Structure of YraM, a protein essential for growth of Haemophilus influenzae

Nontypeable Haemophilus influenzae is an obligate human parasite that often causes middle ear infections in children and exacerbates chronic obstructive pulmonary disorder, the fourth leading cause of death in the United States. There are no effective vaccines available for this strain. The lipoprotein YraM (gene HI1655) was identified as essential for the growth and viability of H. influenzae but its function is unknown. Sequence comparisons showed that YraM is a fusion of two protein modules. We grew crystals of the carboxyl-terminal module of YraM comprising residues 257-573 (YraM-C), phased the diffraction data by the multiwavelength anomalous diffraction technique, and refined the model to a crystallographic R-factor of 0.16 (R(free) = 0.19) with data to 1.35 A resolution. The two-domain structure of YraM-C adopts a fold similar to that observed for the open, unliganded forms of several periplasmic binding proteins (PBPs) involved in bacterial active transport. Sequence alignments of YraM homologues from other Gram-negative species showed that the most conserved residues of YraM-C cluster between the two domains in the location where other PBPs bind their cognate ligand. Modeling of YraM-C into a closed conformation similar to the leucine-bound form of the Leu/Ile/Val-binding protein (LIVBP) shows a putative binding pocket larger than the leucine-binding site in LIVBP. The pocket has both polar and nonpolar surfaces, with the latter located in the same area where a leucine side chain binds to LIVBP. We discuss possible biological functions of YraM considering its predicted location in the outer membrane, a novel place for such a binding protein.     

Prediction of protein structure from ideal forms

For many years it has been accepted that the sequence of a protein can specify its three-dimensional structure. However, there has been limited progress in explaining how the sequence dictates its fold and no attempt to do this computationally without the use of specific structural data has ever succeeded for any protein larger than 100 residues. We describe a method that can predict complex folds up to almost 200 residues using only basic principles that do not include any elements of sequence homology. The method does not simulate the folding chain but generates many thousands of models based on an idealized representation of structure. Each rough model is scored and the best are refined. On a set of five proteins, the correct fold score well and when tested on a set of larger proteins, the correct fold was ranked highest for some proteins more than 150 residues, with others being close topological variants. All other methods that approach this level of success rely on the use of templates or fragments of known structures. Our method is unique in using a database of ideal models based on general packing rules that, in spirit, is closer to an ab initio approach.     

Correlated responses to selection for faster development and early reproduction in Drosophila: the evolution of larval traits

Studies on selection for faster development in Drosophila have typically focused on the trade-offs among development time, adult weight, and adult life span. Relatively less attention has been paid to the evolution of preadult life stages and behaviors in response to such selection. We have earlier reported that four laboratory populations of D. melanogaster selected for faster development and early reproduction, relative to control populations, showed considerably reduced preadult development time and survivorship, dry weight at eclosion, and larval growth rates. Here we study the larval phase of these populations in greater detail. We show here that the reduction in development time after about 50 generations of selection is due to reduced duration of the first and third larval instars and the pupal stage, whereas the duration of the second larval instar has not changed. About 90% of the preadult mortality in the selected populations is due to larval mortality. The third instar larvae, pupae, and freshly eclosed adults of the selected populations weigh significantly less than controls, and this difference appears during the third larval instar. Thereafter, percentage weight loss during the pupal stage does not differ between selected and control populations. The minimum amount of time a larva must feed to subsequently complete development is lower in the selected populations, which also exhibit a syndrome of reduced energy expenditure through reduction in larval feeding rate, larval digging and foraging activity, and pupation height. Comparison of these results with those observed earlier in populations selected for adaptation to larval crowding and faster development under a different protocol from ours reveal differences in the evolved traits that suggest that the responses to selection for faster development are greatly affected by the larval density at which selection acts and on details of the selection pressures acting on the timing of reproduction.     

A Novel Reading Scheme for Assessing the Extent of Radiographic Abnormalities and Its Association with Disease Severity in Sputum Smear-Positive Tuberculosis: An Observational Study in Hyderabad/India

Background

Existing reading schemes for chest X-ray (CXR) used to grade the extent of disease severity at diagnosis in patients with pulmonary tuberculosis (PTB) are often based on numerical scores that summate specific radiographic features. However, since PTB is known to exhibit a wide heterogeneity in pathology, certain features might be differentially associated with clinical parameters of disease severity.

Objective

We aimed to grade disease severity in PTB patients at diagnosis and after completion of DOTS treatment by developing a reading scheme based on five different radiographic manifestations and analyze their association with the clinical parameters of systemic involvement and infectivity.

Methods

141 HIV-negative adults with newly diagnosed sputum smear-positive PTB were enrolled in a prospective observational study in Hyderabad, India. The presence and extent on CXRs of five radiographic manifestations, i.e., lung involvement, alveolar infiltration, cavitation, lymphadenopathy and pleural effusion, were classified using the new reading scheme by using a four-quadrant approach. We evaluated the inter-reader reliability of each manifestation, and its association with BMI and sputum smear positivity at diagnosis. The presence and extent of these radiographic manifestations were further compared with CXRs on completion of DOTS treatment.

