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1.
BMC Bioinformatics ; 15: 339, 2014 Oct 05.
Article in English | MEDLINE | ID: mdl-25282047

ABSTRACT

BACKGROUND: Signatures are short sequences that are unique and not similar to any other sequence in a database that can be used as the basis to identify different species. Even though several signature discovery algorithms have been proposed in the past, these algorithms require the entirety of databases to be loaded in the memory, thus restricting the amount of data that they can process. It makes those algorithms unable to process databases with large amounts of data. Also, those algorithms use sequential models and have slower discovery speeds, meaning that the efficiency can be improved. RESULTS: In this research, we are debuting the utilization of a divide-and-conquer strategy in signature discovery and have proposed a parallel signature discovery algorithm on a computer cluster. The algorithm applies the divide-and-conquer strategy to solve the problem posed to the existing algorithms where they are unable to process large databases and uses a parallel computing mechanism to effectively improve the efficiency of signature discovery. Even when run with just the memory of regular personal computers, the algorithm can still process large databases such as the human whole-genome EST database which were previously unable to be processed by the existing algorithms. CONCLUSIONS: The algorithm proposed in this research is not limited by the amount of usable memory and can rapidly find signatures in large databases, making it useful in applications such as Next Generation Sequencing and other large database analysis and processing. The implementation of the proposed algorithm is available at http://www.cs.pu.edu.tw/~fang/DDCSDPrograms/DDCSD.htm.


Subject(s)
Algorithms , Databases, Nucleic Acid , Genomics/methods , Cluster Analysis , High-Throughput Nucleotide Sequencing , Humans
2.
Int Orthop ; 38(9): 1923-30, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24962292

ABSTRACT

PURPOSE: Our aim was to evaluate whether nonviral vectors can genetically modify primary human juvenile and adult meniscal fibrochondrocytes at low toxicity in vitro and to test the hypothesis that transfected human meniscal fibrochondrocytes transplanted into longitudinal defects and onto human medial meniscus explant cultures are capable of expressing transgene products in vitro. METHODS: Eighteen nonviral gene transfer systems were examined to identify the best suited method for an efficient transfection of primary cultures of juvenile and adult human meniscal fibrochondrocytes using luciferase and lacZ reporter gene constructs and then transplanted to meniscus explant cultures. RESULTS: Gene transfer systems FuGENE 6, GeneJammer, TurboFectin 8, calcium phosphate co-precipitates and GeneJuice led to minimal toxicity in both cell types. Nanofectin 2 and JetPEI resulted in maximal luciferase activity in both cell types. Maximal transfection efficiency based on X-gal staining following lacZ gene transfer was achieved using Lipofectamine 2000, revealing a mean transfection efficiency of 8.6 % in human juvenile and of 8.4 % in adult meniscal fibrochondrocytes. Transfected, transplanted meniscal fibrochondrocytes adhered to the meniscal tissue and continued to express the transgene for at least five days following transfection. CONCLUSIONS: Nonviral gene transfer systems are safe and capable of transfecting both juvenile and adult human meniscal fibrochondrocytes, which, when transplanted to meniscal tissue in vitro, permit the expression of selected transgenes to be maintained. These results are of value for combining gene therapy and cell transplantation approaches as a means to enhance meniscal repair.


Subject(s)
Cell Transplantation/methods , Chondrocytes/transplantation , Gene Transfer Techniques , Menisci, Tibial/surgery , Transfection/methods , Age Factors , Cell Survival , Cells, Cultured , Chondrocytes/cytology , Genetic Therapy/methods , Genetic Vectors , Humans , In Vitro Techniques , Menisci, Tibial/cytology
3.
Int Orthop ; 38(9): 1931-6, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24957484

