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.

Diagnosis of abdominal tuberculosis: Detection of mycobacterial CFP-10 and HspX proteins by gold nanoparticle-PCR amplified immunoassay.

Attempts were made to improve the efficacy of PCR amplified immunoassay (I-PCR) for diagnosing abdominal TB cases by utilizing the gold nanoparticle (AuNP)-based I-PCR, where AuNPs were functionalized with detection antibodies/oligonucleotides that exhibited 84.3% sensitivity and 95.1% specificity. This assay would improve the ongoing algorithms used in abdominal TB diagnosis.

Quantification of mycobacterial proteins in extrapulmonary tuberculosis cases by nano-based real-time immuno-PCR.

Aim: Diagnosis of extrapulmonary tuberculosis (EPTB) is difficult, and a rapid and dependable diagnostic test is urgently needed. Methods: A nano-based assay, SYBR Green magnetic bead-coupled gold nanoparticle-based real-time immuno-polymerase chain reaction (MB-AuNP-RT-I-PCR) was studied for the quantitative detection of Mycobacterium tuberculosis MPT-64+CFP-10 proteins in clinically suspected EPTB patients. Results: A wide range (270 fg/ml-9.9 ng/ml) of MPT-64+CFP-10 was quantified by MB-AuNP-RT-I-PCR in EPTB cases, whereas magneto-ELISA demonstrated a narrow range (1.8-10 ng/ml). Furthermore, high sensitivity (88.2%) and specificity (100%) were attained by MB-AuNP-RT-I-PCR in EPTB (n = 51) and non-TB control (n = 49) subjects, respectively. Both MB-AuNP-I-PCR/magneto-ELISA exhibited significantly lower (p < 0.05-0.01) sensitivities than MB-AuNP-RT-I-PCR. Conclusion: The MB-AuNP-RT-I-PCR described herein shows good diagnostic accuracy, which may translate into a credible diagnostic kit.

Extrapulmonary tuberculosis (EPTB) is a type of tuberculosis disease caused by the bacteria Mycobacterium tuberculosis (Mtb) that affect other regions of the body, rather than the lungs. Detecting EPTB is difficult, and a fast and reliable test is needed. This study developed a test based on a small particle, known as a nanoparticle, to identify Mtb in people with EPTB. The test shows good accuracy and could be used for routine testing.

Diagnosis of genitourinary tuberculosis: detection of mycobacterial lipoarabinomannan and MPT-64 biomarkers within urine extracellular vesicles by nano-based immuno-PCR assay.

We detected a cocktail of Mycobacterium tuberculosis lipoarabinomannan (LAM) and MPT-64 biomarkers within urine extracellular vesicles (EVs) of genitourinary TB (GUTB) patients by nano-based immuno-PCR (I-PCR) assay, i.e., magnetic bead-coupled gold nanoparticle-based I-PCR (MB-AuNP-I-PCR) and compared the results with I-PCR and Magneto-ELISA. The size (s) of urine EVs ranged between 52.6 and 220.4 nm as analyzed by transmission electron microscopy (TEM) and nanoparticle tracking analysis. Functionalized AuNPs (coupled with detection antibodies/oligonucleotides) were characterized by UV-vis spectroscopy, TEM, ELISA, PCR, Atomic Force Microscopy and Fourier Transform Infrared spectroscopy, while conjugation of capture antibodies with MBs was validated by UV-vis spectroscopy and Magneto-ELISA. Our MB-AuNP-I-PCR exhibited sensitivities of 85% and 87.2% in clinically suspected (n = 40) and total (n = 47) GUTB cases, respectively, with 97.1% specificity in non-TB controls (n = 35). These results were further authenticated by the quantitative SYBR Green MB-AuNP-real-time I-PCR (MB-AuNP-RT-I-PCR). Concurrently, I-PCR and Magneto-ELISA showed sensitivities of 68.1% and 61.7%, respectively in total GUTB cases, which were significantly lower (p < 0.05-0.01) than MB-AuNP-I-PCR. Markedly, a wide range (400 fg/mL-11 ng/mL) of LAM+MPT-64 was quantified within urine EVs of GUTB cases by SYBR Green MB-AuNP-RT-I-PCR, which can assess the disease dynamics. This study will certainly improve the current algorithms used in GUTB diagnostics.

Diagnosis of tuberculosis by nanoparticle-based immuno-PCR assay based on mycobacterial MPT64 and CFP-10 detection.

