Apoptosis Resistance in Fibroblasts Precedes Progressive Scarring in Pulmonary Fibrosis and Is Partially Mediated by Toll-Like Receptor 4 Activation.

Inhalation of environmental toxicants such as cigarette smoke, metal or wood dust, silica, or asbestos is associated with increased risk for idiopathic pulmonary fibrosis (IPF). IPF involves progressive scarring of lung tissue, which interferes with normal respiration and is ultimately fatal; however, the complex cellular mechanisms of IPF pathogenesis remain unclear. Fibroblast apoptosis is essential in normal wound healing but is dysregulated in IPF. Recent studies suggest that Toll-like receptor 4 (TLR4) is key in the onset of IPF. Here, radiation-induced PF was used as a model for IPF because it very closely mimics the progressive and intractable nature of IPF. Female C57BL/6J (C57) and C57BL/6J TLR4-/- mice were exposed to a single dose of 13 Gy whole-thorax ionizing radiation. Although both strains showed similar levels of immediate radiation-induced damage, C57 mice exhibited more extensive fibrosis at 22-week postirradiation (PI) than TLR4-/- mice. Isolated C57 primary 1° MLFs showed decreased apoptosis susceptibility as early as 8-week postirradiation, a phenotype that persisted for the remainder of the radiation response. TLR4-/- 1° mouse lung fibroblasts did not exhibit significant apoptosis resistance at any point. Systemic release of high mobility group box 1, a TLR4 agonist, during the pneumonitis phase of the radiation response may act through TLR4 to contribute to fibroblast apoptosis resistance and thus interfere with wound resolution. These findings demonstrate that apoptosis resistance occurs earlier in pulmonary fibrosis pathogenesis than previously assumed, and that TLR4 signaling is a key mediator in this process.

An accessible and high-throughput strategy of continuously monitoring apoptosis by fluorescent detection of caspase activation.

We present a real-time, high-throughput, and cost-effective method of detecting apoptosis in vitro using a previously developed reagent that detects caspase activation by fluorescence. Current methods of assessing apoptosis fail to account for the dimension of time, and thus are limited in data yielded per sample. This reagent allows real-time detection of apoptosis, but until now has been restricted to a costly automated detection system. Here, we describe apoptosis detection with the Essen Bioscience IncuCyte® Caspase-3/7 Reagent using a multimode microplate reader, a common instrument in biological laboratories, which may be used prior to or in lieu of the automated system. This modified microplate reader apoptosis assay was validated against the established automated system, and was shown to detect a strong dose-response relationship (automated system r2 = 0.9968, microplate reader r2 = 0.9924). We also propose a quick and reliable method of quantifying cell density by Hoechst 33342 nuclear staining in microplates (r2 = 0.8812 between Hoechst signal and cell density). We assert that the dimension of time should not be overlooked, and that the method presented here is an accessible strategy for many researchers due to low startup cost and precise detection of apoptosis in real time.

Next generation DNA sequencing technology delivers valuable genetic markers for the genomic orphan legume species, Bituminaria bituminosa.

BACKGROUND: Bituminaria bituminosa is a perennial legume species from the Canary Islands and Mediterranean region that has potential as a drought-tolerant pasture species and as a source of pharmaceutical compounds. Three botanical varieties have previously been identified in this species: albomarginata, bituminosa and crassiuscula. B. bituminosa can be considered a genomic 'orphan' species with very few genomic resources available. New DNA sequencing technologies provide an opportunity to develop high quality molecular markers for such orphan species. RESULTS: 432,306 mRNA molecules were sampled from a leaf transcriptome of a single B. bituminosa plant using Roche 454 pyrosequencing, resulting in an average read length of 345 bp (149.1 Mbp in total). Sequences were assembled into 3,838 isotigs/contigs representing putatively unique gene transcripts. Gene ontology descriptors were identified for 3,419 sequences. Raw sequence reads containing simple sequence repeat (SSR) motifs were identified, and 240 primer pairs flanking these motifs were designed. Of 87 primer pairs developed this way, 75 (86.2%) successfully amplified primarily single fragments by PCR. Fragment analysis using 20 primer pairs in 79 accessions of B. bituminosa detected 130 alleles at 21 SSR loci. Genetic diversity analyses confirmed that variation at these SSR loci accurately reflected known taxonomic relationships in original collections of B. bituminosa and provided additional evidence that a division of the botanical variety bituminosa into two according to geographical origin (Mediterranean region and Canary Islands) may be appropriate. Evidence of cross-pollination was also found between botanical varieties within a B. bituminosa breeding programme. CONCLUSIONS: B. bituminosa can no longer be considered a genomic orphan species, having now a large (albeit incomplete) repertoire of expressed gene sequences that can serve as a resource for future genetic studies. This experimental approach was effective in developing codominant and polymorphic SSR markers for application in diverse genetic studies. These markers have already given new insight into genetic variation in B. bituminosa, providing evidence that a division of the botanical variety bituminosa may be appropriate. This approach is commended to those seeking to develop useful markers for genomic orphan species.

