Human Prominin-1 (CD133) Is Detected in Both Neoplastic and Non-Neoplastic Salivary Gland Diseases and Released into Saliva in a Ubiquitinated Form.

Prominin-1 (CD133) is physiologically expressed at the apical membranes of secretory (serous and mucous) and duct cells of major salivary glands. We investigated its expression in various human salivary gland lesions using two distinct anti-prominin-1 monoclonal antibodies (80B258 and AC133) applied on paraffin-embedded sections and characterized its occurrence in saliva. The 80B258 epitope was extensively expressed in adenoid cystic carcinoma, in lesser extent in acinic cell carcinoma and pleomorphic adenoma, and rarely in mucoepidermoid carcinoma. The 80B258 immunoreactivity was predominately detected at the apical membrane of tumor cells showing acinar or intercalated duct cell differentiation, which lined duct- or cyst-like structures, and in luminal secretions. It was observed on the whole cell membrane in non-luminal structures present in the vicinity of thin-walled blood vessels and hemorrhagic areas in adenoid cystic carcinoma. Of note, AC133 labeled only a subset of 80B258-positive structures. In peritumoral salivary gland tissues as well as in obstructive sialadenitis, an up-regulation of prominin-1 (both 80B258 and AC133 immunoreactivities) was observed in intercalated duct cells. In most tissues, prominin-1 was partially co-expressed with two cancer markers: carcinoembryonic antigen (CEA) and mucin-1 (MUC1). Differential centrifugation of saliva followed by immunoblotting indicated that all three markers were released in association with small membrane vesicles. Immuno-isolated prominin-1-positive vesicles contained CEA and MUC1, but also exosome-related proteins CD63, flotillin-1, flotillin-2 and the adaptor protein syntenin-1. The latter protein was shown to interact with prominin-1 as demonstrated by its co-immunoisolation. A fraction of saliva-associated prominin-1 appeared to be ubiquitinated. Collectively, our findings bring new insights into the biochemistry and trafficking of prominin-1 as well as its immunohistochemical profile in certain types of salivary gland tumors and inflammatory diseases

Gene expression analysis of osteoblastic cells contacted by orthopedic implant particles

Particles generated from orthopedic implants through years of wear play an essential role in the aseptic loosening of a prosthesis. We have investigated the biocompatibility of these orthopedic particles on different osteoblast-like cells representative of different stages of osteoblast maturation. We found the particles induced a caspase-dependent apoptosis of osteoblasts, with less mature osteoblasts being the most susceptible. An analysis of gene expression was performed on the less mature osteoblasts, which were in contact with the particles. We found that the particles had a profound impact on genes that code for inflammatory cytokines and genes involved in controlling the nuclear architecture. Results from this study suggest that the peri-implant osteolysis after a total joint replacement can be due in part to a decrease of bone formation and not solely to an overstimulation of bone resorption as is generally proposed. Development of new drugs that promote normal bone formation and osteoblast survival would possibly control peri-implant osteolysis, resulting in a better prognosis for patients with orthopedic implants

Large-scale gene expression analysis of osteoblasts cultured on three different Ti-6Al-4V surface treatments

To improve implant biocompatibility, we developed a simple cost-effective thermal surface treatment allowing an increase in the oxide layer thickness of a titanium (Ti) alloy used in orthopaedic implants. The goal of this study was to test in vitro the reaction of osteoblasts to the developed surface treatment and to compare it to the osteoblast reaction to two other surface treatments currently used in the practice of implant surgery. Quantification of osteoblast gene expression on a large scale was used in this study. The kinetics of gene expression over 120 h was followed for 58 genes to quantify the effect of the developed surface treatment. Twenty eight genes were further selected to compare the effects of surface treatments on osteoblasts. Based on the genes studied, we could propose a general pathway for the cell reaction according to the surface treatments used: (1) metal ion release changes the time course of gene expression in the FAK pathway; (2) once the accumulation of metal ions released from the Ti surface exceeds a threshold value, cell growth is diminished and apoptosis may be activated; (3) PTK up-regulation is also induced by metal ion release; (4) the expression of Bcl-2 family and Bax may suggest that metal ions induce apoptosis. The developed treatment seems to increase the Ti-6Al-4V biocompatibility as highlighted by the lower impact of this treatment by the different pathways studied, on the lower inflammatory reaction that could be induced, as well as by the lower induced osteoblast apoptosis compared to the two other surface treatments

Effect of micromechanical stimulations on osteoblasts: development of a device simulating the mechanical situation at the bone-implant interface

Many experimental models have been developed to investigate the effects of mechanical stimulation of cells, but none of the existing devices can simulate micromotions at the cellular-mechanical interface with varying amplitudes and loads. Osteoblasts are sensitive to mechanical stimuli, so to study the bone-implant interface it would be important to quantify their reaction in a situation mimicking the mechanical situation arising at that interface. In this study, we present the development of a new device allowing the application of micromotions and load on cells in vitro. The new device allowed the cells to be stimulated with sinusoidal motions of amplitudes comprised between +/- 5 and +/- 50 microm, frequencies between 0.5 and 2 Hz, and loads between 50 and 1000 Pa. The device, with a total length of 20 cm, was designed to work in an incubator at 37 degrees C and 100% humidity. Expression of various bone important genes was monitored by real-time RT-PCR. Micromotions and load were shown to affect the behavior of osteoblasts by down-regulating the expression of genes necessary for the creation of organic extracellular matrix (collagen type I) as well as for genes involved in the mineralization process (osteocalcin, osteonectin). The developed device could then be used to simulate different mechanical situations at the bone-implant interface

