Articular cartilage is indispensable for joint function but has limited capacity for self-repair. Engineering of neocartilage in vitro is therefore a major target for autologous cartilage repair in arthritis. Previous analysis of neocartilage has targeted cellular organization and specific molecular components. However, the complexity of extracellular matrix (ECM) development in neocartilage has not been investigated by proteomics. To redress this, we developed a mouse neocartilage culture system that produces a cartilaginous ECM. Differential analysis of the tissue proteome of 3-week neocartilage and 3-day postnatal mouse cartilage using solubility-based protein fractionation targeted components involved in neocartilage development, including ECM maturation. Initially, SDS-PAGE analysis of sequential extracts revealed the transition in protein solubility from a high proportion of readily soluble (NaCl-extracted) proteins in juvenile cartilage to a high proportion of poorly soluble (guanidine hydrochloride-extracted) proteins in neocartilage. Label-free quantitative mass spectrometry (LTQ-Orbitrap) and statistical analysis were then used to filter three significant protein groups: proteins enriched according to extraction condition, proteins differentially abundant between juvenile cartilage and neocartilage, and proteins with differential solubility properties between the two tissue types. Classification of proteins differentially abundant between NaCl and guanidine hydrochloride extracts (n ‫؍‬ 403) using bioinformatics revealed effective partitioning of readily soluble components from subunits of larger protein complexes. Proteins significantly enriched in neocartilage (n ‫؍‬ 78) included proteins previously not reported or with unknown function in cartilage (integrin-binding protein DEL1; coiled-coil domain-containing protein 80; emilin-1 and pigment epithelium derived factor). Proteins with differential extractability between juvenile cartilage and neocartilage included ECM components (nidogen-2, perlecan, collagen VI, matrilin-3, tenascin and thrombospondin-1), and the relationship between protein extractability and ECM ultrastructural organization was supported by electron microscopy. Additionally, one guanidine extract-specific neocartilage protein, protease nexin-1, was confirmed by immunohistochemistry as a novel component of developing articular cartilage in vivo. The extraction profile and matrix-associated immunostaining implicates protease nexin-1 in cartilage development in vitro and in vivo. Molecular & Cellular Proteomics 9:1296 -1313, 2010.The cartilage of the mammalian skeletal system has two distinct roles. The epiphyseal cartilage of the growth plate drives endochondral bone growth, and the hyaline cartilage at the weight-bearing surfaces of bones facilitates joint articulation. In both environments, chondrocyte-regulated production, assembly, and turnover of the extracellular matrix (ECM) 1 are essential for the tissue to withstand compressive forces and respond to mechanical loading. The m...

IGFBP-2 is highly expressed in both the serum and tumor tissues of most cancers, and is considered one of the most significant genes in the signature of major cancers. IGFBP-2 mainly modulates IGF actions in the pericellular space; however, there is considerable evidence to suggest that IGFBP-2 may also act independently of the IGFs. These IGF-independent actions of IGFBP-2 are exerted either via interactions at the cell surface or intracellularly, via interaction with cytoplasmic or nuclear-binding partners. The precise mechanism underlying the intracellular/intranuclear localization of IGFBP-2 remains unclear. In this study, we investigated IGFBP-2 nuclear localization in several common cancer cells with the aim of dissecting the mechanism of its nuclear trafficking. IGFBP-2 is detected in the nuclei of common cancer cells, including breast, prostate and several neuroblastoma cell lines, using cell fractionation and confocal microscopy. Via nuclear import assays, we show that nuclear entry of IGFBP-2 is mediated by the classical nuclear import mechanisms, primarily through importin-α, as demonstrated by the use of blocking, competition and co-immunoprecipitation assays. Bioinformatics analysis of the IGFBP-2 protein sequence with PSORT II identified a classical nuclear localization signal (cNLS) sequence at 179PKKLRPP185, within the IGFBP-2 linker domain, mutagenesis of which abolishes IGFBP-2 nuclear import. Accordingly, the NLSmutIGFBP-2 fails to activate the VEGF promoter, which would otherwise occur in the presence of wild-type IGFBP-2. As a consequence, no activation of angiogenic processes were observed in NLSmutIGFBP-2 expressing SHEP cells when implanted onto our in vivo quail chorio-allantoic membrane model. Taken together, these data show for the first time that IGFBP-2 possesses a functional NLS sequence and that IGFBP-2 actively translocates into the nucleus by a classical nuclear import mechanism, involving formation of IGFBP-2 complexes with importin-α. Nuclear IGFBP-2 is required for the activation of VEGF expression and consequent angiogenesis.