An integrated genetic and functional analysis of the role of type II transmembrane serine proteases (TMPRSSs) in hearing loss.

Building on our discovery that mutations in the transmembrane serine protease, TMPRSS3, cause nonsyndromic deafness, we have investigated the contribution of other TMPRSS family members to the auditory function. To identify which of the 16 known TMPRSS genes had a strong likelihood of involvement in hearing function, three types of biological evidence were examined: 1) expression in inner ear tissues; 2) location in a genomic interval that contains a yet unidentified gene for deafness; and 3) evaluation of hearing status of any available Tmprss knockout mouse strains. This analysis demonstrated that, besides TMPRSS3, another TMPRSS gene was essential for hearing and, indeed, mice deficient for Hepsin (Hpn) also known as Tmprss1 exhibited profound hearing loss. In addition, TMPRSS2, TMPRSS5, and CORIN, also named TMPRSS10, showed strong likelihood of involvement based on their inner ear expression and mapping position within deafness loci PKSR7, DFNB24, and DFNB25, respectively. These four TMPRSS genes were then screened for mutations in affected members of the DFNB24 and DFNB25 deafness families, and in a cohort of 362 sporadic deaf cases. This large mutation screen revealed numerous novel sequence variations including three potential pathogenic mutations in the TMPRSS5 gene. The mutant forms of TMPRSS5 showed reduced or absent proteolytic activity. Subsequently, TMPRSS genes with evidence of involvement in deafness were further characterized, and their sites of expression were determined. Tmprss1, 3, and 5 proteins were detected in spiral ganglion neurons. Tmprss3 was also present in the organ of Corti. TMPRSS1 and 3 proteins appeared stably anchored to the endoplasmic reticulum membranes, whereas TMPRSS5 was also detected at the plasma membrane. Collectively, these results provide evidence that TMPRSS1 and TMPRSS3 play and TMPRSS5 may play important and specific roles in hearing.

LOCATE: a mammalian protein subcellular localization database.

LOCATE is a curated, web-accessible database that houses data describing the membrane organization and subcellular localization of mouse and human proteins. Over the past 2 years, the data in LOCATE have grown substantially. The database now contains high-quality localization data for 20% of the mouse proteome and general localization annotation for nearly 36% of the mouse proteome. The proteome annotated in LOCATE is from the RIKEN FANTOM Consortium Isoform Protein Sequence sets which contains 58 128 mouse and 64 637 human protein isoforms. Other additions include computational subcellular localization predictions, automated computational classification of experimental localization image data, prediction of protein sorting signals and third party submission of literature data. Collectively, this database provides localization proteome for individual subcellular compartments that will underpin future systematic investigations of these regions. It is available at http://locate.imb.uq.edu.au/

Fast automated cell phenotype image classification.

BACKGROUND: The genomic revolution has led to rapid growth in sequencing of genes and proteins, and attention is now turning to the function of the encoded proteins. In this respect, microscope imaging of a protein's sub-cellular localisation is proving invaluable, and recent advances in automated fluorescent microscopy allow protein localisations to be imaged in high throughput. Hence there is a need for large scale automated computational techniques to efficiently quantify, distinguish and classify sub-cellular images. While image statistics have proved highly successful in distinguishing localisation, commonly used measures suffer from being relatively slow to compute, and often require cells to be individually selected from experimental images, thus limiting both throughput and the range of potential applications. Here we introduce threshold adjacency statistics, the essence which is to threshold the image and to count the number of above threshold pixels with a given number of above threshold pixels adjacent. These novel measures are shown to distinguish and classify images of distinct sub-cellular localization with high speed and accuracy without image cropping. RESULTS: Threshold adjacency statistics are applied to classification of protein sub-cellular localization images. They are tested on two image sets (available for download), one for which fluorescently tagged proteins are endogenously expressed in 10 sub-cellular locations, and another for which proteins are transfected into 11 locations. For each image set, a support vector machine was trained and tested. Classification accuracies of 94.4% and 86.6% are obtained on the endogenous and transfected sets, respectively. Threshold adjacency statistics are found to provide comparable or higher accuracy than other commonly used statistics while being an order of magnitude faster to calculate. Further, threshold adjacency statistics in combination with Haralick measures give accuracies of 98.2% and 93.2% on the endogenous and transfected sets, respectively. CONCLUSION: Threshold adjacency statistics have the potential to greatly extend the scale and range of applications of image statistics in computational image analysis. They remove the need for cropping of individual cells from images, and are an order of magnitude faster to calculate than other commonly used statistics while providing comparable or better classification accuracy, both essential requirements for application to large-scale approaches.