Aim: To improve the diagnostic accuracy of immuno-PCR (I-PCR) in tuberculosis (TB) patients by using functionalized gold nanoparticles (AuNPs) coupled with detection antibodies and oligonucleotides, and magnetic beads (MBs) conjugated with capture antibodies in the liquid phase. Materials & methods: MB-coupled AuNP-based I-PCR (MB-AuNP-I-PCR) assay was designed to detect a cocktail of Mycobacterium tuberculosis MPT64 and CFP-10 proteins in bodily fluids of TB patients. Results: The sensitivities of 89.3 (n = 94) and 78.1% (n = 73) were observed in pulmonary TB and extrapulmonary TB patients, respectively, with specificities of 97.9-98.3%. Notably, the sensitivities attained by MB-AuNP-I-PCR in smear-negative pulmonary TB and extrapulmonary TB patients were significantly higher (p < 0.05-0.001) than Magneto-ELISA and GeneXpert assay. Conclusion: The improved technology, as well as enhanced diagnostic accuracy of MB-AuNP-I-PCR, may lead to development of an attractive diagnostic kit.

Boron-doped carbon quantum dots: a 'turn-off' fluorescent probe for dopamine detection.

Boron-doped carbon quantum dots (size 2.3 nm) were fabricated by a modified hydrothermal carbonization one-pot synthesis protocol using 4-hydroxy phenylboronic acid as the common precursor that provided seed for the formation of carbon quantum dots as well as the dopant. These quantum dots exhibited excellent properties, namely good aqueous dispersion, strong fluorescence emission, good environmental stability, high selectivity and sensitivity towards the neurochemical dopamine even in the absence of any linker, functionalizing agents or enzyme. It is shown that this material can be used as a 'turn-off' fluorescent probe for the detection of even low concentrations of dopamine with a limit of detection (3σ/S) of about 6 µM. The simplicity of the synthesis protocol and the ease of dopamine detection define the novelty of this approach.

Boron Doped Carbon Quantum Dots: Turn-Off Fluorescent Probe for Dopamine Detection.

In this report, Boron-doped carbon quantum dots (BCQD, size = 2.3 nm) were fabricated by modified hydrothermal carbonization method by one-pot synthesis using phenyl boronic acid as the common precursor that provided seed for the formation of carbon quantum dots as well as the dopant. These quantum dots exhibited excellent properties including aqueous dispersibility, strong fluorescence emission, good environmental stability, highselectivity and sensitivity towards the neurochemical, dopamine even in the absence of any linker or functionalizing agents. It is shown that this material can be used as a "turn off" fluorescent probe for the detection of even low concentration of dopamine with a minimum detection limit of ~6 µM. The simplicity of synthesis protocol and the easiness of dopamine detection define the novelty of this approach.

Multifunctional, fluorescent DNA-derived carbon dots for biomedical applications: bioimaging, luminescent DNA hydrogels, and dopamine detection.

Here, we describe the synthesis of 2-3 nm, hydrophilic, blue fluorescence-emitting carbon dots (C-Dots, made using a DNA precursor) by the hydrothermal route from the gelling concentration of 2% (w/v) DNA. These dots exhibited highly efficient internalization in pathogenic fungal cells, negligible cytotoxicity, good PL stability, and high biocompatibility, thus demonstrating their potential as nanotrackers in microbial studies. Bioimaging was performed using Candida albicans as the representative for microbial pathogens. The novelty of these dots is that they formed fluorescent nanocomposite hydrogels with the same DNA much below the gelation concentration (1% w/v) and the tunable gels possessed strength between 20 and 80 Pa with the corresponding gelation temperature Tgel between 40 to 50 °C. The network density and gelation free energy data supported the superior crosslinking ability of these dots. The as-prepared hydrogels can replace the existing toxic quantum dot-based hydrogels for drug delivery. We also demonstrated the use of a DNA hydrogel-fabricated working electrode (DNA-C-Dot/ITO electrode) for the biosensing of dopamine. Our electrochemical biosensor had a detection limit of 5 × 10-3 mM for dopamine. These multifunctional, fluorescent C-Dots and hydrogel after suitable conjugation or loading with molecules and drugs hold promising potential for further exploitation in bioimaging, targeted drug delivery, wound healing, and biosensing applications.