CD133 expression is an independent prognostic marker for low survival in colorectal cancer

Colon cancer cells have previously been demonstrated to contain a subpopulation of CD133+ tumour cells that have the ability to initiate tumour growth and are thus referred to as colon cancer-initiating cells or colon cancer stem cells (CSCs). As CD133 is currently one of the best markers to characterise colon CSCs, we analysed CD133+ tumour cells in colorectal cancer specimens using immunohistochemistry. We show that CD133 detection is specific and that the CD133 antigen is localised on the glandular-luminal surface of colon cancer cells, whereas undifferentiated tumour cells at the front of invasion are CD133−. In addition, CD133+ cells are characterised in situ by lack of CK20 expression, whereas they are positive for EpCAM. Moreover, we show that CD133 expression in colorectal cancer is an independent prognostic marker that correlates with low survival in a stratified patient collective. Our results indicate that in colorectal cancer, the CD133+ tumour cells can be detected by immunohistochemistry, which facilitates their further characterisation in situ

The Importance of the Stem Cell Marker Prominin-1/CD133 in the Uptake of Transferrin and in Iron Metabolism in Human Colon Cancer Caco-2 Cells

As the pentaspan stem cell marker CD133 was shown to bind cholesterol and to localize in plasma membrane protrusions, we investigated a possible function for CD133 in endocytosis. Using the CD133 siRNA knockdown strategy and non-differentiated human colon cancer Caco-2 cells that constitutively over-expressed CD133, we provide for the first time direct evidence for a role of CD133 in the intracellular accumulation of fluorescently labeled extracellular compounds. Assessed using AC133 monoclonal antibody, CD133 knockdown was shown to improve Alexa488-transferrin (Tf) uptake in Caco-2 cells but had no impact on FITC-dextran or FITC-cholera-toxin. Absence of effect of the CD133 knockdown on Tf recycling established a role for CD133 in inhibiting Tf endocytosis rather than in stimulating Tf exocytosis. Use of previously identified inhibitors of known endocytic pathways and the positive impact of CD133 knockdown on cellular uptake of clathrin-endocytosed synthetic lipid nanocapsules supported that CD133 impact on endocytosis was primarily ascribed to the clathrin pathway. Also, cholesterol extraction with methyl-β-cyclodextrine up regulated Tf uptake at greater intensity in the CD133high situation than in the CD133low situation, thus suggesting a role for cholesterol in the inhibitory effect of CD133 on endocytosis. Interestingly, cell treatment with the AC133 antibody down regulated Tf uptake, thus demonstrating that direct extracellular binding to CD133 could affect endocytosis. Moreover, flow cytometry and confocal microscopy established that down regulation of CD133 improved the accessibility to the TfR from the extracellular space, providing a mechanism by which CD133 inhibited Tf uptake. As Tf is involved in supplying iron to the cell, effects of iron supplementation and deprivation on CD133/AC133 expression were investigated. Both demonstrated a dose-dependent down regulation here discussed to the light of transcriptional and post-transciptional effects. Taken together, these data extend our knowledge of the function of CD133 and underline the interest of further exploring the CD133-Tf-iron network

Learning a peptide-protein binding affinity predictor with kernel ridge regression

We propose a specialized string kernel for small bio-molecules, peptides and pseudo-sequences of binding interfaces. The kernel incorporates physico-chemical properties of amino acids and elegantly generalize eight kernels, such as the Oligo, the Weighted Degree, the Blended Spectrum, and the Radial Basis Function. We provide a low complexity dynamic programming algorithm for the exact computation of the kernel and a linear time algorithm for it's approximation. Combined with kernel ridge regression and SupCK, a novel binding pocket kernel, the proposed kernel yields biologically relevant and good prediction accuracy on the PepX database. For the first time, a machine learning predictor is capable of accurately predicting the binding affinity of any peptide to any protein. The method was also applied to both single-target and pan-specific Major Histocompatibility Complex class II benchmark datasets and three Quantitative Structure Affinity Model benchmark datasets. On all benchmarks, our method significantly (p-value < 0.057) outperforms the current state-of-the-art methods at predicting peptide-protein binding affinities. The proposed approach is flexible and can be applied to predict any quantitative biological activity. The method should be of value to a large segment of the research community with the potential to accelerate peptide-based drug and vaccine development.Comment: 22 pages, 4 figures, 5 table

Horizontal gene transfer in Histophilus somni and its role in the evolution of pathogenic strain 2336, as determined by comparative genomic analyses