Differential use of signal peptides and membrane domains is a common occurrence in the protein output of transcriptional units.

Membrane organization describes the orientation of a protein with respect to the membrane and can be determined by the presence, or absence, and organization within the protein sequence of two features: endoplasmic reticulum signal peptides and alpha-helical transmembrane domains. These features allow protein sequences to be classified into one of five membrane organization categories: soluble intracellular proteins, soluble secreted proteins, type I membrane proteins, type II membrane proteins, and multi-spanning membrane proteins. Generation of protein isoforms with variable membrane organizations can change a protein's subcellular localization or association with the membrane. Application of MemO, a membrane organization annotation pipeline, to the FANTOM3 Isoform Protein Sequence mouse protein set revealed that within the 8,032 transcriptional units (TUs) with multiple protein isoforms, 573 had variation in their use of signal peptides, 1,527 had variation in their use of transmembrane domains, and 615 generated protein isoforms from distinct membrane organization classes. The mechanisms underlying these transcript variations were analyzed. While TUs were identified encoding all pairwise combinations of membrane organization categories, the most common was conversion of membrane proteins to soluble proteins. Observed within our high-confidence set were 156 TUs predicted to generate both extracellular soluble and membrane proteins, and 217 TUs generating both intracellular soluble and membrane proteins. The differential use of endoplasmic reticulum signal peptides and transmembrane domains is a common occurrence within the variable protein output of TUs. The generation of protein isoforms that are targeted to multiple subcellular locations represents a major functional consequence of transcript variation within the mouse transcriptome.

Subcellular localization of mammalian type II membrane proteins.

Application of a computational membrane organization prediction pipeline, MemO, identified putative type II membrane proteins as proteins predicted to encode a single alpha-helical transmembrane domain (TMD) and no signal peptides. MemO was applied to RIKEN's mouse isoform protein set to identify 1436 non-overlapping genomic regions or transcriptional units (TUs), which encode exclusively type II membrane proteins. Proteins with overlapping predicted InterPro and TMDs were reviewed to discard false positive predictions resulting in a dataset comprised of 1831 transcripts in 1408 TUs. This dataset was used to develop a systematic protocol to document subcellular localization of type II membrane proteins. This approach combines mining of published literature to identify subcellular localization data and a high-throughput, polymerase chain reaction (PCR)-based approach to experimentally characterize subcellular localization. These approaches have provided localization data for 244 and 169 proteins. Type II membrane proteins are localized to all major organelle compartments; however, some biases were observed towards the early secretory pathway and punctate structures. Collectively, this study reports the subcellular localization of 26% of the defined dataset. All reported localization data are presented in the LOCATE database (http://www.locate.imb.uq.edu.au).

LOCATE: a mouse protein subcellular localization database.

We present here LOCATE, a curated, web-accessible database that houses data describing the membrane organization and subcellular localization of proteins from the FANTOM3 Isoform Protein Sequence set. Membrane organization is predicted by the high-throughput, computational pipeline MemO. The subcellular locations of selected proteins from this set were determined by a high-throughput, immunofluorescence-based assay and by manually reviewing >1700 peer-reviewed publications. LOCATE represents the first effort to catalogue the experimentally verified subcellular location and membrane organization of mammalian proteins using a high-throughput approach and provides localization data for approximately 40% of the mouse proteome. It is available at http://locate.imb.uq.edu.au.

Criterion validity of informants in the diagnosis of disruptive behavior disorders in children: a preliminary study.

The relative validity of the reports of parents, teachers, and children, and combinations of these informants was assessed using measures of concurrent impairment associated with disruptive behavior disorders as criterion variables in a clinic-referred sample of 177 boys ages 7-12 years. For oppositional defiant disorder (ODD) and conduct disorder (CD), the reports from teachers, alone and in combination with parents and children, showed the strongest relations to impairment criteria. The reports of parents and children, alone and in combination, were relatively less valid. For attention-deficit hyperactivity disorder, there were no significant correlations between the reports of any single informant or combination of informants and the impairment criteria. This study provides preliminary support for the validity of multi-informant diagnostic assessment of ODD and CD in children, especially when the teacher is one of the informants.