Co-expression of anti-NFκB RNA aptamers and siRNAs leads to maximal suppression of NFκB activity in mammalian cells

The specific down-regulation of gene expression in cells is a powerful method for elucidating a gene's function. A common method for suppressing gene expression is the elimination of mRNA by RNAi or antisense. Alternatively, oligonucleotide-derived aptamers have been used as protein-directed agents for the specific knock-down of both intracellular and extracellular protein activity. Protein-directed methods offer the advantage of more closely mimicking small molecule therapeutics' mechanism of activity. Furthermore, protein-directed methods may synergize with RNA-directed methods since the two methods attack gene expression at different levels. Here we have knocked down a well-characterized intracellular protein's activity, NFκB, by expressing either aptamers or small interfering RNAs (siRNAs). Both methods can diminish NFκB's activity to similar levels (from 29 to 64%). Interestingly, expression of both aptamers and siRNAs simultaneously, suppressed NFκB activity better than either method alone (up to 90%). These results demonstrate that the expression of intracellular aptamers is a viable alternative to siRNA knock-down. Furthermore, for the first time, we show that the use of aptamers and siRNA together can be the most effective way to achieve maximal knock-down of protein activity.     

Estrogen induces thymic atrophy by eliminating early thymic progenitors and inhibiting proliferation of beta-selected thymocytes

Although it has been established that high levels of estrogen can induce thymic involution, the mechanism by which this happens is not known. We have found that daily i.p. injections of the synthetic estrogen 17-beta-estradiol reduce thymus cellularity by 80% over a period of 4-6 days. Although the atrophy is most strikingly observed in the CD4/CD8 double-positive (DP) thymic subset, the loss of thymocytes is not accompanied by a significant increase in thymocyte apoptosis, suggesting that direct killing of cells may not be the dominant means by which estrogens induce thymic atrophy. Instead, we find that estradiol drastically reduces the lineage-negative, Flt3(+)Sca-1(+)c-Kit(+) population in the bone marrow, a population that contains thymic homing progenitors. Within the thymus, we observe that estradiol treatment results in a preferential depletion of early thymic progenitors. In addition, we find that estradiol leads to a significant reduction in the proliferation of thymocytes responding to pre-TCR signals. Reduced proliferation of DN3 and DN4 cell subsets is likely the major contributor to the reduction in DP thymocytes that is observed. The reduction in early thymic progenitors is also likely to contribute to thymic atrophy, as we show that estradiol treatment can reduce the size of Rag1-deficient thymuses, which lack pre-TCR signals and DP thymocytes.     

Development of a duplex real-time PCR for the detection of Rickettsia spp. and typhus group rickettsia in clinical samples

Molecular diagnosis using real-time polymerase chain reaction (PCR) may allow earlier diagnosis of rickettsiosis. We developed a duplex real-time PCR that amplifies (1) DNA of any rickettsial species and (2) DNA of both typhus group rickettsia, that is, Rickettsia prowazekii and Rickettsia typhi. Primers and probes were selected to amplify a segment of the 16S rRNA gene of Rickettsia spp. for the pan-rickettsial PCR and the citrate synthase gene (gltA) for the typhus group rickettsia PCR. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing human DNA and 22 pathogens or skin commensals. Real-time PCR was applied to 16 clinical samples. Rickettsial DNA was detected in the skin biopsies of three patients. In one patient with severe murine typhus, the typhus group PCR was positive in a skin biopsy from a petechial lesion and seroconversion was later documented. The two other patients with negative typhus group PCR suffered from Mediterranean and African spotted fever, respectively; in both cases, skin biopsy was performed on the eschar. Our duplex real-time PCR showed a good analytical sensitivity and specificity, allowing early diagnosis of rickettsiosis among three patients, and recognition of typhus in one of them.     