ABSTRACT

PURPOSE: Our aim was to study the effect of three-dimensional (3D) environment and overexpression of human fibroblast growth factor 2 (FGF-2) on meniscal fibrochondrocytes in vitro. METHODS: Human meniscal fibrochondrocytes were transfected with expression plasmid vectors carrying the Photinus pyralis luciferase gene, the Escherichia coli ß-galactosidase gene or a human FGF-2 cDNA. Modified fibrochondrocytes were cultivated in 3D alginate hydrogel or cell pellets or in 2D monolayer culture. RESULTS: The levels of luciferase activity showed a peak at day two and returned to baseline levels by day 11, regardless of the type of cultivation. Both 3D environments supported the secretion of human FGF-2 protein upon FGF-2 transfection. Overexpression of human FGF-2 by genetically modified human meniscal fibrochondrocytes stimulated proliferation but not glycosaminoglycan synthesis only in 3D culture. Culture in alginate spheres resulted in a larger difference in cell numbers compared with pellet cultures. CONCLUSIONS: Three-dimensional alginate spheres are well suited for the culture of genetically modified human meniscal fibrochondrocytes. These data are of value for cell-based approaches to meniscal repair using genetically modified human meniscal fibrochondrocytes overexpressing human FGF-2.


Subject(s)
Cell Culture Techniques/methods , Cell Transplantation/methods , Chondrocytes/transplantation , Fibroblast Growth Factor 2/metabolism , Gene Transfer Techniques , Menisci, Tibial/surgery , Alginates , Animals , Cell Survival , Cells, Cultured , Chondrocytes/cytology , Chondrocytes/metabolism , Escherichia coli/genetics , Fibroblast Growth Factor 2/genetics , Fireflies/genetics , Genetic Therapy/methods , Genetic Vectors , Glucuronic Acid , Hexuronic Acids , Humans , In Vitro Techniques , Menisci, Tibial/cytology , Menisci, Tibial/metabolism , Up-Regulation
4.
J Nurs Res ; 32(1): e311, 2024 Jan 01.
Article in English | MEDLINE | ID: mdl-38190326

ABSTRACT

BACKGROUND: Poor functional status relating to heart failure (HF) negatively affects health-related quality of life (HRQOL). Patients with HF, especially those with New York Heart Association (NYHA) Class III or IV HF, often exhibit poor HRQOL because of physical limitations and HF-related symptoms. Although sense of coherence (SOC) has been reported to be a determinant of HRQOL, its role as a mediator between functional status and HRQOL remains unclear, and few studies have explored the prevalence of HF in patients in NYHA Classes I and II. PURPOSE: This study was designed to investigate SOC as a mediator between different functional status classes and HRQOL in patients with HF. METHODS: A cross-sectional study was conducted on patients with HF recruited from a hospital in northern Taiwan from April 2020 to September 2020. The Minnesota Living with Heart Failure Questionnaire and a questionnaire on sociodemographic characteristics; functional classification in terms of NYHA Classes I, II, and III; and SOC were administered. The PROCESS v3.5 (by Andrew F. Hayes) macro was applied to analyze the effects, and Model 4 was used to examine the mediating role of SOC on the relationship between NYHA functional class and HRQOL. RESULTS: Of the 295 participants, SOC was found to mediate the effects of functional status on HRQOL more significantly in patients in Class II than those in Class III but not more significantly in patients in Class I than those in Class III. A weaker mediating effect of SOC was noted on the relationship between functional status and HRQOL in patients with HF in NYHA Class II than those in Class III. CONCLUSIONS: In patients with HF, poor functional status often reduces HRQOL significantly. SOC mediates the relationship between functional status and HRQOL more significantly in those in NYHA Class II than those in Class III. Nursing staff should work to increase patients' SOC by strengthening their coping capacity and improving their functional status to improve their HRQOL.


Subject(s)
Heart Failure , Sense of Coherence , Humans , Quality of Life , Cross-Sectional Studies , Functional Status , Heart Failure/complications
5.
Molecules ; 18(3): 2978-87, 2013 Mar 04.
Article in English | MEDLINE | ID: mdl-23459306

ABSTRACT

The versatile oligosaccharide biopolymers, chitin and chitosan, are typically produced using enzymatic processes. However, these processes are usually costly because chitinases and chitosanases are available in limited quantities. Fortunately, a number of commercial enzymes can hydrolyze chitin and chitosan to produce long chain chitin or chitosan oligosaccharides. Here, a platform to screen for enzymes with chitinase and chitosanase activities using a single gel with glycol chitin or glycol chitosan as a substrate was applied. SDS-resistant chitinase and chitosanase activities were observed for pancreatin. Its chitotriosidase had an optimal hydrolysis pH of 4 in the substrate specificity assay. This activity was thermally unstable, but independent of 2-mercaptoethanol. This is the first time a chitotriosidase has been identified in the hog. This finding suggests that oligochitosaccharides can be mass-produced inexpensively using pancreatin.