Results

At diagnosis, an average lung area of 51.7% +/- 23.3% was affected by radiographic abnormalities. 94% of the patients had alveolar infiltrates, with 89.4% located in the upper quadrants, suggesting post primary PTB and in 34.8% of patients cavities were found. We further showed that the extent of affected lung area was a negative predictor of BMI (β value -0.035, p 0.019). No significant association of BMI with any of the other CXR features was found. The extent of alveolar infiltrates, along with the presence of cavitation, were strongly associated with sputum smear positivity. The microbiological cure rate in our cohort after 6 months of DOTS treatment was 95%. The extent of the affected lung area in these patients decreased from 56.0% +/- 21.5% to 31.0 +/- 20% and a decrease was also observed in the extent of alveolar infiltrates from 98.4% to 25.8% in at least one quadrant, presence of cavities from 34.8% to 1.6%, lymphadenopathy from 46.8% to 16.1%, and pleural effusion from 19.4% to 6.5%.

Conclusions

We established a new assessment scheme for grading disease severity in PTB by specifically considering five radiographic manifestations which were differently associated with the BMI and sputum smear positivity, changed to a different extent after 6 months of treatment and exhibited an excellent agreement between radiologists. Our results suggest that this reading scheme might contribute to the estimation of disease severity with respect to differences in disease pathology. Further studies are needed to determine a correlation with short and long-term pulmonary function impairment and whether there would be any benefit in lengthening or modulating therapy based on this CXR severity assessment.     

Interpolyelectrolyte Complexes of Eudragit? EPO with Hypromellose Acetate Succinate and Eudragit? EPO with Hypromellose Phthalate as Potential Carriers for Oral Controlled Drug Delivery

The objective of this study was to compare a novel controlled release tablet formulation based on interpolyelectrolyte complex (PEC). Interpolymer interactions between the countercharged polymers like Eudragit® EPO (polycation) and hypromellose acetate succinate (polyanion) and Eudragit® EPO and hypromellose phthalate (polyanion) were investigated with a view to their use in per oral controlled release drug delivery systems. The formation of inter-macromolecular ionic bonds between cationic polymer and anionic polymer was investigated using Fourier transform infrared (FT-IR) spectroscopy and differential scanning calorimetry. The FT-IR spectra of the tested polymeric matrices are characterized by visible changes in the observed IR region indicating the interaction between chains of two oppositely charged copolymers. The performance of the in situ formed PEC as a matrix for controlled release of drugs was evaluated, using acetaminophen as a model drug. The dissolution data of these matrices were fitted to different dissolution models. It was found that drug release followed zero-order kinetics and was controlled by the superposition of the diffusion and erosion. These profiles could be controlled by conveniently modifying the proportion of the polymer ratio, polymer type, and polymer concentration the in the tablets.KEY WORDS: Eudragit E, hypromellose acetate succinate, hypromellose phthalate polyelectrolyte complexation     

Bioconversion of isoflavone glycosides to aglycones,mineral bioavailability and vitamin B complex in fermented soymilk by probiotic bacteria and yeast

Aim: To study the role of β‐glucosidase producing probiotic bacteria and yeast in the biotransformation of isoflavone glycosides to aglycones, mineral bioavailability and vitamin B complex in fermented soymilk. Methods and Results: Five isolates of probiotic lactic acid bacteria (LAB), Lactobacillus acidophilus B4496, Lactobacillus bulgaricus CFR2028, Lactobacillus casei B1922, Lactobacillus plantarum B4495 and Lactobacillus fermentum B4655 with yeast Saccharomyces boulardii were used to ferment soymilk to obtain the bioactive isoflavones, genistein and daidzein. High‐performance liquid chromatography was used to analyse the concentration of isoflavones. Bioactive aglycones genistein and daidzein after 24 and 48 h of fermentation ranged from 97·49 to 98·49% and 62·71 to 92·31% respectively with different combinations of LAB with yeast. Increase in bioavailability of minerals and vitamin B complex were also observed in fermented soymilk. Conclusions: LAB in combination with yeast S. boulardii has great potential for the enrichment of bioactive isoflavones, enhancing the viability of LAB strains, decreasing the antinutrient phytic acid and increasing the mineral bioavailability in soymilk fermentation. Significance and Impact of the Study: Fermentation of soymilk with probiotic organisms improves the bioavailability of isoflavones, assists in digestion of protein, provides more soluble calcium, enhances intestinal health and supports immune system. Increased isoflavone aglycone content in fermented soymilk improves the biological functionality of soymilk.     

Effect of glutaraldehyde on hemoglobin: functional aspects and M?ssbauer parameters

Glutaraldehyde is widely used for the cross-linking of hemoglobin for blood substitute research or for technological purposes. The effects of this reagent on the biochemical properties of hemoglobin were correlated with M?ssbauer data. Human hemoglobin was cross-linked by glutaraldehyde as soluble polymers and insoluble particles. Effects of cross-linking on oxygen affinity, oxidation-reduction potential, autoxidation kinetics, and thermal stability were studied. Stability of cross-linked hemoglobin was specifically studied by M?ssbauer spectroscopy. Oxygen affinity is increased, redox potential is decreased, autoxidation rates are increased, and stability towards thermal denaturation is increased. The regeneration of partially denatured hemoglobin by glutaraldehyde cross-linking is shown. Effects of cross-linking on biochemical properties are explained by the hypothesis of the opening of the heme pocket on the distal-histidine side and the concomitant charge transfer from the iron to the oxygen.