Method Validation for Simultaneous Quantification of Olmesartan and Hydrochlorothiazide in Human Plasma Using LC-MS/MS and Its Application Through Bioequivalence Study in Healthy Volunteers.

A new, simple, sensitive, selective, rapid, and high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for simultaneous quantification of Olmesartan and hydrochlorothiazide in human plasma. Simple liquid-liquid extraction procedure was applied for plasma sample pretreatment using a mixture of diethyl ether and dichloromethane, as an extraction solution. Analytes were separated on UNISOL C18 150*4.6 mm, 5 µm column using methanol, and 2 mM ammonium acetate pH 5.5 (80:20, v/v) as a mobile phase and detected by electrospray ionization in the multiple reaction monitoring (MRM) mode. The mass transition ion pairs were followed in negative ion mode as m/z 445.20 → 148.90 for Olmesartan; m/z 451.40 → 154.30 for Olmesartan D6 and m/z 295.80 → 205.10 for hydrochlorothiazide; m/z 298.90 → 206.30 for hydrochlorothiazide 13C D2. The method showed excellent linearity (r 2 > 0.99) over the concentration range of 5.002-2,599.934 ng/ml for Olmesartan and from 3.005 to 499.994 ng/ml for hydrochlorothiazide. Precision (% CV) and accuracy (% bias) for Olmesartan were found in the range of 3.07-9.02% and -5.00-0.00%, respectively. Precision (% CV) and accuracy (% bias) for hydrochlorothiazide were found in the range of 3.32-8.21% and 1.99-3.80%, respectively. This as developed novel and high-throughput liquid-liquid extraction bioanalytical method has substantial innovative value with the benefits of cost effectiveness, good extraction efficiency, shorter analysis run time, low organic solvent consumption, and simpler procedure over the previously reported solid-phase extraction method. The application of this method in pharmacokinetic studies was further demonstrated successfully through a bioequivalence study conducted on healthy human subjects, following oral administration of combined formulation of Olmesartan medoxomil and hydrochlorothiazide in fixed-dose tablet.

Evaluation of in silico designed inhibitors targeting MelF (Rv1936) against Mycobacterium marinum within macrophages.

We recently identified inhibitors targeting Mycobacterium marinum MelF (Rv1936) by in silico analysis, which exhibited bacteriostatic/bactericidal activity against M. marinum and M. tuberculosis in vitro. Herein, we evaluated the effect of best four inhibitors (# 5175552, # 6513745, # 5255829, # 9125618) obtained from the ChemBridge compound libraries, on intracellular replication and persistence of bacteria within IFN-γ activated murine RAW264.7 and human THP-1 macrophages infected with M. marinum. Inhibitors # 5175552 and # 6513745 significantly reduced (p < 0.05) the intracellular replication of bacilli during day 7 post-infection (p.i.) within RAW264.7 and THP-1 macrophages infected at multiplicity of infection (MOI) of ~1.0. These observations were substantiated by electron microscopy, which revealed the protective effect of # 5175552 in clearing the bacilli inside murine macrophages. Strikingly, # 6513745 displayed synergism with isoniazid against M. marinum in murine macrophages, whereas # 5175552 significantly suppressed (p < 0.05) the persistent bacilli during day 10-14 p.i. in infected RAW264.7 and THP-1 macrophages (MOI of ~ 0.1). Moreover, # 5175552 and # 6513745 were non-cytotoxic to host macrophages at both 1X and 5X MIC. Further validation of these inhibitors against M. tuberculosis-infected macrophages and animal models has potential for development as novel anti-tubercular agents.

Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay.