Subject(s)
Hexosaminidases/metabolism , Pancreas/chemistry , Pancreas/enzymology , Pancreatin/metabolism , Animals , Chitin/chemistry , Chitosan/chemistry , Hydrogen-Ion Concentration , Hydrolysis , Substrate Specificity , Swine
6.
J Virol ; 85(14): 6856-66, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21543471

ABSTRACT

Heliothis zea nudivirus 1 (HzNV-1 or Hz-1 virus), previously regarded as a nonoccluded baculovirus, recently has been placed in the Nudivirus genus. This virus generates HzNV-1 HindIII-I 1 (hhi1) and many other transcripts during productive viral infection; during latent viral infection, however, persistency-associated gene 1 (pag1) is the only gene expressed. In this report, we used transient expression assays to show that hhi1 can trigger strong apoptosis in transfected cells, which can be blocked, at least partially, by the inhibitor of apoptosis genes Autographa californica iap2 (Ac-iap2) and H. zea iap2 (Hz-iap2). In addition to these two genes, unexpectedly, pag1, which encodes a noncoding RNA with no detectable protein product, was found to efficiently suppress hhi1-induced apoptosis. The assay of pro-Sf-caspase-1 processing by hhi1 transfection did not detect the small P12 subunit at any of the time intervals tested, suggesting that hhi1 of HzNV-1 induces apoptosis through alternative caspase pathways.


Subject(s)
Apoptosis/physiology , Genes, Viral , Inhibitor of Apoptosis Proteins/genetics , Nucleopolyhedroviruses/genetics , Animals , Base Sequence , Cell Line , DNA Primers , In Situ Nick-End Labeling , Reverse Transcriptase Polymerase Chain Reaction , Spodoptera
7.
BMC Bioinformatics ; 11: 132, 2010 Mar 16.
Article in English | MEDLINE | ID: mdl-20230647

ABSTRACT

BACKGROUND: DNA signatures are distinct short nucleotide sequences that provide valuable information that is used for various purposes, such as the design of Polymerase Chain Reaction primers and microarray experiments. Biologists usually use a discovery algorithm to find unique signatures from DNA databases, and then apply the signatures to microarray experiments. Such discovery algorithms require to set some input factors, such as signature length l and mismatch tolerance d, which affect the discovery results. However, suggestions about how to select proper factor values are rare, especially when an unfamiliar DNA database is used. In most cases, biologists typically select factor values based on experience, or even by guessing. If the discovered result is unsatisfactory, biologists change the input factors of the algorithm to obtain a new result. This process is repeated until a proper result is obtained. Implicit signatures under the discovery condition (l, d) are defined as the signatures of length < or = l with mismatch tolerance > or = d. A discovery algorithm that could discover all implicit signatures, such that those that meet the requirements concerning the results, would be more helpful than one that depends on trial and error. However, existing discovery algorithms do not address the need to discover all implicit signatures. RESULTS: This work proposes two discovery algorithms - the consecutive multiple discovery (CMD) algorithm and the parallel and incremental signature discovery (PISD) algorithm. The PISD algorithm is designed for efficiently discovering signatures under a certain discovery condition. The algorithm finds new results by using previously discovered results as candidates, rather than by using the whole database. The PISD algorithm further increases discovery efficiency by applying parallel computing. The CMD algorithm is designed to discover implicit signatures efficiently. It uses the PISD algorithm as a kernel routine to discover implicit signatures efficiently under every feasible discovery condition. CONCLUSIONS: The proposed algorithms discover implicit signatures efficiently. The presented CMD algorithm has up to 97% less execution time than typical sequential discovery algorithms in the discovery of implicit signatures in experiments, when eight processing cores are used.


