Reactive phenotypes after acute and chronic NK-cell activation

Several phenotypic changes have been shown to occur after NK-cell stimulation, involving molecules that have been proved to regulate NK-cell migration into tissues and NK-cell activation and proliferation as well as target cell recognition and killing. Here, we review the reactive phenotypes observed in vivo after acute and chronic NK-cell activation.     

Determination of protein concentration in downstream biomanufacturing processes by in-line index of refraction

Protein concentration determination is a necessary in-process control for the downstream operations within biomanufacturing. As production transitions from batch mode to an integrated continuous bioprocess paradigm, there is a growing need to move protein concentration quantitation from off-line to in-line analysis. One solution to fulfill this process analytical technology need is an in-line index of refraction (IoR) sensor to measure protein concentration in real time. Here the performance of an IoR sensor is evaluated through a series of experiments to assess linear response, buffer matrix effects, dynamic range, sensor-to-sensor variability, and the limits of detection and quantitation. The performance of the sensor was also tested in two bioprocessing scenarios, ultrafiltration and capture chromatography. The implementation of this in-line IoR sensor for real-time protein concentration analysis and monitoring has the potential to improve continuous bioprocess manufacturing.     

Nutrient resorption in tropical savanna forests and woodlands of central Brazil

Nutrient limitation in Brazilian savanna (known as cerrado) presumably causes trees to maximize nutrient resorption from senesced leaves to reduce their dependence on nutrient availability. To assess patterns between nutrient resorption and soil fertility, we measured community-level nitrogen (N), phosphorus (P), and potassium (K) concentrations in mature and senesced leaves and soil fertility in the upper 50 cm soil layer in structurally diverse cerrado ecosystems in the Cuiaba Basin (CB) and Pantanal (PAN) of Mato Grosso, Brazil. Foliar nutrient concentration data were used to estimate resorption efficiency and proficiency, and correlation was used to determine whether resorption efficiency and proficiency varied across soil fertility gradients. We found that N and P resorption proficiency (NRP and PRP, respectively) and P resorption efficiency (PRE) increased significantly as total soil N (NRP) and extractable P (PRP and PRE) declined. In contrast, K resorption efficiency (KRE) declined as soil sand content and bulk density increased, which was likely due to a reduction in soil water-holding capacity. Leaf N/P ratios indicate potential N limitation and/or N + P co-limitation for ecosystems in the PAN and P limitation and/or N + P co-limitation for ecosystems in the CB, while trends in leaf N/K ratios indicate possible K or K + P co-limitation for the CB only. Our results illustrate that cerrado forests and woodlands have highly variable nutrient resorption capacities that vary predictably across soil fertility or textural gradients and indicate that cerrado communities have flexible nutrient resorption that can reduce their dependence on soil nutrient availability.     

Patients' costs and cost-effectiveness of tuberculosis treatment in DOTS and non-DOTS facilities in Rio de Janeiro, Brazil

Background

Costs of tuberculosis diagnosis and treatment may represent a significant burden for the poor and for the health system in resource-poor countries.

Objectives

The aim of this study was to analyze patients'' costs of tuberculosis care and to estimate the incremental cost-effectiveness ratio (ICER) of the directly observed treatment (DOT) strategy per completed treatment in Rio de Janeiro, Brazil.

Methods

We interviewed 218 adult patients with bacteriologically confirmed pulmonary tuberculosis. Information on direct (out-of-pocket expenses) and indirect (hours lost) costs, loss in income and costs with extra help were gathered through a questionnaire. Healthcare system additional costs due to supervision of pill-intake were calculated considering staff salaries. Effectiveness was measured by treatment completion rate. The ICER of DOT compared to self-administered therapy (SAT) was calculated.

Principal Findings

DOT increased costs during the treatment phase, while SAT increased costs in the pre-diagnostic phase, for both the patient and the health system. Treatment completion rates were 71% in SAT facilities and 79% in DOT facilities. Costs per completed treatment were US$ 194 for patients and U$ 189 for the health system in SAT facilities, compared to US$ 336 and US$ 726 in DOT facilities. The ICER was US$ 6,616 per completed DOT treatment compared to SAT.

Conclusions

Costs incurred by TB patients are high in Rio de Janeiro, especially for those under DOT. The DOT strategy doubles patients'' costs and increases by fourfold the health system costs per completed treatment. The additional costs for DOT may be one of the contributing factors to the completion rates below the targeted 85% recommended by WHO.     

Probing the Interaction of Brain Fatty Acid Binding Protein (B-FABP) with Model Membranes

Brain fatty acid-binding protein (B-FABP) interacts with biological membranes and delivers polyunsaturated fatty acids (FAs) via a collisional mechanism. The binding of FAs in the protein and the interaction with membranes involve a motif called “portal region”, formed by two small α-helices, A1 and A2, connected by a loop. We used a combination of site-directed mutagenesis and electron spin resonance to probe the changes in the protein and in the membrane model induced by their interaction. Spin labeled B-FABP mutants and lipidic spin probes incorporated into a membrane model confirmed that B-FABP interacts with micelles through the portal region and led to structural changes in the protein as well in the micelles. These changes were greater in the presence of LPG when compared to the LPC models. ESR spectra of B-FABP labeled mutants showed the presence of two groups of residues that responded to the presence of micelles in opposite ways. In the presence of lysophospholipids, group I of residues, whose side chains point outwards from the contact region between the helices, had their mobility decreased in an environment of lower polarity when compared to the same residues in solution. The second group, composed by residues with side chains situated at the interface between the α-helices, experienced an increase in mobility in the presence of the model membranes. These modifications in the ESR spectra of B-FABP mutants are compatible with a less ordered structure of the portal region inner residues (group II) that is likely to facilitate the delivery of FAs to target membranes. On the other hand, residues in group I and micelle components have their mobilities decreased probably as a result of the formation of a collisional complex. Our results bring new insights for the understanding of the gating and delivery mechanisms of FABPs.     

