Talaromyces marneffei Outside Endemic Areas in India: an Emerging Infection with Atypical Clinical Presentations and Review of Published Reports from India

Mycopathologia - Talaromycosis is a disseminated disease caused by Talaromyces (Penicillium) marneffei, mainly seen in acquired immunodeficiency syndrome (AIDS) patients. Its distribution is...     

Microbial Delignification with White Rot Fungi Improves Forage Digestibility

Three wild-type white rot fungi and two cellulase-less mutants developed from Phanerochaete chrysosporium K-3 (formerly Sporotrichum pulverulentum) were tested for their ability to delignify grass cell walls and improve biodegradation by rumen microorganisms. Fungal-treated and control stems of Bermuda grass were analyzed for their content of ester- and ether-linked aromatics by using alkali extraction and gas chromatography, for in vitro dry weight digestion and production of volatile fatty acids in in vitro fermentations with mixed ruminal microorganisms, for loss of lignin and other aromatics from specific cell wall types by using microspectrophotometry, and for structural changes before and after in vitro degradation by rumen microorganisms by using transmission electron microscopy. P. chrysosporium K-3 and Ceriporiopsis subvermispora FP 90031-sp produced the greatest losses in lignin and improved the biodegradation of Bermuda grass over that of untreated control substrate. However, C. subvermispora removed the most lignin and significantly improved biodegradation over all other treatments. Phellinus pini RAB-83-19 and cellulase-less mutants 3113 and 85118 developed from P. chrysosporium K-3 did not improve the biodegradation of Bermuda grass lignocellulose. Results indicated that C. subvermispora extensively removed ester-linked p-coumaric and ferulic acids and also removed the greatest amount of non-ester-linked aromatics from plant cell walls. Microscopic observations further indicated that C. subvermispora removed esters from parenchyma cell walls as well as esters and lignin from the more recalcitrant cell walls (i.e., sclerenchyma and vascular tissues). C. subvermispora improved in vitro digestion and volatile fatty acid production by ruminal microorganisms by about 80%, while dry matter loss due to fungi was about 20% greater than loss in untreated control stems. The chemical and structural studies used identified sites of specific fungal attack and suggested mechanisms whereby improvement occurred.     

Isotope-coded affinity tag (ICAT) approach to redox proteomics: identification and quantitation of oxidant-sensitive cysteine thiols in complex protein mixtures

An approach is described for the simultaneous identification and quantitation of oxidant-sensitive cysteine thiols in a complex protein mixture using a thiol-specific, acid-cleavable isotope-coded affinity tag (ICAT) reagent (Applied Biosystems, USA). The approach is based on the fact that only free cysteine thiols are susceptible to labeling by the iodoacetamide-based ICAT, and that mass spectrometry can be used to quantitate the relative labeling of free thiols. Applying this approach, we have identified cysteine thiols of proteins in a rabbit heart membrane fraction that are sensitive to a high concentration of hydrogen peroxide. Previously known and some novel proteins with oxidant-sensitive cysteines were identified. Of the many protein thiols labeled by the ICAT, only relatively few were oxidized more than 50% despite the high concentration of oxidant used, indicating that oxidant-sensitive thiols are relatively rare, and denoting their specificity and potential functional relevance.     

Powder properties and their influence on dry powder inhaler delivery of an antitubercular drug

