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.     

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...     

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.     

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.     

Molecular genetic approach for identifying markers associated with yield traits in the silkworm, Bombyx mori using RFLP-STS primers

Bombyx mori, the mulberry silkworm, exhibits wide variability in yield and developmental attributes. The genetics of yield expression, shown to be of polygenic nature, is poorly studied in silkworm. To identify markers associated with 10 selected yield traits, multiple regression analysis (MRA) and discriminant function analysis (DFA) were applied on 64 markers generated with eight RFLP-derived sequence-tagged-site (STS) primers on the genomic DNA of 20 silkworm stocks of different origin and diverse yield potential. The analyses led to the identification of ten markers showing significant association with the different yield traits. The markers could classify the stocks according to yield potential, irrespective of their origin and status of diapause. Trait means were significantly different for stocks with and with out the associated marker. The inheritance of a marker G2(1300bp), selected at the first step of MRA for five yield traits was shown to segregate in 1:1 ratio in the F2 progeny from a cross between two divergent stocks. The relevance of the STS primers is discussed in the context of applying multiple regression model for identifying markers associated with yield expression and suitability for molecular breeding work in B. mori for yield improvement.     

Optimization of humanized IgGs in glycoengineered Pichia pastoris

As the fastest growing class of therapeutic proteins, monoclonal antibodies (mAbs) represent a major potential drug class. Human antibodies are glycosylated in their native state and all clinically approved mAbs are produced by mammalian cell lines, which secrete mAbs with glycosylation structures that are similar, but not identical, to their human counterparts. Glycosylation of mAbs influences their interaction with immune effector cells that kill antibody-targeted cells. Here we demonstrate that human antibodies with specific human N-glycan structures can be produced in glycoengineered lines of the yeast Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. Glycoengineered P. pastoris provides a general platform for producing recombinant antibodies with human N-glycosylation.