive, 11 resistant) and 3 standard laboratory strains of Candida. Growth of the organisms was significantly effected in both solid and liquid media at different test compound concentrations. The minimum inhibitory concentrations (MICs) of Isoeugenol (compound 1) against 31 strains of Candida ranged 100-250 µg/ml and those of o -methoxy cinnamaldehyde (compound 2) ranged 200-500 µg/ml, respectively. Insight studies to mechanism suggested that these compounds exert antifungal activity by targeting H(+)-ATPase located in the membranes of pathogenic Candida species. At their respective MIC(90) average inhibition of H(+)-efflux for standard, clinical and resistant isolates caused by compound 1 and compound 2 was 70%, 74%, 82% and 42%, 42% and 43%. Respective inhibition of H(+)-efflux by fluconazole (5 µg/ml) was 94%, 92% and 10%. Inhibition of H(+)-ATPase leads to intracellular acidification and cell death. SEM analysis of Candida cells showed cell membrane breakage and alterations in morphology. Haemolytic activity on human erythrocytes was studied to exclude the possibility of further associated cytotoxicity.

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.

Interesting anticandidal effects of anisic aldehydes on growth and proton-pumping-ATPase-targeted activity.

Attention has been drawn to evaluate the antifungal activity of p-anisaldehyde (1), o-anisaldehyde (2) and m-anisaldehyde (3). To put forward this approach, antifungal activity has been assessed in thirty six fluconazole-sensitive and eleven fluconazole-resistant Candida isolates. Growth and sensitivity of the organisms were significantly effected by test compounds at different concentrations. The rapid irreversible action of compound-1, compound-2 and compound-3 on fungal cells suggested a membrane-located target for their action. We investigated their effect on H(+) ATPase mediated H(+)-pumping by various Candida species. All the compounds inhibit H(+)- ATPase activity at their respective MIC(90) values. Inhibition of H(+) ATPase leads to intracellular acidification and cell death. Scanning electron microscopy analysis revealed deep wrinkles, deformity and flowed content. Furthermore, it was also observed that position of methoxy group attached to the benzene ring decides antifungal activity of the compound. The present study indicates that compound-1, compound-2 and compound-3 have significant antifungal activity against Candida, including azole-resistant strains, advocating further investigation for clinical applications in the treatment of fungal infections.

Spice oil cinnamaldehyde exhibits potent anticandidal activity against fluconazole resistant clinical isolates.

Fluconazole resistance is becoming an important clinical concern. We studied the in vitro effects of cinnamaldehyde against 18 fluconazole-resistant Candida isolates. MIC(90) of cinnamaldehyde against different Candida isolates ranged 100-500 µg/ml. Growth and sensitivity of the organisms were significantly affected by cinnamaldehyde at different concentrations. The rapid irreversible action of this compound on fungal cells suggested membrane-located targets for its action. Insight studies to mechanism suggested that cinnamaldehyde exerts its antifungal activity by targeting sterol biosynthesis and plasma membrane ATPase activity. Inhibition of H(+) (-)ATPase leads to intracellular acidification and cell death. Toxicity against H9c2 rat cardiac myoblasts was studied to exclude the possibility of further associated cytotoxicity. The observed selectively fungicidal characteristics against fluconazole-resistant Candida isolates signify a promising candidature of this essential oil as an antifungal agent in treatments for candidosis.