Development of a conjugate of (99m)Tc-EC with aminomethylenediphosphonate in the search for a bone tracer with fast clearance from soft tissue.

For the currently used (99m)Tc-labeled diphosphonates such as (99m)Tc-MDP and (99m)Tc-HDP, the required interval of 2.5 to 3 h between injection and the scintigraphic bone imaging is an inconvenience. The present study was set up in an attempt to develop a technetium-99m-labeled diphosphonate with efficient bone uptake and more rapid clearance from blood and soft tissue by renal extraction and excretion so that it would be possible to start imaging as early as 1 h after injection. A conjugate of the new renal tracer agent (99m)Tc-ethylene dicysteine ((99m)Tc-L,L-EC), covalently bound via one of its carboxylates with aminomethylenediphosphonic acid (AMDP), was synthesized in seven steps. EC-AMDP could be labeled easily and efficiently with (99m)Tc at pH > or = 12 and room temperature. Analysis using ion pair reversed phase high performance liquid chromatography showed the formation of a mixture of two main compounds with reproducible relative ratios, which were stable as a function of time. In a baboon, the scintigraphic images obtained with the new agent showed good quality bone scans, with clear visualization of the skeleton and low soft tissue activity at respectively 1 and 2 h after injection.     

Plankton composition,biomass, phylogeny and toxin genes in Lake Big Momela,Tanzania

Lake Big Momela, one of the East African soda lakes in Northern Tanzania characterised by highly saline-alkaline conditions, making them inhospitable to a range of organisms, although supporting massive growths of some adapted planktonic microorganisms that serve as food for birds, such as Lesser Flamingo. The temporal dynamics of plankton, with an emphasis on cyanobacteria, were examined in 2007 using morphological traits and ribosomal genetic markers (16S and 18S rRNA). Cyanobacterial genes encoding for hepatotoxins (mcyE and ndaF) were also screened. Rotifers and copepods dominated the zooplankton, whereas cyanobacteria, such as Anabaenopsis elenkinii and Arthrospira fusiformis dominated the phytoplankton community, and these being related to representatives in other East African soda lakes. The cyanobacteria community also showed distinct seasonal patterns influenced by environmental parameters, mainly salinity, pH and nitrate. Significant positive correlations were found between phytoplankton abundance and nitrate concentrations (r = 0.617, p = 0.033). No signals of the hepatotoxin synthetase genes mcyE and ndaF were retrieved from cyanobacteria during the whole year. In general, our data illustrate the presence of rich planktonic communities, including some unique and potentially endemic cyanobacteria.     

Strategies to fit pattern-mixture models

Whereas most models for incomplete longitudinal data are formulated within the selection model framework, pattern-mixture models have gained considerable interest in recent years (Little, 1993, 1994). In this paper, we outline several strategies to fit pattern-mixture models, including the so-called identifying restrictions strategy. Multiple imputation is used to apply this strategy to realistic settings, such as quality-of-life data from a longitudinal study on metastatic breast cancer patients.     

Identification of genes encoding exported Mycobacterium tuberculosis proteins using a Tn552'phoA in vitro transposition system

Secreted and cell envelope-associated proteins are important to both Mycobacterium tuberculosis pathogenesis and the generation of protective immunity to M. tuberculosis. We used an in vitro Tn552'phoA transposition system to identify exported proteins of M. tuberculosis. The system is simple and efficient, and the transposon inserts randomly into target DNA. M. tuberculosis genomic libraries were targeted with Tn552'phoA transposons, and these libraries were screened in M. smegmatis for active PhoA translational fusions. Thirty-two different M. tuberculosis open reading frames were identified; eight contain standard signal peptides, six contain lipoprotein signal peptides, and seventeen contain one or more transmembrane domains. Four of these proteins had not yet been assigned as exported proteins in the M. tuberculosis databases. This collection of exported proteins includes factors that are known to participate in the immune response of M. tuberculosis and proteins with homologies, suggesting a role in pathogenesis. Nine of the proteins appear to be unique to mycobacteria and represent promising candidates for factors that participate in protective immunity and virulence. This technology of creating comprehensive fusion libraries should be applicable to other organisms.     

