XNA ligation using T4 DNA ligase in crowding conditions

T4 DNA ligase is capable of ligating 2’OMe-RNA duplexes, HNA, LNA and FANA mixed sequences in the presence of 10% w/v PEG8000 and 3 M betaine. The enzymatic joining of oligonucleotides containing multiple consecutive XNA nucleotides at the ligation site has not been reported before

Macromolecular crowding alone or in combination with other in vitro microenvironment modulators to maintain tenogenic phenotype in vitro

Tendon injuries are very common among the different musculoskeletal disorders affecting millions of people annually. Given that current treatments have failed to restore injured tendons to the prior to injury state, cell-based therapies hold a great promise to engineer functional tissue surrogates in vitro in order to restore the damaged tissues. However, the development of a rich in extracellular matrix tenocyte-assembled tendon equivalent requires prolonged in vitro culture, which is associated with phenotypic drift. Recapitulation of tendon tissue microenvironment in vitro with cues that enhance and accelerate extracellular matrix synthesis and deposition, whilst maintaining tenocyte phenotype, may lead to functional tissue engineering cell-based therapies. Herein, we assessed the synergistic effect of macromolecular crowding with low oxygen tension or growth factor supplementation or surface topography as in vitro microenvironmental modulators on the behaviour of human tenocytes. Firstly, it was demonstrated that human tenocytes cultured at 2 % oxygen tension and with 50 μg/ml carrageenan (macromolecular crowder used) significantly increased synthesis and deposition of collagen types I, III, V and VI. Gene analysis at day 7 illustrated that human tenocytes at 2 % oxygen tension and with 50 μg/ml carrageenan significantly increased expression of the extracellular matrix-related genes prolyl 4-hydroxylase subunit alpha 1, procollagen-lysine 2-oxoglutarate 5-dioxygenase 2, the tendon-related markers scleraxis, tenomodulin and elastin, whilst chondrogenic (e.g. runt-related transcription factor 2, cartilage oligomeric matrix protein, aggrecan) and osteogenic (e.g. secreted phosphoprotein 1, bone gamma-carboxyglutamate protein) trans-differentiation markers were significantly down-regulated or remained unchanged. Then, we assessed the synergistic effect of simultaneous and serial growth factor (insulin growth factor-1, platelet-derived growth factor ββ, growth differentiation factor 5 and transforming growth factor β3) supplementation to carrageenan in human tenocyte function, collagen synthesis and deposition and gene expression. Transforming growth factor β3 supplementation (without / with carrageenan) induced the highest (among all groups) DNA content. In all cases, tenocyte proliferation was significantly increased as a function of time in culture, whilst metabolic activity was not affected. Carrageenan supplementation induced significantly higher collagen deposition than groups without carrageenan (without / with any growth factor). Of all the growth factors used, transforming growth factor β3 induced the highest collagen XXVI deposition when used together with carrageenan in both simultaneous and serial fashion. At day 13, gene expression analysis revealed that transforming growth factor β3 in serial supplementation to carrageenan upregulated the most and downregulated the least collagen-and tendon-related genes and upregulated the least and downregulated the most osteo-, chondro-, fibrosis-and adipose-related trans-differentiation genes. Finally, the combining effect of surface topography and macromolecular crowding on human tenocyte culture was analysed. Our data demonstrated that bidirectionally aligned electrospun fibres induced physiological tenocyte growth and alignment (without / with carrageenan), whilst macromolecular crowding enhanced and accelerated tendon-specific extracellular matrix deposition, which was further oriented to the direction of the electrospun fibres, recapitulating extracellular matrix orientation in native tendons. Collectively, these data advocate the use of multifactorial approaches for the accelerated development of functional tissue-like surrogates in vitro

Macromolecular crowding alone or in combination with other in vitro microenvironment modulators to maintain tenogenic phenotype in vitro