Subject(s)
Algorithms , Computational Biology/methods , Databases, Nucleic Acid , DNA/chemistry , Gene Expression Profiling/methods , Pattern Recognition, Automated/methods , Sequence Analysis, DNA/methods
8.
J Gene Med ; 11(4): 302-12, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19194979

ABSTRACT

BACKGROUND: Baculovirus is an effective vector for gene delivery into primary chondrocytes and repeated baculovirus transduction (i.e. supertransduction) appears to be promising for prolonging transgene expression, but how supertransduction may influence baculovirus-mediated gene delivery is unknown. METHODS: We first investigated whether prior baculovirus transduction suppressed the ensuing transgene expression mediated by the supertransduced baculovirus, and then examined whether baculovirus triggered the expression of various cytokines. Whether interferon-alpha and -beta (IFN-alpha/beta) suppressed the transgene expression as well as the pivotal step responsible for the attenuated transgene expression were examined. RESULTS: Baculovirus transduction of chondrocytes elicited an immediate yet transient expression of IFN-alpha/beta, which repressed the transgene expression in a dose-dependent manner. The attenuation was observed for transgene expression driven by different promoters and resulted neither from internalization or nuclear import of baculovirus. Moreover, the attenuation was alleviated if supertransduction was performed when IFN-alpha/beta responses diminished. Baculovirus transduction also triggered the expression of tumor necrosis factor-alpha and interleukin (IL)-6, but not IL-1beta. Despite the induction of these responses, supertransduction of chondrocytes with a baculovirus expressing bone morphogenetic protein-2 successfully enhanced the chondrogenic differentiation and matrix synthesis. CONCLUSIONS: Baculovirus transduction of primary chondrocytes elicits antiviral effects that suppress transgene expression. Nonetheless, baculovirus supertransduction comprises a feasible approach to extend transgene expression for cartilage engineering.


Subject(s)
Baculoviridae/genetics , Chondrocytes/metabolism , Interferon-alpha/genetics , Interferon-beta/genetics , Transduction, Genetic/methods , Transgenes/genetics , Animals , Baculoviridae/immunology , Cells, Cultured , Cytokines/genetics , Genetic Vectors/immunology , Promoter Regions, Genetic , Rabbits , Rats , Tissue Engineering , Transcriptional Activation
9.
Biomaterials ; 28(14): 2294-305, 2007 May.
Article in English | MEDLINE | ID: mdl-17298844

ABSTRACT

Conjugating a single glycosaminoglycan (GAG) species such as chondroitin-6-sulfate (CSC) to chitosan is beneficial to chondrocyte culture and extracellular matrix (ECM) production, but whether fabrication of 3D chitosan scaffolds with additional minor GAG species such as dermatan sulfate (DS) further improves the ECM production is unknown. In this study, Response Surface Methodology (RSM) was employed to design CSC/DS/chitosan scaffolds of various formulations for cartilage engineering and to investigate the roles of individual GAG species in cartilage formation. The CSC/DS formulation affected neither the physical properties of scaffolds nor cell adhesion, but influenced cell morphology, GAGs and collagen production and chondrocytic gene expression. The linear effects elucidated by RSM analysis suggested that within the level range higher CSC levels favored GAGs and collagen production, whereas lower DS levels were desired for these responses. Nonetheless, the quadratic effects of DS and two-way interactions between CSC and DS also contributed to the GAGs and collagen production. Accordingly, the optimal formulation, as predicted by RSM and validated by experiments, comprised 2.8 mg CSC and 0.01 mg DS per scaffold. This study confirmed the importance of DS in cartilage tissue engineering and implicated the feasibility of rational CSC/DS/chitosan scaffold design with the aid of RSM.