<p>Abstract</p> <p>Background</p> <p>Pneumonia and myocarditis are the most commonly reported diseases due to <it>Histophilus somni</it>, an opportunistic pathogen of the reproductive and respiratory tracts of cattle. Thus far only a few genes involved in metabolic and virulence functions have been identified and characterized in <it>H. somni </it>using traditional methods. Analyses of the genome sequences of several <it>Pasteurellaceae </it>species have provided insights into their biology and evolution. In view of the economic and ecological importance of <it>H. somni</it>, the genome sequence of pneumonia strain 2336 has been determined and compared to that of commensal strain 129Pt and other members of the <it>Pasteurellaceae</it>.</p> <p>Results</p> <p>The chromosome of strain 2336 (2,263,857 bp) contained 1,980 protein coding genes, whereas the chromosome of strain 129Pt (2,007,700 bp) contained only 1,792 protein coding genes. Although the chromosomes of the two strains differ in size, their average GC content, gene density (total number of genes predicted on the chromosome), and percentage of sequence (number of genes) that encodes proteins were similar. The chromosomes of these strains also contained a number of discrete prophage regions and genomic islands. One of the genomic islands in strain 2336 contained genes putatively involved in copper, zinc, and tetracycline resistance. Using the genome sequence data and comparative analyses with other members of the <it>Pasteurellaceae</it>, several <it>H. somni </it>genes that may encode proteins involved in virulence (<it>e.g</it>., filamentous haemaggutinins, adhesins, and polysaccharide biosynthesis/modification enzymes) were identified. The two strains contained a total of 17 ORFs that encode putative glycosyltransferases and some of these ORFs had characteristic simple sequence repeats within them. Most of the genes/loci common to both the strains were located in different regions of the two chromosomes and occurred in opposite orientations, indicating genome rearrangement since their divergence from a common ancestor.</p> <p>Conclusions</p> <p>Since the genome of strain 129Pt was ~256,000 bp smaller than that of strain 2336, these genomes provide yet another paradigm for studying evolutionary gene loss and/or gain in regard to virulence repertoire and pathogenic ability. Analyses of the complete genome sequences revealed that bacteriophage- and transposon-mediated horizontal gene transfer had occurred at several loci in the chromosomes of strains 2336 and 129Pt. It appears that these mobile genetic elements have played a major role in creating genomic diversity and phenotypic variability among the two <it>H. somni </it>strains.</p

Experimental neck muscle pain impairs standing balance in humans

Impaired postural control has been reported in patients with chronic neck pain of both traumatic and non-traumatic etiologies, but whether painful stimulation of neck muscle per se can affect balance control during quiet standing in humans remains unclear. The purpose of the present experiment was thus to investigate the effect of experimental neck muscle pain on standing balance in young healthy adults. To achieve this goal, 16 male university students were asked to stand upright as still as possible on a force platform with their eyes closed in two conditions of No pain and Pain of the neck muscles elicited by experimental painful electrical stimulation. Postural control and postural performance were assessed by the displacements of the center of foot pressure (CoP) and of the center of mass (CoM), respectively. The results showed increased CoP and CoM displacements variance, range, mean velocity, and mean and median frequencies in the Pain relative to the No pain condition. The present findings emphasize the destabilizing effect of experimental neck muscle pain per se, and more largely stress the importance of intact neck neuromuscular function on standing balance

The Stem Cell Marker CD133 Associates with Enhanced Colony Formation and Cell Motility in Colorectal Cancer

CD133 is a membrane molecule that has been, controversially, reported as a CSC marker in colorectal cancer (CRC). In this study, we sought to clarify the expression and role of CD133 in CRC. Initially the size of the CD133−expressing (CD133+) population in eight well-described CRC cell lines was measured by flow cytometry and was found to range from 0% to >95%. The cell line HT29 has a CD133+ population of >95% and was chosen for functional evaluation of CD133 after gene knockdown by RNA interference. A time course assay showed that CD133 inhibition had no significant effect on cell proliferation or apoptosis. However, CD133 knockdown did result in greater susceptibility to staurosporine-induced apoptosis (p = 0.01) and reduction in cell motility (p<0.04). Since gene knockdown may cause “off-target” effects, the cell line SW480 (which has a CD133+ population of 40%) was sorted into pure CD133+ and CD133− populations to allow functional comparison of isogenic populations separated only by CD133 expression. In concordance with the knockdown experiments, a time course assay showed no significant proliferative differences between the CD133+/CD133− populations. Also greater resistance to staurosporine-induced apoptosis (p = 0.008), greater cell motility (p = 0.03) and greater colony forming efficiency was seen in the CD133+ population than the CD133− population in both 2D and 3D culture (p<0.0001 and p<0.003 respectively). Finally, the plasticity of CD133 expression in tumour cells was tested. Quantitative PCR analysis showed there was transcriptional repression in the CD133− population of SW480. Prolonged culture of a pure CD133− population resulted in re-emergence of CD133+ cells. We conclude that CD133 expression in CRCs is associated with some features attributable to stemness and that there is plasticity of CD133 expression. Further studies are necessary to delineate the mechanistic basis of these features