Tendon injuries are very common among the different musculoskeletal disorders affecting millions of people annually. Given that current treatments have failed to restore injured tendons to the prior to injury state, cell-based therapies hold a great promise to engineer functional tissue surrogates in vitro in order to restore the damaged tissues. However, the development of a rich in extracellular matrix tenocyte-assembled tendon equivalent requires prolonged in vitro culture, which is associated with phenotypic drift. Recapitulation of tendon tissue microenvironment in vitro with cues that enhance and accelerate extracellular matrix synthesis and deposition, whilst maintaining tenocyte phenotype, may lead to functional tissue engineering cell-based therapies. Herein, we assessed the synergistic effect of macromolecular crowding with low oxygen tension or growth factor supplementation or surface topography as in vitro microenvironmental modulators on the behaviour of human tenocytes. Firstly, it was demonstrated that human tenocytes cultured at 2 % oxygen tension and with 50 μg/ml carrageenan (macromolecular crowder used) significantly increased synthesis and deposition of collagen types I, III, V and VI. Gene analysis at day 7 illustrated that human tenocytes at 2 % oxygen tension and with 50 μg/ml carrageenan significantly increased expression of the extracellular matrix-related genes prolyl 4-hydroxylase subunit alpha 1, procollagen-lysine 2-oxoglutarate 5-dioxygenase 2, the tendon-related markers scleraxis, tenomodulin and elastin, whilst chondrogenic (e.g. runt-related transcription factor 2, cartilage oligomeric matrix protein, aggrecan) and osteogenic (e.g. secreted phosphoprotein 1, bone gamma-carboxyglutamate protein) trans-differentiation markers were significantly down-regulated or remained unchanged. Then, we assessed the synergistic effect of simultaneous and serial growth factor (insulin growth factor-1, platelet-derived growth factor ββ, growth differentiation factor 5 and transforming growth factor β3) supplementation to carrageenan in human tenocyte function, collagen synthesis and deposition and gene expression. Transforming growth factor β3 supplementation (without / with carrageenan) induced the highest (among all groups) DNA content. In all cases, tenocyte proliferation was significantly increased as a function of time in culture, whilst metabolic activity was not affected. Carrageenan supplementation induced significantly higher collagen deposition than groups without carrageenan (without / with any growth factor). Of all the growth factors used, transforming growth factor β3 induced the highest collagen XXVI deposition when used together with carrageenan in both simultaneous and serial fashion. At day 13, gene expression analysis revealed that transforming growth factor β3 in serial supplementation to carrageenan upregulated the most and downregulated the least collagen-and tendon-related genes and upregulated the least and downregulated the most osteo-, chondro-, fibrosis-and adipose-related trans-differentiation genes. Finally, the combining effect of surface topography and macromolecular crowding on human tenocyte culture was analysed. Our data demonstrated that bidirectionally aligned electrospun fibres induced physiological tenocyte growth and alignment (without / with carrageenan), whilst macromolecular crowding enhanced and accelerated tendon-specific extracellular matrix deposition, which was further oriented to the direction of the electrospun fibres, recapitulating extracellular matrix orientation in native tendons. Collectively, these data advocate the use of multifactorial approaches for the accelerated development of functional tissue-like surrogates in vitro

Hypoxia preconditioning of bone marrow mesenchymal stem cells before implantation in orthopaedics

[No abstract available]This work is supported by the: European Commission, Horizon 2020, Marie Skłodowska-Curie Actions, Innovative Training Networks (ITN) Programme Tendon Therapy Train, under the grant agreement number 676338; Science Foundation Ireland, Career Development Award Programme, under the grant agreement number 15/CDA/3629; and Science Foundation Ireland and the European Regional Development Fund, under the grant agreement number 13/RC/2073

Hypoxia preconditioning of bone marrow mesenchymal stem cells before implantation in orthopaedics

[No abstract available]This work is supported by the: European Commission, Horizon 2020, Marie Skłodowska-Curie Actions, Innovative Training Networks (ITN) Programme Tendon Therapy Train, under the grant agreement number 676338; Science Foundation Ireland, Career Development Award Programme, under the grant agreement number 15/CDA/3629; and Science Foundation Ireland and the European Regional Development Fund, under the grant agreement number 13/RC/2073.peer-reviewe

Polyadenylate polymerase modulations in human epithelioid cervix and breast cancer cell lines, treated with etoposide or cordycepin, follow cell cycle rather than apoptosis induction

