Apaf-1 and caspase-9 do not act as tumor suppressors in myc-induced lymphomagenesis or mouse embryo fibroblast transformation

Based on experiments with cultured fibroblasts, the apoptosis regulators caspase-9 and Apaf-1 are hypothesized to function as tumor suppressors. To investigate their in vivo role in lymphomagenesis, an IgH enhancer-driven c-myc transgene was crossed onto Apaf-1−/− and caspase-9−/− mice. Due to perinatal lethality, Eμ-myc transgenic Apaf-1−/− or caspase-9−/− fetal liver cells were used to reconstitute lethally irradiated recipient mice. Surprisingly, no differences were seen in rate, incidence, or severity of lymphoma with loss of Apaf-1 or caspase-9, and Apaf-1 was not a critical determinant of anticancer drug sensitivity of c-myc–induced lymphomas. Moreover, loss of Apaf-1 did not promote oncogene-induced transformation of mouse embryo fibroblasts. Thus, Apaf-1 and caspase-9 do not suppress c-myc–induced lymphomagenesis and embryo fibroblast transformation

Domains in human splicing factors SF3a60 and SF3a66 required for binding to SF3a120, assembly of the 17S U2 snRNP, and prespliceosome formation

The active 17S U2 small nuclear ribonucleoprotein particle (snRNP), which binds to the intron branch site during the formation of the prespliceosome, is assembled in vitro by sequential interactions of the essential splicing factors SF3b and SF3a with the 12S U2 snRNP. We have analyzed the function of individual subunits of human SF3a (SF3a60, SF3a66, and SF3a120) by testing recombinant proteins, expressed in insect cells, in various in vitro assays. The recombinant subunits readily form the SF3a heterotrimer, where SF3a60 and SF3a66 interact with SF3a120, but not with each other. All SF3a subunits are essential for the formation of the mature 17S U2 snRNP and the prespliceosome. Single subunits engage in interactions with the 15S U2 snRNP (consisting of the 12S U2 snRNP and SF3b), and SF3a60 appears to play a major role in recruiting SF3a120 into the U2 particle. Analysis of functional domains in SF3a60 and SF3a66 identified interaction sites for SF3a120 in their N-terminal portions. C(2)H(2)-type zinc finger domains mediate the integration of SF3a60 and SF3a66 into the U2 snRNP, and we propose a model in which protein-protein interactions between the zinc finger domains and the Sm proteins, common to all spliceosomal snRNPs, contribute to the assembly of the 17S U2 snRNP. Finally, we demonstrate that all domains required for interactions within the SF3a heterotrimer and the formation of the 17S U2 snRNP are also necessary to assemble the prespliceosome

The nuclear factor-kappaB and p53 pathways function independently in primary cells and transformed fibroblasts responding to genotoxic damage

With nuclear factor-kappaB (NF-kappaB) and p53 functions generally having disparate outcomes for cell survival and cell division, understanding how these pathways are coordinated following a common activation signal such as DNA damage has important implications for cancer therapy. Conflicting reports concerning NF-kappaB and p53 interplay in different cell line models prompted a reexamination of this issue using mouse primary thymocytes and embryonic fibroblasts, plus fibroblasts transformed by E1A12S. Here, we report that following the treatment of these cells with a range of stress stimuli, p53 and NF-kappaB were found to regulate cell cycling and survival independently

A role for Cajal bodies in the final steps of U2 snRNP biogenesis

The biogenesis of Sm-type small nuclear ribonucleoproteins (snRNPs) involves the export of newly transcribed small nuclear RNAs (snRNAs) to the cytoplasm, assembly with seven common proteins and modification at the 5' and 3' termini. Binding of snRNP-specific proteins and snRNA modification complete the maturation process. This is thought to occur after reimport of the core snRNPs into the nucleus. The heterotrimeric splicing factor SF3a converts a pre-mature 15S U2 snRNP into the functional 17S particle. To analyze cellular aspects of this process, we studied domains in SF3a60 and SF3a66 that are required for their localization to nuclear speckles. Regions in SF3a60 and SF3a66 that mediate the binding to SF3a120 are necessary for nuclear import of the proteins, suggesting that the SF3a heterotrimer forms in the cytoplasm. SF3a60 and SF3a66 deleted for zinc finger domains required for the incorporation of SF3a into the U2 snRNP are nuclear, indicating that the 17S U2 snRNP is assembled in the nucleus. However, these proteins show an aberrant nuclear distribution. Endogenous SF3a subunits colocalize with U2 snRNP in nuclear speckles, but cannot be detected in Cajal bodies, unlike core U2 snRNP components. By contrast, SF3a60 and SF3a66 lacking the zinc finger domains accumulate in Cajal bodies and are diffusely distributed in the cytoplasm, suggesting a function for Cajal bodies in the final maturation of the U2 snRNP

3D Printing Approach in Dentistry: The Future for Personalized Oral Soft Tissue Regeneration.