Subject(s)
Cartilage, Articular/cytology , Chitosan/chemistry , Chondrocytes/cytology , Chondroitin Sulfates/chemistry , Dermatan Sulfate/chemistry , Tissue Engineering , Animals , Cell Adhesion , Cell Culture Techniques , Cell Proliferation , Cells, Cultured , Chondrocytes/metabolism , Chondrocytes/physiology , Chondroitin Sulfates/ultrastructure , Collagen/analysis , Collagen/biosynthesis , Dermatan Sulfate/ultrastructure , Gene Expression , Glycosaminoglycans/analysis , Glycosaminoglycans/biosynthesis , Histocytochemistry , Rats , Rats, Wistar , Reproducibility of Results
10.
Biomaterials ; 28(23): 3437-47, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17467793

ABSTRACT

Baculovirus has emerged as a new gene delivery vector thanks to a number of advantages. This study demonstrated that baculovirus conferred efficient gene delivery and mediated expression of growth factors (TGF-beta1, IGF-1 and BMP-2) to therapeutic levels in rabbit chondrocytes. Interestingly, the cellular response to growth factor stimulation was dependent on the cell passage. The highly de-differentiated passage 5 (P5) chondrocytes failed to respond to the stimulation by either growth factor. The de-differentiated P3 cells also failed to maintain the chondrocyte phenotype, but baculovirus-mediated BMP-2 expression remarkably reversed the de-differentiation and enhanced the aggrecan and collagen II production in 2D and 3D cultures, as evidenced by cell morphology, histological staining and gene expression analyses. Baculovirus-mediated TGF-beta1 expression modestly enhanced the cartilage-specific matrix production, although to a lesser extent. Intriguingly, IGF-1, a well-known chondroinductive protein, failed to stimulate the P3 cells likely due to the loss of IGF-1 receptor expression. In summary, this study proved for the first time the potentials of baculovirus in modulating the differentiation status of chondrocytes in the context of cartilage tissue engineering, but also highlighted the importance of selecting appropriate cell passage and growth factor for genetic manipulation.


Subject(s)
Bone Morphogenetic Proteins/metabolism , Chondrocytes/cytology , Chondrocytes/metabolism , Insulin-Like Growth Factor I/metabolism , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta/metabolism , Aggrecans/metabolism , Animals , Baculoviridae/genetics , Bone Morphogenetic Protein 2 , Bone Morphogenetic Proteins/genetics , Bone Morphogenetic Proteins/pharmacology , Cartilage, Articular/cytology , Cell Differentiation/drug effects , Cells, Cultured , Collagen Type II/biosynthesis , Gene Expression/drug effects , Immunohistochemistry , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/pharmacology , Phenotype , Rabbits , Transduction, Genetic , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/pharmacology , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/pharmacology
11.
Biomaterials ; 27(16): 3154-62, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16457882

ABSTRACT

We have previously demonstrated efficient baculovirus transduction of rat chondrocytes in 6-well plates. To further explore the potential of baculovirus in cartilage tissue engineering, the baculovirus-transduced chondrocytes were seeded into porous scaffolds and cultivated in a rotating-shaft bioreactor (RSB) which was developed for two-phase cultivation of tissue engineered cartilage. The baculovirus transduction resulted in efficiencies up to 90%, and affected neither cell adhesion to the scaffolds nor cell survival in the RSB. After 4-week RSB cultivation, the transduced cells remained highly differentiated and grew into constructs that resembled the untransduced constructs with regard to gross appearance, construct size, cell morphology, cell spatial distribution, glycosaminoglycan and collagen production and deposition. Importantly, baculovirus transduction did not alter the expression of chondrocytic genes. These data confirmed that baculovirus transduction neither harms chondrocytes nor retards the formation of cartilage-like tissues in the RSB, thus implicating the potentials of combining baculovirus-mediated gene transfer with RSB cultivation in in vitro cartilage tissue engineering.