Proteasome inhibition in glyoxal-treated fibroblasts and resistance of glycated glucose-6-phosphate dehydrogenase to 20 S proteasome degradation in vitro

Glycation and glycoxidation protein products are formed upon binding of sugars to NH(2) groups of lysine and arginine residues and have been shown to accumulate during aging and in pathologies such as Alzheimer's disease and diabetes. Because the proteasome is the major intracellular proteolytic system involved in the removal of altered proteins, the effect of intracellular glycation on proteasome function has been analyzed in human dermal fibroblasts subjected to treatment with glyoxal that promotes the formation of N epsilon-carboxymethyl-lysine adducts on proteins. The three proteasome peptidase activities were decreased in glyoxal-treated cells as compared with control cells, and glyoxal was also found to inhibit these peptidase activities in vitro. In addition, the activity of glucose-6-phosphate dehydrogenase, a crucial enzyme for the regulation of the intracellular redox status, was dramatically reduced in glyoxal-treated cells. Further analysis was performed to determine whether glycated proteins are substrates for proteasome degradation. In contrast to the oxidized glucose-6-phosphate dehydrogenase, both N epsilon-carboxymethyl-lysine- and fluorescent-glycated enzymes were resistant to degradation by the 20 S proteasome in vitro, and this resistance was correlated with an increased conformational stability of the glycated proteins. These results provide one explanation for why glycated proteins build up both as a function of disease and aging. Finally, N epsilon-carboxymethyl-lysine-modified proteins were found to be ubiquitinated in glyoxal-treated cells suggesting a potential mechanism by which these modified proteins may be marked for degradation.     

Value of a newly sequenced bacterial genome

Next-generation sequencing(NGS) technologies have made high-throughput sequencing available to medium- and small-size laboratories, culminating in a tidal wave of genomic information. The quantity of sequenced bacterial genomes has not only brought excitement to the field of genomics but also heightened expectations that NGS would boost antibacterial discovery and vaccine development. Although many possible drug and vaccine targets have been discovered, the success rate of genome-based analysis has remained below expectations. Furthermore, NGS has had consequences for genome quality, resulting in an exponential increase in draft(partial data) genome deposits in public databases. If no further interests are expressed for a particular bacterial genome, it is more likely that the sequencing of its genome will be limited to a draft stage, and the painstaking tasks of completing the sequencing of its genome and annotation will not be undertaken. It is important to know what is lost when we settle for a draft genome and to determine the "scientific value" of a newly sequenced genome. This review addresses the expected impact of newly sequenced genomes on antibacterial discovery and vaccinology. Also, it discusses the factors that could be leading to the increase in the number of draft deposits and the consequent loss of relevant biological information.     

Thermoanaerobacter thermohydrosulfuricus WC1 Shows Protein Complement Stability during Fermentation of Key Lignocellulose-Derived Substrates

Thermoanaerobacter spp. have long been considered suitable Clostridium thermocellum coculture partners for improving lignocellulosic biofuel production through consolidated bioprocessing. However, studies using “omic”-based profiling to better understand carbon utilization and biofuel producing pathways have been limited to only a few strains thus far. To better characterize carbon and electron flux pathways in the recently isolated, xylanolytic strain, Thermoanaerobacter thermohydrosulfuricus WC1, label-free quantitative proteomic analyses were combined with metabolic profiling. SWATH-MS proteomic analysis quantified 832 proteins in each of six proteomes isolated from mid-exponential-phase cells grown on xylose, cellobiose, or a mixture of both. Despite encoding genes consistent with a carbon catabolite repression network observed in other Gram-positive organisms, simultaneous consumption of both substrates was observed. Lactate was the major end product of fermentation under all conditions despite the high expression of gene products involved with ethanol and/or acetate synthesis, suggesting that carbon flux in this strain may be controlled via metabolite-based (allosteric) regulation or is constrained by metabolic bottlenecks. Cross-species “omic” comparative analyses confirmed similar expression patterns for end-product-forming gene products across diverse Thermoanaerobacter spp. It also identified differences in cofactor metabolism, which potentially contribute to differences in end-product distribution patterns between the strains analyzed. The analyses presented here improve our understanding of T. thermohydrosulfuricus WC1 metabolism and identify important physiological limitations to be addressed in its development as a biotechnologically relevant strain in ethanologenic designer cocultures through consolidated bioprocessing.