Synthesis and glycosidase inhibitory activity of 5-thioglucopyranosylamines. Molecular modeling of complexes with glucoamylase

The synthesis of a series of 5-thio-D-glucopyranosylarylamines by reaction of 5-thio-D-glucopyranose pentaacetate with the corresponding arylamine and mercuric chloride catalyst is reported. The products were obtained as anomeric mixtures of the tetraacetates which can be separated and crystallized. The tetraacetates were deprotected to give alpha/beta mixtures of the parent compounds which were evaluated as inhibitors of the hydrolysis of maltose by glucoamylase G2 (GA). A transferred NOE NMR experiment with an alpha/beta mixture of 7 in the presence of GA showed that only the alpha isomer is bound by the enzyme. The Ki values, calculated on the basis of specific binding of the alpha isomers, are 0.47 mM for p-methoxy-N-phenyl-5-thio-D-glucopyranosylamine (7), 0.78 mM for N-phenyl-5-thio-D-glucopyranosylamine (8), 0.27 mM for p-nitro-N-phenyl-5-thio-D-glucopyranosylamine (9) and 0.87 mM for p-trifluoromethyl-N-phenyl-5-thio-D-glucopyranosylamine (10), and the K(m) values for the substrates maltose and p-nitrophenyl alpha-D-glucopyranoside are 1.2 and 3.7 mM, respectively. Methyl 4-amino-4-deoxy-4-N-(5'-thio-alpha-D-glucopyranosyl)-alpha-D-glucopyrano side (11) is a competitive inhibitor of GA wild-type (Ki 4 microM) and the active site mutant Trp120-->Phe GA (Ki 0.12 mM). Compounds 7, 8, and 11 are also competitive inhibitors of alpha-glucosidase from brewer's yeast, with Ki values of 1.05 mM, > 10 mM, and 0.5 mM, respectively. Molecular modeling of the inhibitors in the catalytic site of GA was used to probe the ligand-enzyme complementary interactions and to offer insight into the differences in inhibitory potencies of the ligands.     

Mapping of the human Voltage-Dependent Anion Channel isoforms 1 and 2 reconsidered

Eukaryotic porins or VDACs (Voltage-Dependent Anion-selective Channels) are integral membrane proteins forming large hydrophilic pores. Three functioning genes for VDAC isoforms have been detected in mouse and the corresponding cDNAs are known also in humans. Tissue-specific VDAC isoform 1 (HVDAC1) deficiency in human skeletal muscle is responsible of a rare mitochondrial encephalomyopathy, fatal in childhood. Since coding sequences are not affected in the patient, we focused our interest in the gene structure. HVDAC1 and 2 have been previously mapped at chromosomes Xq13-21 and 21, respectively. Screening of an human chromosome X cosmid library resulted only in the isolation of processed pseudogenes, finely mapped at Xq22 and Xp11.2. Here, we report the mapping of HVDAC1 to chromosome 5q31 and HVDAC2 to chromosome 10q22 by FISH. Exon/intron probes, designed on the basis of the mouse gene structures, were obtained by long extension PCR amplification using the whole genomic DNA as a template. The sequence of the probe extremities clearly pointed to a genuine VDAC genomic sequence. Human and mouse regions where VDAC 1 and 2 genes were mapped are known to be synthetic, thus reinforcing the mapping of the human homologues.     

GAA repeat instability in Friedreich ataxia YAC transgenic mice

Friedreich ataxia (FRDA) is primarily caused by an unstable GAA repeat-expansion mutation within intron 1 of the FRDA gene. However, the exact mechanisms leading to this expansion and its consequences are not fully understood. To study the dynamics of this mutation, we have generated two lines of human FRDA YAC transgenic mice that contain GAA repeat expansions within the appropriate genomic context. We have detected intergenerational instability and age-related somatic instability in both lines, with pronounced expansions found in the cerebellum. The dynamic nature of our transgenic GAA repeats is comparable with previous FRDA patient somatic tissue data. However, there is a difference between our FRDA YAC transgenic mice and other trinucleotide-repeat mouse models, which do not show pronounced repeat instability in the cerebellum. This represents the first mouse model of FRDA GAA repeat instability that will help to dissect the mechanism of this repeat.     

Enantiomeric resolution of kielcorin derivatives by HPLC on polysaccharide stationary phases using multimodal elution

Analytical HPLC methods using carbamate chiral stationary phases of polysaccharide derivatives were developed for the enantiomeric resolution of five racemic mixtures of xanthonolignoids: rac-trans-kielcorin C, rac-cis-kielcorin C, rac-trans-kielcorin D, rac-trans-isokielcorin D, and rac-trans-kielcorin E. The separations were evaluated with the stationary phases cellulose tris-3,5-dimethylphenylcarbamate, amylose tris-3,5-dimethylphenylcarbamate, amylose tris-(S)-1-phenylethylcarbamate, and amylose tris-3,5-dimethoxyphenylcarbamate under normal, reversed-phase, and polar organic elution conditions. Chiral recognition of those chiral stationary phases, the influence of mobile phases on the enantiomers separation, and the effects of structural features of the solutes on the chiral discrimination observed are discussed. The best performance was achieved on an amylose tris-3,5-dimethylphenylcarbamate phase. Polar organic conditions gave shorter retention factors and better resolutions and were a valuable alternative to the alcohol-hexane or reversed-phase conditions.