The biological basis of degenerative disc disease: proteomic and biomechanical analysis of the canine intervertebral disc

IntroductionIn the present study, we sought to quantify and contrast the secretome and biomechanical properties of the non-chondrodystrophic (NCD) and chondrodystrophic (CD) canine intervertebral disc (IVD) nucleus pulposus (NP).MethodsWe used iTRAQ proteomic methods to quantify the secretome of both CD and NCD NP. Differential levels of proteins detected were further verified using immunohistochemistry, Western blotting, and proteoglycan extraction in order to evaluate the integrity of the small leucine-rich proteoglycans (SLRPs) decorin and biglycan. Additionally, we used robotic biomechanical testing to evaluate the biomechanical properties of spinal motion segments from both CD and NCD canines.ResultsWe detected differential levels of decorin, biglycan, and fibronectin, as well as of other important extracellular matrix (ECM)-related proteins, such as fibromodulin and HAPLN1 in the IVD NP obtained from CD canines compared with NCD canines. The core proteins of the vital SLRPs decorin and biglycan were fragmented in CD NP but were intact in the NP of the NCD animals. CD and NCD vertebral motion segments demonstrated significant differences, with the CD segments having less stiffness and a more varied range of motion.ConclusionsThe CD NP recapitulates key elements of human degenerative disc disease. Our data suggest that at least some of the compromised biomechanical properties of the degenerative disc arise from fibrocartilaginous metaplasia of the NP secondary to fragmentation of SLRP core proteins and associated degenerative changes affecting the ECM. This study demonstrates that the degenerative changes that naturally occur within the CD NP make this animal a valuable animal model with which to study IVD degeneration and potential biological therapeutics.

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The online version of this article (doi:10.1186/s13075-015-0733-z) contains supplementary material, which is available to authorized users.     

Identification of proteins secreted by head and neck cancer cell lines using LC-MS/MS: Strategy for discovery of candidate serological biomarkers

In search of blood-based biomarkers that would enhance the ability to diagnose head and neck/oral squamous cell carcinoma (HNOSCC) in early stages or predict its prognosis, we analyzed the HNOSCC secretome (ensemble of proteins secreted and/or shed from the tumor cells) for potential biomarkers using proteomic technologies. LC-MS/MS was used to identify proteins in the conditioned media of four HNOSCC cell lines (SCC4, HSC2, SCC38, and AMOSIII); 140 unique proteins were identified on the basis of 5% global false discovery rate, 122 of which were secretory proteins, with 29 being previously reported to be overexpressed in HNOSCC in comparison to normal head and neck tissues. Of these, five proteins including α-enolase, peptidyl prolyl isomerase A/cyclophilin A, 14-3-3 ζ, heterogeneous ribonucleoprotein K, and 14-3-3 σ were detected in the sera of HNOSCC patients by Western blot analysis. Our study provides the evidence that analysis of head and neck cancer cells' secretome is a viable strategy for identifying candidate serological biomarkers for HNOSCC. In future, these biomarkers may be useful in predicting the likelihood of transformation of oral pre-malignant lesions, prognosis of HNOSCC patients and evaluate response to therapy using minimally invasive tests.     

DONNÉES EXPÉRIMENTALES SUR LES CHANGEMENTS DE GALERIE DES LARVES MINEUSES DE PHILOPHYLLA HERACLEI (DIPTERA,TEPHRITIDAE)

Résumé Les larves de la mouche du céleri peuvent quitter leur galerie d'origine et en creuser une nouvelle dans le parenchyme d'une autre foliole. Ces changements se produisent lorsque la quantité de nourriture est insuffisante pour assurer le développement complet ou lorsque l'état physiologique de la feuille est mauvais.Des changements peuvent être induits expérimentalement en transférant les larves d'une foliole sur une autre, ou en augmentant la pression de la compétition intraspécifique; dans ce dernier cas le comportement d'alimentation peut être changé, les larves pouvant se nourrir aux dépens des pétioles.Les changements de galerie sont plus nombreux et la mortalité larvaire plus faible lorsque le déficit de saturation en eau du milieu ambiant est faible. Lorsque l'humidité relative est basse (HR=50–60%) la mortalíté larvaire est importante à l'intérieur des galeries d'origine et le nombre de changements est par conséquent limité.

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Summary The mining larvae of the celery fly, Philophylla heraclei, normally achieve their full development in a single mine inside a celery leaflet. If the amount of food is not sufficient, or in the case of a bad physiological condition of the leaflet (desiccation, celery leafspot, etc...), the larvae can leave their original mine and move to another leaflet to bore another mine.Such migrations can be induced experimentally by removing the larvae from their mine and placing them on another leaflet or leafstalk. They can also be induced by creating acute competition for food on celery plants offering a limited number of small leaves. In this case, larvae are frequently observed feeding on the tissues of the petiole of leaflets or the leafstalk, which behaviour is not observed in nature.There are few cases of migrations and larval mortality inside the leaf tissue is high (85%) when the humidity of the chamber is far removed from saturation (RH=50–60%). When humidity is near saturation (RH=95%), there are many cases of larval migration; about 50% of the larvae then reach the pupal stage, most of the larval mortality being due to the lack of utilisable vegetable tissue.In nature, this possibility of migration in a humid atmosphere enables the larvae of P. heraclei to resist competition and to escape leaf-spot which causes a rapid decrease in the amount of healthy utilisable tissue.

     

Lactococcus piscium: a psychrotrophic lactic acid bacterium with bioprotective or spoilage activity in food—a review

The genus Lactococcus comprises 12 species, some known for decades and others more recently described. Lactococcus piscium, isolated in 1990 from rainbow trout, is a psychrotrophic lactic acid bacterium, probably disregarded because most of the strains are unable to grow at 30°C. During the last 10 years, this species has been isolated from a large variety of food: meat, seafood and vegetables, mostly packed under vacuum (VP) or modified atmosphere (MAP) and stored at chilled temperature. Recently, culture‐independent techniques used for characterization of microbial ecosystems have highlighted the importance of Lc. piscium in food. Its role in food spoilage varies according to the strain and the food matrix. However, most studies have indicated that Lc. piscium spoils meat, whereas it does not degrade the sensory properties of seafood. Lactococcus piscium strains have a large antimicrobial spectrum, including Gram‐positive and negative bacteria. In various seafoods, some strains have a protective effect against spoilage and can extend the sensory shelf‐life of the products. They can also inhibit the growth of Listeria monocytogenes, by a cell‐to‐cell contact‐dependent. This article reviews the physiological and genomic characteristics of Lc. piscium and discusses its spoilage or protective activities in food.     

Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.