Cancer results from an imbalance between cell cycle progression and apoptosis. Therefore, most anticancer drugs exert their anti proliferative and cytotoxic activity via cell cycle arrest and induction of apoptosis, a controlled form of cell death that is dysregulated in cancer. Many polyadenylation trans-acting factors, including polyadenylate polymerase (PAP), are increasingly found to be involved in cell cycle, apoptosis and cancer prognosis. The objective of the present study was to identify PAP modulations in the response of two epithelial cancer cell lines (HeLa and MCF-7) to apoptosis induction by the anticancer drugs etoposide and cordycepin. Cells were assessed for PAP activity and isoforms by the highly sensitive PAP activity assay and Western blotting, respectively. Induction of apoptosis was determined by endonucleosomal DNA cleavage, 4’6-diamidino-2-phenylindol (DAPI) staining and caspase-6 activity assay, whereas cytotoxicity and cell cycle status were assessed by trypan blue staining, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay and flow cytometry. Our results indicate that PAP changes very early in response to either etoposide or cordycepin treatment, even prior to the hallmarks of apoptosis (chromatin condensation and cleavage), in both cell lines tested, but in a different mode. Our results suggest, for the first time, that in the epithelial cancer cell lines used, PAP modulations follow cell cycle progression rather than the course of apoptosis

The effect of the polyadenylation inhibitor cordycepin on human Molt-4 and Daudi leukaemia and lymphoma cell lines

Purpose: Posttranscriptional modifications, such as polyadenylation, are very often implicated in the regulation and dysregulation of cell death, through regulation of the expression of specific genes. Based on the fact that an increasing number of adenosine analogues show their antiproliferative and cytotoxic activity via induction of apoptosis, we assessed the effect of cordycepin, a polyadenylation specific inhibitor, an adenosine analogue and a well-known chemotherapeutic drug, on two human leukemia and lymphoma cell lines. Methods: Cells were treated with the anticancer drug cordycepin and assessed for poly(A) polymerase (PAP) activity and isoforms by the highly sensitive PAP activity assay and western blotting, respectively. Induction of apoptosis was determined by endonucleosomal DNA cleavage, DAPI staining and Δψm reduction, whereas cytotoxicity and cell cycle status were assessed by Trypan blue staining, MTT assay and flow cytometry. Results and conclusions: The results showed that the differentiated modulations of PAP in the two cell lines may be a result of the additive effect of the changes in cell cycle and apoptotic pathway induced. © 2007 Springer-Verlag

The role of cordycepin in cancer treatment via induction or inhibition of apoptosis: Implication of polyadenylation in a cell type specific manner

Purpose: Most anticancer drugs show their antiproliferative and cytotoxic activity via induction of apoptosis. In the present study we assessed the implication and role of cordycepin, a polyadenylation-specific inhibitor and a well-known chemotherapeutic drug, in apoptosis, induced by the anticancer drug etoposide. Methods: For this purpose, a variety of leukemia and lymphoma cell lines (U937, K562, HL-60, Daudi, Molt-4) were treated with the anticancer drugs etoposide and/or cordycepin and assessed for poly(A) polymerase (PAP) activity and isoforms by the highly sensitive PAP activity assay and western blotting, respectively. Induction of apoptosis was determined by endonucleosomal DNA cleavage, DAPI staining, caspase-6 activity assay and ΔΨm reduction, whereas cytotoxicity and cell cycle status were assessed by Trypan blue staining, MTT assay and flow cytometry. Results and conclusions: The results showed that PAP changes in all cell lines, in response to apoptosis induced by etoposide, in many cases even prior to hallmarks of apoptosis (endonucleosomal cleavage of DNA, ΔΨm reduction). A further elucidation to this apoptosis-polyadenylation correlation was added, by cell treatment with cordycepin, resulting in either suppression (U937, K562) or induction (HL-60) of the apoptotic process, according to the cell type. However, inhibition of polyadenylation did not influence the cell lines Daudi and Molt-4 used, where alternative apoptotic pathways are induced through cleavage of DNA into high molecular weight fragments. © 2007 Springer-Verlag