Effects of FGF-2 on human adipose tissue derived adult stem cells morphology and chondrogenesis enhancement in Transwell culture

Injured cartilage is difficult to repair due to its poor vascularisation. Cell based therapies may serve as tools to more effectively regenerate defective cartilage. Both adult mesenchymal stem cells (MSCs) and human adipose derived stem cells (hADSCs) are regarded as potential stem cell sources able to generate functional cartilage for cell transplantation. Growth factors, in particular the TGF-b superfamily, influence many processes during cartilage formation, including cell proliferation, extracellular matrix synthesis, maintenance of the differentiated phenotype, and induction of MSCs towards chondrogenesis. In the current study, we investigated the effects of FGF-2 on hADSC morphology and chondrogenesis in Transwell culture. hADSCs were obtained from patients undergoing elective surgery, and then cultured in expansion medium alone or in the presence of FGF-2 (10 ng/ml). mRNA expression levels of SOX-9, aggrecan and collagen type II and type X were quantified by real-time polymerase chain reaction. The morphology, doubling time, trypsinization time and chondrogenesis of hADSCs were also studied. Expression levels of SOX-9, collagen type II, and aggrecan were all significantly increased in hADSCs expanded in presence of FGF-2. Furthermore FGF-2 induced a slender morphology, whereas doubling time and trypsinization time decreased. Our results suggest that FGF-2 induces hADSCs chondrogenesis in Transwell culture, which may be beneficial in cartilage tissue engineering.     

Cdc48/p97, a key actor in the interplay between autophagy and ubiquitin/proteasome catabolic pathways

The AAA-ATPase Cdc48/p97 controls a large array of cellular functions including protein degradation, cell division, membrane fusion through its ability to interact with and control the fate of ubiquitylated proteins. More recently, Cdc48/p97 also appeared to be involved in autophagy, a catabolic cell response that has long been viewed as completely distinct from the Ubiquitine/Proteasome System. In particular, conjugation by ubiquitin or ubiquitin-like proteins as well as ubiquitin binding proteins such as Cdc48/p97 and its cofactors can target degradation by both catabolic pathways. This review will focus on the recently described functions of Cdc48/p97 in autophagosome biogenesis as well as selective autophagy.     

beta-Catenin has sequential roles in the survival and specification of ventral dermis

The dermis promotes the development and maintains the functional components of skin, such as hair follicles, sweat glands, nerves and blood vessels. The dermis is also crucial for wound healing and homeostasis of the skin. The dermis originates from the somites, the lateral plate mesoderm and the cranial neural crest. Despite the importance of the dermis in the structural and functional integrity of the skin, genetic analysis of dermal development in different parts of the embryo is incomplete. The signaling requirements for ventral dermal cell development have not been established in either the chick or the mammalian embryo. We have shown previously that Wnt signaling specifies the dorsal dermis from the somites. In this study, we demonstrate that Wnt/beta-catenin signaling is necessary for the survival of early ventral dermal progenitors. In addition, we show that, at later stages, Wnt/beta-catenin signaling is sufficient for ventral dermal cell specification. Consistent with the different origins of dorsal and ventral dermal cells, our results demonstrate both conserved and divergent roles of beta-catenin/Wnt signaling in dermal development.     

Tissue engineering: Dentin – pulp complex regeneration approaches (A review)

Dental pulp is a highly specialized tissue that preserves teeth. It is important to maintain the capabilities of dental pulp before a pulpectomy by creating a local restoration of the dentin-pulp complex from residual dental pulp. The articles identified were selected by two reviewers based on entry and exit criteria. All relevant articles indexed in PubMed, Springer, Science Direct, and Scopus with no limitations from 1961 to 2016 were searched. Factors investigated in the selected articles included the following key words: Dentin-Pulp Complex, Regeneration, Tissue Engineering, Scaffold, Stem Cell, and Growth Factors. Of the 233 abstracts retrieved, the papers which were selected had evaluated the clinical aspects of the application of dentin-pulp regeneration. Generally, this study has introduced a new approach to provoke the regeneration of the dentin-pulp complex after a pulpectomy, so that exogenous growth factors and the scaffold are able to induce cells and blood vessels from the residual dental pulp in the tooth root canal. This study further presents a new strategy for local regeneration therapy of the dentin-pulp complex. This review summarizes the current knowledge of the potential beneficial effects derived from the interaction of dental materials with the dentin-pulp complex as well as potential future developments in this exciting field.     

Novel functional polymorphism on PADI-4 gene and its association with arthritis onset