Subject(s)
Baculoviridae/genetics , Bioreactors , Cartilage/cytology , Tissue Engineering/methods , Transfection/methods , Aggrecans , Animals , Cell Count , Cell Differentiation/genetics , Cell Line , Cell Survival/genetics , Chondrocytes/cytology , Chondrocytes/metabolism , Chondroitin Sulfate Proteoglycans/analysis , Chondroitin Sulfate Proteoglycans/genetics , Collagen/analysis , Collagen/genetics , Collagen Type II/genetics , Extracellular Matrix/chemistry , Extracellular Matrix Proteins/analysis , Extracellular Matrix Proteins/genetics , Flow Cytometry , Gene Expression/genetics , Genes, Immediate-Early/genetics , Glycolates/chemistry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Lactic Acid , Lectins, C-Type/analysis , Lectins, C-Type/genetics , Polyglycolic Acid , Polylactic Acid-Polyglycolic Acid Copolymer , Promoter Regions, Genetic/genetics , Rats , Rats, Wistar , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Spodoptera
12.
Biomaterials ; 27(10): 2222-32, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16305805

ABSTRACT

To develop a novel biomaterial for chondrocyte culture, 8 glycosaminoglycan (GAG)/chitosan membranes (groups N1-N8) were prepared, with the aid of a 2-level 2(4-1) fractional factorial design, by co-immobilizing chondroitin-4-sulfate (CSA), chondroitin-6-sulfate (CSC), dermatan sulfate (DS), and heparin to chitosan membranes. The fractional factorial design allowed us to partly interpret the effects of individual GAGs and two-way interactions between GAGs. Within the level range of -1 and +1, low CSA level (2.6 mg) is favorable for collagen synthesis but not for cell proliferation. High CSC level (1.3 mg) is favorable for GAG production but not for cell proliferation. Conversely, high heparin (0.33 mg) and DS (0.13 mg) levels are desired for cell proliferation but not for the production of collagen and GAG. Moreover, the two-way interactions between GAGs influence the cell behavior. Among the 8 GAG/chitosan membranes, N1 and N4 (containing low CSA and heparin levels) lead to the maintenance of proper chondrocyte phenotype, as judged by the chondrocyte-like morphology, modest cell expansion, higher GAG and collagen production and proper cartilage marker gene expression. In conclusion, this approach provides a means of rationally predicting and evaluating the proper formulation of GAG/chitosan membranes and may facilitate the rational design of other tissue engineering scaffolds.


Subject(s)
Biocompatible Materials/chemical synthesis , Chitosan/chemical synthesis , Glycosaminoglycans/chemical synthesis , Animals , Biocompatible Materials/chemistry , Cell Adhesion , Cell Proliferation , Cell Shape , Chitosan/chemistry , Chondrocytes/cytology , Chondrocytes/physiology , Collagen/genetics , Collagen/metabolism , Gene Expression , Glycosaminoglycans/chemistry , Materials Testing , Rats , Rats, Wistar , Tissue Engineering
13.
Biotechnol Prog ; 20(6): 1802-9, 2004.
Article in English | MEDLINE | ID: mdl-15575715

ABSTRACT

A novel rotating-shaft bioreactor (RSB) was developed for two-phase cultivation of tissue-engineered cartilage. The reactor consisted of a rotating shaft on which the chondrocyte/scaffold constructs (7.5 mm diameter x 3.5 mm thickness) were fixed and a reactor vessel half-filled with medium. The horizontal rotation of the shaft resulted in alternating exposure of the constructs to gas and liquid phases, thus leading to efficient oxygen and nutrient transfer, as well as periodically changing, mild shear stress exerting on the construct surfaces (0-0.32 dyn/cm2 at 10 rpm), as revealed by computer simulation. Strategic operation of the RSB (maintaining rotating speed at 10 rpm for 3 weeks and lowering the speed to 2 rpm in week 4) in combination with higher seeding density (6 x 10(6) chondrocytes/scaffold) and medium perfusion resulted in uniform cell distribution and increased glycosaminoglycan (3.1 mg/scaffold) and collagen (7.0 mg/scaffold) deposition. The 4-week constructs resembled native cartilages in terms of not only gross appearance and cell morphology but also distributions of glycosaminoglycan, total collagen, and type II collagen, confirming the maintenance of chondrocyte phenotype and formation of cartilage-like constructs in the RSB cultures. In summary, the novel RSB may be implicated for in vitro study of chondrogenesis and de novo cartilage development under periodic mechanical loading. With proper optimization of the culture conditions, a RSB may be employed for the production of cartilage-like constructs.