Modular bioreactor for primary human hepatocyte culture: medium flow stimulates expression and activity of detoxification genes

Down-regulation of detoxification genes, notably cytochrome P450 (CYPs), in primary hepatocyte cultures is a long-standing and major concern. We evaluated the influence of medium flow in this model. Hepatocytes isolated from 12 different liver donors were cultured either in a multichamber modular bioreactor (MCmB, flow rate 250-500 μL/min) or under standard/static conditions, and the expression of 32 genes, enzyme activities and biological parameters were measured 7-21 days later. mRNA expression of genes involved in xenobiotic/drug metabolism and transport, including CYP1A1, 1A2, 2B6, 2C9, 3A4 (and activities for some of them), UDP-glucuronosyltransferase (UGT) 1A1, UGT2B4, UGT2B7, glutathione S-transferase (GSTα), and multidrug resistance protein 1 (MDR1) and MRP2, were specifically up-regulated by medium flow as compared with static controls in all cultures tested. In 2-week-old cultures, expression of detoxification genes reached levels close to or higher than those measured in freshly isolated hepatocytes. In contrast, CYP2D6 and most of other tested genes were not affected by medium flow. We conclude that medium flow specifically interferes with, and up-regulates, the activity of xenosensors and/or the expression of detoxification genes in primary human hepatocytes. Down-regulation of detoxification genes in conventional (static) cultures is therefore partly a consequence of the absence of medium circulation.     

Regulation of the xylan-degrading apparatus of Cellvibrio japonicus by a novel two-component system

The microbial degradation of lignocellulose biomass is not only an important biological process but is of increasing industrial significance in the bioenergy sector. The mechanism by which the plant cell wall, an insoluble composite structure, activates the extensive repertoire of microbial hydrolytic enzymes required to catalyze its degradation is poorly understood. Here we have used a transposon mutagenesis strategy to identify a genetic locus, consisting of two genes that modulate the expression of xylan side chain-degrading enzymes in the saprophytic bacterium Cellvibrio japonicus. Significantly, the locus encodes a two-component signaling system, designated AbfS (sensor histidine kinase) and AbfR (response regulator). The AbfR/S two-component system is required to activate the expression of the suite of enzymes that remove the numerous side chains from xylan, but not the xylanases that hydrolyze the beta1,4-linked xylose polymeric backbone of this polysaccharide. Studies on the recombinant sensor domain of AbfS (AbfS(SD)) showed that it bound to decorated xylans and arabinoxylo-oligosaccharides, but not to undecorated xylo-oligosaccharides or other plant structural polysaccharides/oligosaccharides. The crystal structure of AbfS(SD) was determined to a resolution of 2.6A(.) The overall fold of AbfS(SD) is that of a classical Per Arndt Sim domain with a central antiparallel four-stranded beta-sheet flanked by alpha-helices. Our data expand the number of molecules known to bind to the sensor domain of two-component histidine kinases to include complex carbohydrates. The biological rationale for a regulatory system that induces enzymes that remove the side chains of xylan, but not the hydrolases that cleave the backbone of the polysaccharide, is discussed.     

A high-confidence human plasma proteome reference set with estimated concentrations in PeptideAtlas

Human blood plasma can be obtained relatively noninvasively and contains proteins from most, if not all, tissues of the body. Therefore, an extensive, quantitative catalog of plasma proteins is an important starting point for the discovery of disease biomarkers. In 2005, we showed that different proteomics measurements using different sample preparation and analysis techniques identify significantly different sets of proteins, and that a comprehensive plasma proteome can be compiled only by combining data from many different experiments. Applying advanced computational methods developed for the analysis and integration of very large and diverse data sets generated by tandem MS measurements of tryptic peptides, we have now compiled a high-confidence human plasma proteome reference set with well over twice the identified proteins of previous high-confidence sets. It includes a hierarchy of protein identifications at different levels of redundancy following a clearly defined scheme, which we propose as a standard that can be applied to any proteomics data set to facilitate cross-proteome analyses. Further, to aid in development of blood-based diagnostics using techniques such as selected reaction monitoring, we provide a rough estimate of protein concentrations using spectral counting. We identified 20,433 distinct peptides, from which we inferred a highly nonredundant set of 1929 protein sequences at a false discovery rate of 1%. We have made this resource available via PeptideAtlas, a large, multiorganism, publicly accessible compendium of peptides identified in tandem MS experiments conducted by laboratories around the world.     