Three-dimensional (3D) printing technology allows the production of an individualized 3D object based on a material of choice, a specific computer-aided design and precise manufacturing. Developments in digital technology, smart biomaterials and advanced cell culturing, combined with 3D printing, provide promising grounds for patient-tailored treatments. In dentistry, the "digital workflow" comprising intraoral scanning for data acquisition, object design and 3D printing, is already in use for manufacturing of surgical guides, dental models and reconstructions. 3D printing, however, remains un-investigated for oral mucosa/gingiva. This scoping literature review provides an overview of the 3D printing technology and its applications in regenerative medicine to then describe 3D printing in dentistry for the production of surgical guides, educational models and the biological reconstructions of periodontal tissues from laboratory to a clinical case. The biomaterials suitable for oral soft tissues printing are outlined. The current treatments and their limitations for oral soft tissue regeneration are presented, including "off the shelf" products and the blood concentrate (PRF). Finally, tissue engineered gingival equivalents are described as the basis for future 3D-printed oral soft tissue constructs. The existing knowledge exploring different approaches could be applied to produce patient-tailored 3D-printed oral soft tissue graft with an appropriate inner architecture and outer shape, leading to a functional as well as aesthetically satisfying outcome

Human Gingival Fibroblast Proliferation on Materials Used for Dental Implant Abutments: A Systematic Review

Purpose: To conduct a systematic review to evaluate the influence of materials and surfaces used for dental implant abutments on the proliferation of human gingival fibroblasts. Materials and methods: The focus question of this review was: Which material/surface characteristics used for dental implant abutments influence/enhance proliferation of human gingival fibroblasts? The Medline/PubMed, Embase, and Cochrane Library databases were searched using "gingiva," "fibroblasts," "proliferation," and "dental implant abutments" as main keywords with AND/OR as Boolean operators. In vitro studies reporting 3 to 4 or 6 to 7 days of cell proliferation, surface hydrophilicity, and roughness were included. A quality assessment of the selected studies was performed using the web-based Science in Risk Assessment and Policy (SciRAP) tool. Results: The search identified 1,144 studies, and 44 were eligible for inclusion. The average reporting quality SciRAP score was 82.87 ± 10.68, and the average methodologic quality SciRAP score was 87.35 ± 10.55. Machined, polished, and coated titanium and zirconia surfaces were most commonly investigated. Several studies analyzed aluminum oxide, cobalt-chrome-molybdenum alloy, lithium disilicate, polyether ether ketone, polymer-infiltrated ceramic network, and bioglass. The best cell proliferation was observed on zirconia and on titanium harboring nanotubules or microgrooves. UV treatment, polydopamine, and nitride coatings also improved cell proliferation. Due to the heterogeneity of the data, no correlation could be established between cell profileration and surface hydrophilicity or roughness. However, surface roughness in the range of Ra = 15 to 145 nm and Sa = 19 to 500 nm on titanium and zirconia proved most suitable. Conclusion: Titanium surfaces with directional guidance patterning and zirconia surfaces best supported cell proliferation during the first week of cell culture. Lack of standardization in surface definitions (machined or polished), methodology, and reporting prevented analytical comparison and should be imposed in future studies.</p

Cartilage tissue engineering for degenerative joint disease

Pain in the joint is often due to cartilage degeneration and represents a serious medical problem affecting people of all ages. Although many, mostly surgical techniques, are currently employed to treat cartilage lesions, none has given satisfactory results in the long term. Recent advances in biology and material science have brought tissue engineering to the forefront of new cartilage repair techniques. The combination of autologous cells, specifically designed scaffolds, bioreactors, mechanical stimulations and growth factors together with the knowledge that underlies the principles of cell biology offers promising avenues for cartilage tissue regeneration. The present review explores basic biology mechanisms for cartilage reconstruction and summarizes the advances in the tissue engineering approaches. Furthermore, the limits of the new methods and their potential application in the osteoarthritic conditions are discussed

Multilineage differentiation potential of equine blood-derived fibroblast-like cells

Tissue engineering (TE) has emerged as a promising new therapy for the treatment of damaged tissues and organs. Adult stem cells are considered as an attractive candidate cell type for cell-based TE. Mesenchymal stem cells (MSC) have been isolated from a variety of tissues and tested for differentiation into different cell lineages. While clinical trials still await the use of human MSC, horse tendon injuries are already being treated with autologous bone marrow-derived MSC. Given that the bone marrow is not an optimal source for MSC due to the painful and risk-containing sampling procedure, isolation of stem cells from peripheral blood would bring an attractive alternative. Adherent fibroblast-like cells have been previously isolated from equine peripheral blood. However, their responses to the differentiation conditions, established for human bone marrow MSC, were insufficient to fully confirm their multilineage potential. In this study, differentiation conditions were optimized to better evaluate the multilineage capacities of equine peripheral blood-derived fibroblast-like cells (ePB-FLC) into adipogenic, osteogenic, and chondrogenic pathways. Adipogenic differentiation using rabbit serum resulted in a high number of large-size lipid droplets three days upon induction. Cells' expression of alkaline phosphatase and calcium deposition upon osteogenic induction confirmed their osteogenic differentiation capacities. Moreover, an increase of dexamethasone concentration resulted in faster osteogenic differentiation and matrix mineralization. Finally, induction of chondrogenesis in pellet cultures resulted in an increase in cartilage-specific gene expression, namely collagen II and aggrecan, followed by protein deposition after a longer induction period. This study therefore demonstrates that ePB-FLC have the potential to differentiate into adipogenic, osteogenic, and chondrogenic mesenchymal lineages. The presence of cells with confirmed multilineage capacities in peripheral blood has important clinical implications for cell-based TE therapies in horses