Subject(s)
Bioreactors , Cartilage, Articular/cytology , Cartilage, Articular/growth & development , Cell Culture Techniques/instrumentation , Mechanotransduction, Cellular/physiology , Physical Stimulation/instrumentation , Tissue Engineering/instrumentation , Animals , Cell Culture Techniques/methods , Cell Differentiation/physiology , Cell Proliferation , Cell Size , Equipment Design , Equipment Failure Analysis , Physical Stimulation/methods , Rats , Rats, Wistar , Rotation , Shear Strength , Tissue Engineering/methods
14.
J Gene Med ; 9(6): 470-8, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17431924

ABSTRACT

BACKGROUND: Baculovirus transduction of cultured mammalian cells is typically performed by incubating the cells with virus using culture medium (e.g. Dulbecco's modified Eagle's medium (DMEM)) as the surrounding solution. However, we previously uncovered that DMEM hinders the baculovirus-mediated gene transfer. METHODS: In this study, we systematically explored the influences of promoter and medium constituents on the transduction efficiency by using different recombinant viruses and surrounding solutions for transduction, followed by flow cytometric analyses. Whether the key medium component impeded baculovirus binding to the cells and subsequent virus entry was investigated by immunofluorescence/confocal microscopy and quantitative real-time polymerase chain reaction (Q-PCR). RESULTS: We demonstrated that the poorer transduction by using DMEM as the surrounding solution is independent of the promoter. Examination of the medium constituents group by group revealed that the balanced salt solution suppresses the baculovirus transduction. By omitting individual salt species in the balanced salt solution, we surprisingly uncovered that NaHCO(3), a common buffering agent, exerts the inhibitory effects in a concentration-dependent manner. Intriguingly, NaHCO(3) did not debilitate the baculovirus, nor did it inhibit virus binding to the cells. Instead, NaHCO(3) inhibited baculovirus transduction by reducing the intracellular virus number. CONCLUSIONS: To our best knowledge, this is the first report unraveling the significance of NaHCO(3) in gene transfer. Our finding suggests that baculovirus-mediated gene transfer can be readily enhanced by omitting NaHCO(3) from the medium during the transduction period.


Subject(s)
Baculoviridae/drug effects , Baculoviridae/metabolism , Sodium Bicarbonate/pharmacology , Animals , Cell Line , Culture Media , Potassium/pharmacology , Promoter Regions, Genetic/genetics , Salts/pharmacology , Sodium/pharmacology , Transduction, Genetic , Virus Attachment/drug effects
15.
J Gene Med ; 9(1): 33-43, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17167815

ABSTRACT

BACKGROUND: We have previously demonstrated highly efficient baculovirus transduction of primary rat articular chondrocytes, thus implicating the possible applications of baculovirus in gene-based cartilage tissue engineering. However, baculovirus-mediated gene expression in the chondrocytes is transient. METHODS: In this study, we attempted to prolong the expression by supertransduction, but uncovered that after long-term culture the chondrocytes became more refractory to baculovirus transduction. Therefore, the correlation between baculovirus-mediated enhanced green fluorescent protein (EGFP) expression and cell cycle was investigated by comparing the cycling chondrocytes and chondrocytes rich in quiescent cells, in terms of EGFP expression, virus uptake, cell cycle distribution, nuclear import and methylation of viral DNA. RESULTS: We demonstrated, for the first time, that baculovirus-mediated transduction of chondrocytes is correlated with the cell cycle. The chondrocytes predominantly in G2/M phase were approximately twice as efficient in EGFP expression as the cycling cells, while the cells in S and G1 phases expressed EGFP as efficiently as the cycling cells. Notably, the chondrocyte populations rich in quiescent cells resulted in efficient virus uptake, but less effective nuclear transport of baculoviral DNA and higher degree of methylation, and hence poorer transgene expression. CONCLUSIONS: These findings unravel the practical limitations when employing baculovirus in cartilage tissue engineering. The implications and possible solutions are discussed.