PURPOSE: Immuno-PCR (I-PCR), an ultrasensitive method, combines the versatility of ELISA with the exponential amplification capacity of PCR. Coupling of detection antibodies with the reporter DNA is a critical step of I-PCR. Gold nanoparticles (GNPs) and magnetic beads (MBs) are relatively easy to attach with the antibodies and DNA. Therefore, we designed MB-coupled GNP-based I-PCR (MB-GNP-I-PCR) assay for the detection of Mycobacterium tuberculosis antigen. METHODS: GNPs were synthesized by chemical reduction and seed-mediated synthesis. Functionalized GNPs were prepared by coupling GNPs with the detection antibodies and reporter DNA and were characterized. Detection limit of M. tuberculosis-specific purified early secreted antigenic target-6 (ESAT-6) (Rv3875) was determined by MB-GNP-I-PCR. RESULTS: Transmission electron microscopy revealed spherical and slightly polydispersed GNPs of ~20 and ~60 nm size. Coupling of antibodies to GNPs was indicated by a shift in absorption maxima from 524 to 534 nm, which was confirmed by transmission electron microscopy. A color reaction with ELISA and the presence of 76 bp product by PCR further validated the coupling of detection antibodies and signal DNA to the functionalized GNPs. Also, attachment of capture antibodies with MBs was confirmed by magneto-ELISA. Detection limit of purified ESAT-6 by MB-GNP-I-PCR was determined to be 10 fg/mL, 105-fold lower than analogous ELISA. Notably, no sample matrix effect was observed in the saliva samples of healthy individuals spiked with the purified ESAT-6. CONCLUSION: Unlike conventional I-PCR (solid format), MB-GNP-I-PCR (liquid format) is relatively simple with the reduced background signals, which can be further exploited for the clinical diagnosis of tuberculosis.

Bandgap Tunable AgInS based Quantum Dots for High Contrast Cell Imaging with Enhanced Photodynamic and Antifungal Applications.

Herein, we report a facile microwave-assisted synthesis of cadmium-free water-soluble silver indium sulfide (AgInS2 or AIS) and AgInS@ZnS (or AIS@ZnS) core-shell quantum dots (QDs) using glutathione (GSH) as stabilizer. The core and core-shell nanocrystals exhibit tunable bandgap ranging of 2.3-3.1 and 2.4-3.5 eV, mean particle size of 2.5 and 3.25 nm, quantum yield of 26% and 49%, and fluorescence lifetimes of 326 and 438 ns, respectively. The core-shell QDs exhibit color-tunable emission in the visible region (500 to 600 nm), where the tunability was achieved by varying the molar ratio of Ag:In in the precursors. In vitro evaluation of antifungal activity of these water/ buffer stable QDs against the fungal pathogen, Candida albicans demonstrated that these were not toxic to the fungal cells upto a concentration of 100 µg/ml for 16 hours of incubation. Confocal imaging and spectrofluorometric studies showed enhanced fluorescence inside the microbial cells suggesting that AIS@ZnS particles had the capability to easily penetrate the cells. The increased generation of reactive oxygen species was evaluated for the core-shell QDs (photosensitizers) by using 9, 10-anthracenediyl-bis(methylene)dimalonic acid (ABMDMA) as singlet oxygen (1O2) scavenger molecule. These QDs have the potential for use as high contrast cell imaging, photodynamic and antifungal agents.

Silver nanoparticles induced alterations in multiple cellular targets, which are critical for drug susceptibilities and pathogenicity in fungal pathogen (Candida albicans).

PURPOSE: A significant increase in the incidence of fungal infections and drug resistance has been observed in the past decades due to limited availability of broad-spectrum antifungal drugs. Nanomedicines have shown significant antimicrobial potential against various drug-resistant microbes. Silver nanoparticles (AgNps) are known for their antimicrobial properties and lower host toxicity; however, for clinical applications, evaluation of their impact at cellular and molecular levels is essential. The present study aims to understand the cellular and molecular mechanisms of AgNp-induced toxicity in a common fungal pathogen, Candida albicans. METHODS: AgNps were synthesized by chemical reduction method and characterized using UV-visible spectroscopy, X-ray powder diffraction, transmission electron microscopy, scanning electron microscopy-energy dispersive X-ray spectroscopy, energy dispersive X-ray fluorescence, and zeta potential. The anti-Candida activity of AgNps was assessed by broth microdilution and spot assays. Effects of AgNps on cellular and molecular targets were assessed by monitoring the intracellular reactive oxygen species (ROS) production in the absence and presence of natural antioxidant, changes in surface morphology, cellular ultrastructure, membrane microenvironment, membrane fluidity, membrane ergosterol, and fatty acids. RESULTS: Spherical AgNps (10-30 nm) showed minimum inhibitory concentration (minimum concentration required to inhibit the growth of 90% of organisms) at 40 µg/mL. Our results demonstrated that AgNps induced dose-dependent intracellular ROS which exerted antifungal effects; however, even scavenging ROS by antioxidant could not offer protection from AgNp mediated killing. Treatment with AgNps altered surface morphology, cellular ultrastructure, membrane microenvironment, membrane fluidity, ergosterol content, and fatty acid composition, especially oleic acid. CONCLUSION: To summarize, AgNps affected multiple cellular targets crucial for drug resistance and pathogenicity in the fungal cells. The study revealed new cellular targets of AgNps which include fatty acids like oleic acid, vital for hyphal morphogenesis (a pathogenic trait of Candida). Yeast to hypha transition being pivotal for virulence and biofilm formation, targeting virulence might emerge as a new paradigm for developing nano silver-based therapy for clinical applications in fungal therapeutics.