Variation analysis and gene annotation of eight MHC haplotypes: The MHC Haplotype Project

The human major histocompatibility complex (MHC) is contained within about 4 Mb on the short arm of chromosome 6 and is recognised as the most variable region in the human genome. The primary aim of the MHC Haplotype Project was to provide a comprehensively annotated reference sequence of a single, human leukocyte antigen-homozygous MHC haplotype and to use it as a basis against which variations could be assessed from seven other similarly homozygous cell lines, representative of the most common MHC haplotypes in the European population. Comparison of the haplotype sequences, including four haplotypes not previously analysed, resulted in the identification of >44,000 variations, both substitutions and indels (insertions and deletions), which have been submitted to the dbSNP database. The gene annotation uncovered haplotype-specific differences and confirmed the presence of more than 300 loci, including over 160 protein-coding genes. Combined analysis of the variation and annotation datasets revealed 122 gene loci with coding substitutions of which 97 were non-synonymous. The haplotype (A3-B7-DR15; PGF cell line) designated as the new MHC reference sequence, has been incorporated into the human genome assembly (NCBI35 and subsequent builds), and constitutes the largest single-haplotype sequence of the human genome to date. The extensive variation and annotation data derived from the analysis of seven further haplotypes have been made publicly available and provide a framework and resource for future association studies of all MHC-associated diseases and transplant medicine. Horton and Gibson contributed equally to this work.     

Unraveling G protein-coupled receptor endocytosis pathways using real-time monitoring of agonist-promoted interaction between beta-arrestins and AP-2

The most widely studied pathway underlying agonist-promoted internalization of G protein-coupled receptors (GPCRs) involves beta-arrestin and clathrin-coated pits. However, both beta-arrestin- and clathrin-independent processes have also been reported. Classically, the endocytic routes are characterized using pharmacological inhibitors and various dominant negative mutants, resulting sometimes in conflicting results and interpretational difficulties. Here, taking advantage of the fact that beta-arrestin binding to the beta2 subunit of the clathrin adaptor AP-2 (beta2-adaptin) is needed for the beta-arrestin-mediated targeting of GPCRs to clathrin-coated pits, we developed a bioluminescence resonance energy transfer-based approach directly assessing the molecular steps involved in the endocytosis of GPCRs in living cells. For 10 of the 12 receptors tested, including some that were previously suggested to internalize via clathrin-independent pathways, agonist stimulation promoted beta-arrestin 1 and 2 interaction with beta2-adaptin, indicating a beta-arrestin- and clathrin-dependent endocytic process. Detailed analyses of beta-arrestin interactions with both the receptor and beta2-adaptin also allowed us to demonstrate that recruitment of beta-arrestins to the receptor and the ensuing conformational changes are the leading events preceding AP-2 engagement and subsequent clathrin-mediated endocytosis. Among the receptors tested, only the endothelin A and B receptors failed to promote interaction between beta-arrestins and beta2-adaptin. However, both receptors recruited beta-arrestins upon agonist stimulation, suggesting a beta-arrestin-dependent but clathrin-independent route of internalization for these two receptors. In addition to providing a new tool to dissect the molecular events involved in GPCR endocytosis, the bioluminescence resonance energy transfer-based beta-arrestin/beta2-adaptin interaction assay represents a novel biosensor to assess receptor activation.     

Harvesting of Algae by Froth Flotation

A highly efficient froth flotation procedure has been developed for harvesting algae from dilute suspensions. The method does not depend upon the addition of flotants. Harvesting is carried out in a long column containing the feed solution which is aerated from below. A stable column of foam is produced and harvested from a side arm near the top of the column.

The cell concentration of the harvest is a function of pH, aeration rate, aerator porosity, feed concentration, and height of foam in the harvesting column. The economic aspects of this process seem favorable for mass harvesting of algae for food or other purposes.