The purpose of this study was to determine if aerosol delivery of drug loaded microparticles to lungs infected withMycobacterium tuberculosis may be achieved by predicting dispersion of dry powders through knowledge of particle surface properties. Particle sizes of rifampicin-loaded poly(lactide-co-glycolide) microparticles (R-PLGA), rifampicin alone, and lactose and maltodextrin carrier particles (bulk and 75-125-μm sieved fractions) were determined by electron microscopy for the projected area diameter (D_p) and laser diffraction for the volume diameter (D_v). Surface energies (Y) of R-PLGA, rifampicin alone, lactose, and maltodextrin were obtained by inverse phase gas chromatography, surface areas (S_a) by N₂ adsorption, and cohesive energy densities by calculation. Particle dispersion was evaluated (Andersen nonviable impactor) for 10% blends of R-PLGA and rifampicin alone with bulk and sieved fractions of the carriers. D_p for R-PLGA and rifampicin alone was 3.02 and 2.83 μm, respectively. D_v was 13±1 and 2±1 μm for R-PLGA and rifampicin alone, respectively, indicating that R-PLGA was more aggregated. This was evident in Y of 35±1 and 19±6 mJ/m² for R-PLGA and rifampicin alone. D_p for lactose and maltodextrin (sieved and bulk) was approximately 40 mm. Bulk maltodextrin (D_v=119±6 mm) was more aggregated than bulk lactose (D_v=54±2 mm). This was a result of the higher S_a for maltodextrin (0.54 m²/g) than for lactose (0.21 m²/g). The Y of bulk lactose and maltodextrin was 40±4 and 60±6 mJ/m² and of sieved lactose and maltodextrin was 39±1 and 50±1 mJ/m². Impaction studies yielded higher fine particle fractions of R-PLGA from sieved lactose, 13%±3%, than from sieved maltodextrin, 7%±1%, at 90 L/min. An expression, based on these data, is proposed as a predictor of drug dispersion from carrier particles. Delivery of dry powder formulations can be achieved by characterizing particle surfaces and predicting impact on dispersion.     

Roles of ring-hydroxylating dioxygenases in styrene and benzene catabolism in Rhodococcus jostii RHA1

Proteomics and targeted gene disruption were used to investigate the catabolism of benzene, styrene, biphenyl, and ethylbenzene in Rhodococcus jostii RHA1, a well-studied soil bacterium whose potent polychlorinated biphenyl (PCB)-transforming properties are partly due to the presence of the related Bph and Etb pathways. Of 151 identified proteins, 22 Bph/Etb proteins were among the most abundant in biphenyl-, ethylbenzene-, benzene-, and styrene-grown cells. Cells grown on biphenyl, ethylbenzene, or benzene contained both Bph and Etb enzymes and at least two sets of lower Bph pathway enzymes. By contrast, styrene-grown cells contained no Etb enzymes and only one set of lower Bph pathway enzymes. Gene disruption established that biphenyl dioxygenase (BPDO) was essential for growth of RHA1 on benzene or styrene but that ethylbenzene dioxygenase (EBDO) was not required for growth on any of the tested substrates. Moreover, whole-cell assays of the ΔbphAa and etbAa1::cmrA etbAa2::aphII mutants demonstrated that while both dioxygenases preferentially transformed biphenyl, only BPDO transformed styrene. Deletion of pcaL of the β-ketoadipate pathway disrupted growth on benzene but not other substrates. Thus, styrene and benzene are degraded via meta- and ortho-cleavage, respectively. Finally, catalases were more abundant during growth on nonpolar aromatic compounds than on aromatic acids. This suggests that the relaxed specificities of BPDO and EBDO that enable RHA1 to grow on a range of compounds come at the cost of increased uncoupling during the latter's initial transformation. The stress response may augment RHA1's ability to degrade PCBs and other pollutants that induce similar uncoupling.     

Semi-targeted plasma proteomics discovery workflow utilizing two-stage protein depletion and off-line LC-MALDI MS/MS

A quantitative proteomics workflow was implemented that provides extended plasma protein coverage by extensive protein depletion in combination with the sensitivity and breadth of analysis of two-dimensional LC-MS/MS shotgun analysis. Abundant proteins were depleted by a two-stage process using IgY and Supermix depletion columns in series. Samples are then extensively fractionated by two-dimensional chromatography with fractions directly deposited onto MALDI plates. Decoupling sample fractionation from mass spectrometry facilitates a targeted MS/MS precursor selection strategy that maximizes measurement of a consistent set of peptides across experiments. Multiplexed stable isotope labeling provides quantification relative to a common reference sample and ensures an identical set of peptides measured in the set of samples (set of eight) combined in a single experiment. The more extensive protein depletion provided by the addition of the Supermix column did not compromise overall reproducibility of the measurements or the ability to reliably detect changes in protein levels between samples. The implementation of this workflow is presented for a case study aimed at generating molecular signatures for prediction of first heart attack.