Skeletal growth by endochondral ossification involves tightly coordinated chondrocyte differentiation that creates reserve, proliferating, prehypertrophic, and hypertrophic cartilage zones in the growth plate. Many human skeletal disorders result from mutations in cartilage extracellular matrix (ECM) components that compromise both ECM architecture and chondrocyte function. Understanding normal cartilage development, composition, and structure is therefore vital to unravel these disease mechanisms. To study this intricate process in vivo by proteomics, we analyzed mouse femoral head cartilage at developmental stages enriched in either immature chondrocytes or maturing/hypertrophic chondrocytes (postnatal days 3 and 21, respectively). Using LTQ-Orbitrap tandem mass spectrometry, we identified 703 cartilage proteins. Differentially abundant proteins (q < 0.01) included prototypic markers for both early and late chondrocyte differentiation (epiphycan and collagen X, respectively) and novel ECM and cell adhesion proteins with no previously described roles in cartilage development (tenascin X, vitrin, Urb, emilin-1, and the sushi repeat-containing proteins SRPX and SRPX2). Meta-analysis of cartilage development in vivo and an in vitro chondrocyte culture model (Wilson, R., Diseberg, A. F., Gordon, L., Zivkovic, S., Tatarczuch, L., Mackie, E. J., Gorman, J. J., and Bateman, J. Cartilage is a unique tissue characterized by an abundant extracellular matrix (ECM) 1 and a single cell type, the chondrocyte. However, the permanent hyaline cartilage, which provides the articulating surfaces of long bones and vertebrae, and the transient growth plate cartilage responsible for endochondral bone growth are uniform in neither cellular phenotype nor protein composition. In articular cartilage, the chondrocytes form morphologically distinct regions comprising a superficial region of flattened cells, a sparsely populated middle layer, and a deep zone of hypertrophic chondrocytes embedded in calcified cartilage at the chondro-osseous junc-

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Biodiesel as renewable, environmental friendly, less toxic, and biodegradable is an attractive alternative to fossil fuels and is produced mainly from vegetable oils and animal fats. It is expected, globally, that the use of renewable biofuels, in general, will increase rapidly in the near future. The growing biodiesel production and usage have encouraged assessment of its impact on the environment. The present paper reviews various aspects of biodiesel production using commercial processing technology and biodiesel use through evaluation and analysis of the studies concerning environmental impacts of biodiesel. As a general conclusion, it can be said that biodiesel has the potential to offer a series of perceived benefits such as political, economical, and agricultural, as well as environmental (due to its biodegradability, less toxicity, renewability) and health (greenhouse gas-saving, less harmful exhaust emissions).

BackgroundDespite the increase in cancer incidence in the last years in Serbia, no nation-wide, population-based cancer epidemiology data have been reported. In this study cancer incidence and mortality rates for Serbia are presented using nation-wide data from two population-based cancer registries. These rates are additionally compared to European and global cancer epidemiology estimates. Finally, predictions on Serbian cancer incidence and mortality rates are provided.MethodsCancer incidence and mortality was collected from the cancer registries of Central Serbia and Vojvodina from 1999 to 2009. Using age-specific regression models, we estimated time trends and predictions for cancer incidence and mortality for the following five years (2010–2014). The comparison of Serbian with European and global cancer incidence/mortality rates, adjusted to the world population (ASR-W) was performed using Serbian population-based data and estimates from GLOBOCAN 2008.ResultsIncreasing trends in both overall cancer incidence and mortality rates were identified for Serbia. In men, lung cancer showed the highest incidence (ASR-W 2009: 70.8/100,000), followed by colorectal (ASR-W 2009: 39.9/100,000), prostate (ASR-W 2009: 29.1/100,000) and bladder cancer (ASR-W 2009: 16.2/100,000). Breast cancer was the most common form of cancer in women (ASR-W 2009: 70.8/100,000) followed by cervical (ASR-W 2009: 25.5/100,000), colorectal (ASR-W 2009: 21.1/100,000) and lung cancer (ASR-W 2009: 19.4/100,000). Prostate and colorectal cancers have been significantly increasing over the last years in men, while this was also observed for breast cancer incidence and lung cancer mortality in women. In 2008 Serbia had the highest mortality rate from breast cancer (ASR-W 2008: 22.7/100,000), among all European countries while incidence and mortality of cervical, lung and colorectal cancer were well above European estimates.ConclusionCancer incidence and mortality in Serbia has been generally increasing over the past years. For a number of cancer sites, incidence and mortality is alarmingly higher than in the majority of European regions. For this increasing trend to be controlled, the management of risk factors that are present among the Serbian population is necessary. Additionally, prevention and early diagnosis are areas where significant improvements could still be made.