In vitro studies on oxidative stress-independent, Ag nanoparticles-induced cell toxicity of Candida albicans, an opportunistic pathogen.

Silver nanoparticles (AgNps) have attracted maximal attention among all metal nanoparticles, and the study of their biological properties has gained impetus for further medical adoption. This study evaluated the cellular and molecular mechanisms associated with the action of AgNps against an opportunistic pathogen, Candida albicans. Spherical, stable AgNp (average size 21.6 nm) prepared by a chemical reduction method showed minimum inhibitory concentration (required to inhibit the growth of 90% of organisms) at 40 µg/mL. AgNps have been reported to induce oxidative stress-mediated programmed cell death through the accumulation of intracellular reactive oxygen species (ROS). However, this study demonstrated that intracellular levels of AgNp-induced ROS could be reversed by using antioxidant ascorbic acid, but the sensitivity of AgNp-treated Candida cells could not be completely reversed. Moreover, in addition to the generation of ROS, the AgNps were found to affect other cellular targets resulting in altered membrane fluidity, membrane microenvironment, ergosterol content, cellular morphology, and ultrastructure. Thus, the generation of ROS does not seem to be the sole major cause of AgNp-mediated cell toxicity in Candida. Rather, the multitargeted action of AgNps, generation of ROS, alterations in ergosterol content, and membrane fluidity together seem to have potentiated anti-Candida action. Thus, this "nano-based drug therapy" is likely to favor broad-spectrum activity, multiple cellular targets, and minimum host toxicity. AgNps, therefore, appear to have the potential to address the challenges in multidrug resistance and fungal therapeutics.

Antifungal efficacy of Au@ carbon dots nanoconjugates against opportunistic fungal pathogen, Candida albicans.

In the current study, we have investigated the toxicological effect of a novel hydrophilic nanoconjugate gold@carbon dot (Au@CD) and carbon dots (CDs) on the opportunistic fungal pathogen, Candida albicans. A homogenous experimental analysis was conducted for determining the toxicity of Au@CDs nanoconjugates of five different sizes ranging from 22 ±â€¯2 to 35 ±â€¯3 nm prepared using the carbon dots of mean hydrodynamic radius 12 ±â€¯1 nm. The smallest size of nanoconjugate was synthesized using 0.3 mg ml-1 HAuCl4 precursor. Our study for the first time, conclusively establishes the size-dependent toxicity effect of these characterized nanoconjugates against the abovementioned fungal pathogen. The MIC80 value of smaller sized Au@CDs nanoconjugates, S1-S3 samples were 250, 500 and 500 µg ml-1, respectively, while nanoconjugates of Rh diameter greater than 30 nm (S4 and S5 samples) did not show any toxicity. The results thus demonstrate that alteration in composition (carbon vs Au@CDs) exhibits a profound effect on the susceptibility of Candida albicans cells. While a size-dependent toxicity was observed for the nanoconjugates, CDs were found to be quite toxic owing to their small size which facilitated their entry into the cells and challenged the biocompatibility of carbon allotropes.

Identification of Novel Raft Marker Protein, FlotP in Bacillus anthracis.

