Haptoglobin Genotype-dependent Differences in Macrophage Lysosomal Oxidative Injury

The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb.     

NINJA-OPS: Fast Accurate Marker Gene Alignment Using Concatenated Ribosomes

The explosion of bioinformatics technologies in the form of next generation sequencing (NGS) has facilitated a massive influx of genomics data in the form of short reads. Short read mapping is therefore a fundamental component of next generation sequencing pipelines which routinely match these short reads against reference genomes for contig assembly. However, such techniques have seldom been applied to microbial marker gene sequencing studies, which have mostly relied on novel heuristic approaches. We propose NINJA Is Not Just Another OTU-Picking Solution (NINJA-OPS, or NINJA for short), a fast and highly accurate novel method enabling reference-based marker gene matching (picking Operational Taxonomic Units, or OTUs). NINJA takes advantage of the Burrows-Wheeler (BW) alignment using an artificial reference chromosome composed of concatenated reference sequences, the “concatesome,” as the BW input. Other features include automatic support for paired-end reads with arbitrary insert sizes. NINJA is also free and open source and implements several pre-filtering methods that elicit substantial speedup when coupled with existing tools. We applied NINJA to several published microbiome studies, obtaining accuracy similar to or better than previous reference-based OTU-picking methods while achieving an order of magnitude or more speedup and using a fraction of the memory footprint. NINJA is a complete pipeline that takes a FASTA-formatted input file and outputs a QIIME-formatted taxonomy-annotated BIOM file for an entire MiSeq run of human gut microbiome 16S genes in under 10 minutes on a dual-core laptop.     

A comparison of conventional culture and three rapid methods for the detection of Salmonella in poultry feeds and environmental samples

Three rapid methods, an impedance method (Malthus 2000 Analyzer), a colorimetric DNA hybridization method (Gene-Trak) and a post-enrichment enzyme-linked immunosorbent assay (Salmonella-Tek) were compared with conventional culture for the detection of Salmonella in poultry feeds, and in fluff and dust samples from poultry housing. The percentage positive samples for Salmonella by each of the methods were 25.5% for conventional culture, 38.4% for the Malthus, 28.9% for the Gene-Trak and 28.5% for the Salmonella-Tek. By any method 60/153 (39.2%) of the samples tested were positive on confirmed culture.     

Biodegradation of BTEX by bacteria on powdered activated carbon

Aerobic degradation of a mixture of benzene, toluene, ethylbenzene and the mixed xylenes (BTEX) by a mixed bacterial population was studied in a continuously fed, completely mixed bioreactor in the presence of powdered activated carbon (PAC). Adsorption was characterized in the presence and in the absence of bacteria on PAC, and the affinity of virgin PAC to individual BTEX components was shown to be inversely correlated to their solubility in water. Bacteria colonizing the PAC particles are essential for simultaneous adsorption–biodegradation processes. In order to restrict biofilm formation and thereby mass transfer resistance of pollutants from the bulk to the PAC, the slurry was recirculated in the reactor system using a high shear pump. The bacteria on the PAC surface were constantly in a flux between the adsorbed and free phase, a phenomenon that prevented the formation of a biofilm on the PAC surface and thereby extended the life of the PAC.     

Sepsis,complement and the dysregulated inflammatory response

Sepsis in human beings is a major problem involving many individuals and with a high death rate. Except for a single drug (recombinant activated protein C) that has been approved for treatment of septic patients, supportive measures represent the main clinical approach. There are many models of experimental sepsis, mostly in rodents. A commonly used model is cecal ligation and puncture (CLP). In this model, robust activation of complement occurs together with up-regulation of C5a receptors (C5aR, C5L2) in a variety of different organs (lungs, kidneys, liver, heart). In septic human beings there is abundant evidence for complement activation. Interception of C5a or its receptors in the CLP model greatly improves survival in septic rodents. There is compelling evidence that CLP causes an intense pro-inflammatory state and that C5a interaction with its receptors can be linked to apoptosis of the lymphoid system and cells of the adrenal medulla, loss of innate immune functions of blood neutrophils, consumptive coagulopathy and cardiac dysfunction. These findings may have implications for therapeutic interventions in human beings with sepsis.     

Analysis and visualization of H7 influenza using genomic,evolutionary and geographic information in a modular web service

We have reported previously on use of a web‐based application, Supramap ( http://supramap.org ) for the study of biogeographic, genotypic, and phenotypic evolution. Using Supramap we have developed maps of the spread of drug‐resistant influenza and host shifts in H1N1 and H5N1 influenza and coronaviruses such as SARS. Here we report on another zoonotic pathogen, H7 influenza, and provide an update on the implementation of Supramap as a web service. We find that the emergence of pathogenic strains of H7 is labile with many transitions from high to low pathogenicity, and from low to high pathogenicity. We use Supramap to put these events in a temporal and geospatial context. We identify several lineages of H7 influenza with biomarkers of high pathogenicity in regions that have not been reported in the scientific literature. The original implementation of Supramap was built with tightly coupled client and server software. Now we have decoupled the components to provide a modular web service for POY ( http://poyws.org ) that can be consumed by a data provider to create a novel application. To demonstrate the web service, we have produced an application, Geogenes ( http://geogenes.org ). Unlike in Supramap, in which the user is required to create and upload data files, in Geogenes the user works from a graphical interface to query an underlying dataset. Geogenes demonstrates how the web service can provide underlying processing for any sequence and metadata database. © The Willi Hennig Society 2012.     

Interglacial high sea levels—an absolute chronology derived from shoreline elevations

The heights of some stranded shores in Gippsland, Australia, greatly exceed the level to which the ocean would rise if all present-day ice were to melt, and uplift is thus implied. Differences in level shown by comparison of these interglacial shorelines with others in South Carolina may therefore be the result of movement of one area relative to the other. Contemporary shorelines in the two localities can nevertheless be recognized from certain physiographic similarities and from the meagre measured age data presently available. We believe, furthermore, that there is sufficient information to permit estimation of the average rate of relative movement, and we attempt to analyse the field observations by applying a simple model that relates shoreline elevation to implied rate of uplift. This model is based on the supposition that the earth movements actually experienced were continuous, or can be reasonably represented as small fluctuations about a regular long-term trend. use of the model permits estimation of absolute ages (calculated from differences in altitude of correlated levels) for each high sea level stage that is recognized. Although the calculated or “altimetric” ages derived in this way are quite speculative, they agree with radiometric ages of Alaskan transgressions, interglacial littoral faunas, and warm intervals identified in deep-sea cores, and seem therefore to be verified by these comparisons. Particular correlations between high sea levels and warm core stages are implied: if these pairings are accepted, the altimetric ages can be re-estimated using the ages of the core stages. Predictions which use the calculated ages also provide tests of their value.