BackgroundCitrullinated proteins formed by peptidyl arginine deiminases (PADIs) deimination of arginine residues in proteins are of particular interest in arthritis pathogenesis. Polymorphisms on the PADI-4 gene lead to the malfunctioning of PADIs leading to the onset of arthritis.ObjectiveThe present study was conducted to determine the polymorphisms on the PADI-4 gene and their association with rheumatoid arthritis (RA) as well as Osteoarthritis (OA).MethodologyTo achieve the above-mentioned objective a case-control study was conducted. Blood samples were collected from RA, OA, and control subjects. DNA was extracted from each blood sample by modified organic method and was quantified as well as qualified by DNA gel electrophoresis and Nanodrop. Patients were tested for rs874881, rs11203366, rs11203367, rs2240336, rs2240337, rs2240339, rs1748033 and rs2240340 polymorphic sites by amplifying targeted regions through PCR with site-specific primers. Genotyping was performed by Restriction Fragment Length Polymorphism and direct sequencing method. Mutations were identified by analyzing sequences on BioEdit software. Allelic, genetic, and multiple site analysis were performed by SHEsis and PLINK software. Change in the amino acid sequence was identified by MEGA 6.0 software.ResultsPolymorphisms were identified on all targeted polymorphic sites except rs2240337 in both RA and OA individuals. In addition, two novel mutations were also identified in exon 4 identified i-e SCV000804840: c.218T > C and SCV000807675: c.241G > T. All the SNPs except rs11203366 were found to be significantly associated with RA at an allelic level whereas all SNP’s have been significant risk factors in the onset of OA. At genotypic level rs874881, rs11203366, rs2240339, SCV000804840 and SCV000807675 were significantly associated to RA development whereas rs874881, rs11203366, rs11203367, rs2240339, SCV000804840 and SCV000807675 were genetic risk factors in OA onset. Haplotype analysis indicated that GACCACGCC and GACCACGCT were highly significant in disease development. Polymorphisms identified altered the functioning of PADIs by altering their amino acid sequence.ConclusionIn conclusion, it was found that PADI-4 gene polymorphism was not only involved in the onset of RA but was also found to be a significant risk factor in OA onset.     

Structural basis for interaction between the Ubp3 deubiquitinating enzyme and its Bre5 cofactor

The Bre5 protein is a cofactor for the deubiquitinating enzyme Ubp3, and it contains a nuclear transfer factor 2 (NTF2)-like protein recognition module that is essential for Ubp3 activity. In this study, we report the x-ray crystal structure of the Bre5 NTF2-like domain and show that it forms a homodimeric structure that is similar to other NTF2-like domains, except for the presence of an intermolecular disulfide bond in the crystals. Sedimentation equilibrium studies reveal that under non-reducing conditions, the Bre5 NTF2-like domain is exclusively dimeric, whereas a disulfide bond-deficient mutant undergoes a monomer-dimer equilibrium with a dissociation constant in the midnanomolar range, suggesting that dimer formation and possibly also disulfide bond formation may modulate Bre5 function in vivo. Using deletion analysis, we also identify a novel N-terminal domain of Ubp3 that is necessary and sufficient for interaction with Bre5 and use isothermal titration calorimetry to show that Bre5 and Ubp3 form a 2:1 complex, in contrast to other reported NTF2-like domain/protein interactions that form 1:1 complexes. Finally, we employ structure-based mutagenesis to map the Ubp3 binding surface of Bre5 to a region near the Bre5 dimer interface and show that this binding surface of Bre5 is important for Ubp3 function in vivo. Together, these studies provide novel insights into protein recognition by NTF2-like domains and provide a molecular scaffold for understanding how Ubp3 function is regulated by Bre5 cofactor binding.     

Role of pollination in yield and physicochemical properties of tomatoes (Lycopersicon esculentum)

Very little is known about pollination and its effects on the yield and physicochemical properties of flowering plants in tropical countries. Wind and insect pollinators are among our natural resources because pollination is the most important ecosystem service performed by wind and insects, and is vital to the socio-economic status of human beings. In this experiment, different pollination methods for tomato plants were examined. Self-pollination was encouraged by covering the plants with a plastic sheet. Wind and insects were excluded from these plants, and thus only self-pollination was possible. The experiment occurred during the flowering stage. Wind-pollinated plants were covered with a muslin cloth, which excluded insects, and only wind could pass through the cloth. For insect pollination, plants remained uncovered, allowing free access to insects to pollinate the flowers. At fruit maturity, when fruits were completely red, fruits from each treatment were harvested on the same date and under the same conditions. Results illustrated the substantial importance of insects as pollinators of tomato crops. Open field had greater tomato yield and positive effects on physicochemical properties on fruit than under self and wind pollination.     

Neural differentiation of human umbilical cord matrix-derived mesenchymal cells under special culture conditions

Many neural disorders are characterized by the loss of one or several types of neural cells. Human umbilical cord-derived mesenchymal cells (hUCMs) are capable of differentiating into neuron, astroglia-like and oligodendrocyte cell types. However, a reliable means of inducing the selective differentiation of hUCMs into neural cells in vitro has not yet been established. For induction of neural differentiation, hUCMs were seeded onto sterile glass slides and six various cocktails using a base medium (DMEM/LG) supplemented with 10 % FBS, retinoic acid (RA), dimethyl sulfoxide (DMSO), epidermal growth factor (EGF) and fibroblast growth factor (FGF) were used to compare their effect on neuronal, astrocyte and oligodandrocyte differentiation. The hUCMs were positive for mesenchymal markers, while they were negative for hematopoietic markers. Differentiation to adipogenic and osteogenic lineage was detected in these cells. Our data revealed that the cocktail consisting of DMEM/LG, FBS, RA, FGF, and EGF (DF/R/Fg/E group) induced hUCM cells to express the highest percentage of nestin, ß-tubulin III, neurofilament, and CNPase. The DF/Ds/Fg/E group led to the highest percentage of GFAP expression. While the expression levels of NF, GFAP, and CNPase were the lowest in the DF group. The least percentage of nestin and ß-tubulin III expression was observed in the DF/Ds group. We may conclude that FGF and EGF are important inducers for differentiation of hUCMs into neuron, astrocyte and oligodendrocyte. RA can induce hUCMs to differentiate into neuron and oligodendrocyte while for astrocyte differentiation DMSO had a pivotal role.