Lipid rafts are dynamic, nanoscale assemblies of specific proteins and lipids, distributed heterogeneously on eukaryotic membrane. Flotillin-1, a conserved eukaryotic raft marker protein (RMP) harbor SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C) and oligomerization domains to regulate various cellular processes through its interactions with other signaling or transport proteins. Rafts were thought to be absent in prokaryotes hitherto, but recent report of its presence and significance in physiology of Bacillus subtilis prompted us to investigate the same in pathogenic bacteria (PB) also. In prokaryotes, proteins of SPFH2a subfamily show highest identity to SPFH domain of Flotillin-1. Moreover, bacterial genome organization revealed that Flotillin homolog harboring SPFH2a domain exists in an operon with an upstream gene containing NFeD domain. Here, presence of RMP in PB was initially investigated in silico by analyzing the presence of SPFH2a, oligomerization domains in the concerned gene and NfeD domain in the adjacent upstream gene. After investigating 300 PB, four were found to harbor RMP. Among them, domains of Bas0525 (FlotP) of Bacillus anthracis (BA) showed highest identity with characteristic domains of RMP. Considering the global threat of BA as the bioterror agent, it was selected as a model for further in vitro characterization of rafts in PB. In silico and in vitro analysis showed significant similarity of FlotP with numerous attributes of Flotillin-1. Its punctate distribution on membrane with exclusive localization in detergent resistant membrane fraction; strongly favors presence of raft with RMP FlotP in BA. Furthermore, significant effect of Zaragozic acid (ZA), a raft associated lipid biosynthesis inhibitor, on several patho-physiological attributes of BA such as growth, morphology, membrane rigidity etc., were also observed. Specifically, a considerable decrease in membrane rigidity, strongly recommended presence of an unknown raft associated lipid molecule on membrane of BA. In addition, treatment with ZA decreased secretion of anthrax toxins and FlotP expression, suggesting potential role of raft in pathogenesis and physiology of BA. Thus, the present study not only suggest the existence and role of raft like entity in pathophysiology of BA but also its possible use for the development of novel drugs or vaccines against anthrax.

Vitellogenin from the silkworm, Bombyx mori: an effective anti-bacterial agent.

Silkworm, Bombyx mori, vitellogenin (Vg) was isolated from perivisceral fat body of day 3 of pupa. Both Vg subunits were co-purified as verified by mass spectrometry and immunoblot. Purified Vg responded to specific tests for major posttranslational modifications on native gels indicating its nature as lipo-glyco-phosphoprotein. The Vg fraction had strong antibacterial activity against Gram negative bacterium Escherichia coli and Gram positive bacterium Bacillus subtilis. Microscopic images showed binding of Vg to bacterial cells and their destruction. When infected silkworm larvae were treated with purified Vg they survived the full life cycle in contrast to untreated animals. This result showed that Vg has the ability to inhibit the proliferation of bacteria in the silkworm fluid system without disturbing the regular metabolism of the host.

Phospholipidome of Candida: each species of Candida has distinctive phospholipid molecular species.

By employing electrospray ionization tandem mass spectrometry (ESI-MS/MS), the phospholipidomes of eight hemiascomycetous human pathogenic Candida species have been characterized. Over 200 phospholipid molecular species were identified and quantified. There were no large differences among Candida species in phosphoglyceride class composition; however, differences in phosphoglycerides components (i.e., fatty acyl chains) were identified. In contrast, differences in sphingolipid class composition as well as in molecular species were quite evident. The phospholipid compositions of C. albicans, C. glabrata, C. parapsilosis, C. kefyr, C. tropicalis, C. dubliniensis, C. krusei, and C. utilis could be further discriminated by principal component analysis. Notwithstanding that a single strain of each species was analyzed, our data do point to a typical molecular species imprint of Candida strains.

Morphogenic regulator EFG1 affects the drug susceptibilities of pathogenic Candida albicans.

This study shows that the morphogenic regulator EFG1 level affects the drug susceptibilities of Candida albicans when grown on solid growth media. The Deltaefg1 mutant showed sensitivity particularly to those drugs that target ergosterol or its metabolism. Efg1p disruption showed a gene-dosage effect on drug susceptibilities and resulted in enhanced susceptibility to drugs in the homozygous mutant as compared with the wild type, heterozygous and revertant strains. The enhanced sensitivity to drugs was independent of the status of ATP-binding cassette and MFS multidrug efflux pumps of C. albicans. The Deltaefg1 mutant displayed increased membrane fluidity that coincided with the downregulation of ERG11 and upregulation of OLE1 and ERG3, leading to enhanced passive diffusion of drugs. Interestingly, Deltaefg1 mutant cells displayed enhanced levels of endogenous ROS levels. Notably, the higher levels of ROS in the Deltaefg1 mutant could be reversed by the addition of antioxidants. However, the restoration of ROS levels did not reverse the drug sensitivities of the Deltaefg1 mutant. Taken together, we, for the first time, establish a new role to EFG1 in affecting the drug susceptibilities of C. albicans cells, independent of ROS and known drug efflux mechanisms.