Subject(s)
Baculoviridae/genetics , Cartilage, Articular/cytology , Cell Cycle , Chondrocytes/physiology , Transduction, Genetic , Active Transport, Cell Nucleus , Animals , Cells, Cultured , Chondrocytes/cytology , DNA Methylation , Gene Expression , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Rats , Rats, Wistar , Tissue Engineering , Transgenes
16.
Biotechnol Bioeng ; 97(3): 649-55, 2007 Jun 15.
Article in English | MEDLINE | ID: mdl-17115444

ABSTRACT

We have previously demonstrated that baculovirus can efficiently transduce human mesenchymal stem cells (MSCs) and MSCs-derived adipogenic, chondrogenic, and osteogenic progenitors without compromising the differentiation capacity. Remarkably, the transgene expression level and duration varied widely with the differentiation states at which the progenitors were transduced. However, whether the variation was a general phenomenon and what caused the variation were unclear. Here we demonstrated that transduction of the MSCs and MSC-derived progenitors using baculoviruses carrying egfp driven by CMV, EF-1alpha or CAG promoter resulted in a general trend of varied expression, that is, the chondrogenic progenitors allowed for the poorest expression while the adipogenic progenitors conferred the best expression. Quantification of the nuclear and cytoplasmic egfp gene copy numbers by quantitative real-time PCR revealed that the varied expression did not arise from the discrepancies in gene delivery efficiency nor was it due to the disparities in nuclear transport efficiency. In contrast, the transcription levels paralleled the overall expression levels. These data suggested that although the egfp genes could be efficiently delivered into the nuclei of chondrogenic progenitors, they were not transcribed as well as they were in the adipogenic progenitors. In conclusion, the rapidly altering cellular transcription machinery in the course of differentiation progression predominantly led to the varied expression levels.


Subject(s)
Baculoviridae/genetics , Mesenchymal Stem Cells/metabolism , Transduction, Genetic/methods , Transgenes/physiology , Cell Differentiation , Gene Expression , Genetic Therapy/methods , Genetic Vectors/genetics , Mesenchymal Stem Cells/virology , Transcription, Genetic , Viral Proteins/genetics , Viral Proteins/metabolism
17.
Biotechnol Bioeng ; 93(3): 564-71, 2006 Feb 20.
Article in English | MEDLINE | ID: mdl-16255040

ABSTRACT

Baculovirus has emerged as a promising vector for in vivo or ex vivo gene therapy. To date, the infectious titer and multiplicity of infection (MOI) based on the ability of baculovirus to infect insect cells are commonly adopted to indicate the virus dosage. However, the infectious titer and MOI do not reliably represent the baculovirus transducing ability, making the comparison of baculovirus-mediated gene transfer difficult. To determine the baculovirus transducing ability more rapidly and reliably, we developed a protocol to evaluate the transducing titers of baculovirus stocks. The virus was diluted twofold serially and used to transduce HeLa cells. The resultant transduction efficiencies were measured by flow cytometry for the calculation of transducing titers. Compared to the infectious titer, the determination of transducing titer is more reproducible as the standard deviations among measurements are smaller. Also, the transducing titers can be obtained in 24 h, which is significantly faster as opposed to 4-7 days to obtain the infectious titer. More importantly, we demonstrated that baculoviruses with higher transducing titers could transduce cells at higher efficiency and yield stronger and longer transgene expression, confirming that the transducing titer was representative of the baculovirus transducing ability. This finding is particularly significant for ex vivo gene delivery whereby unconcentrated viruses are used for transduction and long-term transgene expression is desired. In this regard, our titration protocol provides a simple, fast, and reliable measure to evaluate the quality of virus stocks during virus production and purification, and is helpful to predict the performance of vector supernatants and ensure reproducible gene delivery experiments.


Subject(s)
Baculoviridae/genetics , Transduction, Genetic , Baculoviridae/metabolism , Flow Cytometry , Gene Expression , Genetic Therapy , Genetic Vectors , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , HeLa Cells , Humans
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