Search

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. 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. 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. 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.

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.Human meniscal fibrochondrocytes were transfected with expression plasmid vectors carrying the Photinus pyralis luciferase gene, the Escherichia coli β-galactosidase gene or a human FGF-2 cDNA. Modified fibrochondrocytes were cultivated in 3D alginate hydrogel or cell pellets or in 2D monolayer culture.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.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.

The printing equipments of Information Technology (IT) have grown rapidly and diversely coping with heterogeneous requirements today, especially for those printers. but dot-matrix printers break those rules. Although laser-printers or inkjet printers ,etc. have become very popular and useful for printing issues. But the dot-printers are still not faded out from IT-users, especially for those commercial enterprises. The central characteristics are the carbon-papers that used for some formal and financial information printed on them to offer the customers, suppliers, users, official staffs to sign their signatures by their handwriting. Those carbon-papers printing with signature written one time can be divided over two copies with the same handwriting repeated automatically. Today, the laser-printers or inkjet-printers with high-technology can make printing colorful, high-quality, high-speed and multi-functions, but they are impossible to do the carbon-papers printing like the dot-matrix printers. The previous researches proposed in "Template Printing Script Language (TPSL)" and " Virtual Printing Server (VPS)" have solved the Template Printing (TP) problems in heterogeneous environments including heterogeneous OS version, hardware, different brand printers or different model printers. But there are some problems for mobile environment unsolved with template-printing problems. More and more users like to use mobile devices like mobile-phone or tablets PC equipped i-OS or Android-OS installed everywhere. Mobile applications working on the platform of i-OS or Android still meet the template printing requirements. If mobile-users sign their signatures on the template-printing papers like PC-users, the touch screen and writable characteristic of mobile devices become useless and unnecessary. The convenience, simplicity and mobility for mobile devices must enrich new functions based on the mentioned issues above. The study proposes an architecture to extend the Visual Printer Server (VPS) to facilitate the mobile-users to preview the results of the template-printing on mobile environment to confirm the correct information and to write signatures by using theirs mobile devices. Finally the Instant-Preview Virtual Printing Server (IPVPS) merges the mobile-users' signatures into the formal printing results and sends them back to the mobile-users.

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.

Detection engines capable of inspecting packet payloads for application-layer network information are urgently required. The most important technology for fast payload inspection is an efficient multipattern matching algorithm, which performs exact string matching between packets and a large set of predefined patterns. This paper proposes a novel Enhanced Hierarchical Multipattern Matching Algorithm (EHMA) for packet inspection. Based on the occurrence frequency of grams, a small set of the most frequent grams is discovered and used in the EHMA. EHMA is a two-tier and cluster-wise matching algorithm, which significantly reduces the amount of external memory accesses and the capacity of memory. Using a skippable scan strategy, EHMA speeds up the scanning process. Furthermore, independent of parallel and special functions, EHMA is very simple and therefore practical for both software and hardware implementations. Simulation results reveal that EHMA significantly improves the matching performance. The speed of EHMA is about 0.89-1,161 times faster than that of current matching algorithms. Even under real-life intense attack, EHMA still performs well.

Inspection engines that can inspect network content for application-layer information are urgently required. In-depth packet inspection engines, which search the whole packet payload, can identify the interested packets that contain certain patterns. Network equipment then utilizes the searching results from the inspection engines for application-oriented management. The most important technology for fast packet inspection is an efficient multi-pattern matching algorithm to perform exact string matching between packets and a large set of patterns. This paper proposes a novel hierarchical multi-pattern matching algorithm (HMA) for packet inspection. HMA builds hierarchical index tables from the most frequent common-codes, and efficiently reduces the amount of external memory accesses and memory space by two-tier and cluster-wise matching. Analysis and simulation results reveal that HMA performs much better than state-of-the-art matching algorithms. In particular, HMA can update patterns incrementally, thus creating a reliable network system.

This work presents a robust environmental sound recognition system for home automation. Specific home automation services can be activated based on identified sound classes. Additionally, when the sound category is human speech, such speech can be recognized for detecting human intentions as in conventional research on home automation. To attain this ambitious goal, this study uses two key techniques: signal-to-noise ratio-aware subspace-based signal enhancement and sound recognition with independent component analysis mel-frequency cepstral coefficients and a frame-based multiclass support vector machines, respectively. Simulations and an experiment in a real-world environment are given to illustrate the performance of the proposed robust sound recognition system.

In this paper, a new subspace-based speech enhancement algorithm is presented. First, we construct a perceptual filterbank from psycho-acoustic model and incorporate it in the subspace-based enhancement approach. This filterbank is created through a five-level wavelet packet decomposition. The masking properties of the human auditory system are then derived based on the perceptual filterbank. Finally, the prior SNR and the masking threshold of each critical band are taken to decide the attenuation factor of the optimal linear estimator. Five different types of in-car noises in TAICAR database were used in our evaluation. The experimental results demonstrated that our approach outperformed conventional subspace and spectral subtraction methods.

Acoustic characteristics have played an essential role in biometrics. In this article, we introduce a robust, text-independent speaker identification/verification system. This system is mainly based on a subspace-based enhancement technique and probabilistic support vector machines (SVMs). First, a perceptual filterbank is created from a psycho-acoustic model into which the subspace-based enhancement technique is incorporated. We use the prior SNR of each subband within the perceptual filterbank to decide the estimator's gain to effectively suppress environmental background noises. Then, probabilistic SVMs identify or verify the speaker from the enhanced speech. The superiority of the proposed system has been demonstrated by twenty speaker data taken from AURORA-2 database with added background noises

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.

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 NaHCO3, a common buffering agent, exerts the inhibitory effects in a concentration-dependent manner. Intriguingly, NaHCO3 did not debilitate the baculovirus, nor did it inhibit virus binding to the cells. Instead, NaHCO3 inhibited baculovirus transduction by reducing the intracellular virus number. Conclusions To our best knowledge, this is the first report unraveling the significance of NaHCO3 in gene transfer. Our finding suggests that baculovirus-mediated gene transfer can be readily enhanced by omitting NaHCO3 from the medium during the transduction period. Copyright © 2007 John Wiley & Sons, Ltd.

We have previously demonstrated that baculovirus can efficiently transduce human mesenchymal stem cells (MSCs). In this study, we further demonstrated, for the first time, that baculovirus can transduce adipogenic, chondrogenic and osteogenic progenitors originating from MSCs. The transduction efficiency (21-90%), transgene expression level and duration (7-41 days) varied widely with the differentiation lineages and stages of the progenitors, as determined by flow cytometry. The variation stemmed from differential transgene transcription (as revealed by real-time reverse transcription-polymerase chain reaction), rather than from variability in virus entry or cell cycle (as determined by quantitative real-time PCR and flow cytometry). Nonetheless, the baculovirus-transduced cells remained capable of differentiating into adipogenic, osteogenic and chondrogenic pathways. The susceptibility to baculovirus transduction was higher for adipogenic and osteogenic progenitors, but was lower for chondrogenic progenitors. In particular, the duration of transgene expression was prolonged in the transduced adipogenic and osteogenic progenitors (as opposed to the MSCs), implicating the possibility of extending transgene expression via a proper transduction strategy design. Taken together, baculovirus may be an attractive alternative to genetically modify adipogenic and osteogenic progenitors in the ex vivo setting for cell therapy or tissue engineering.

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/cm 2 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.

Heliothis zea nudivirus 1 ( Hz NV-1 or Hz -1 virus), previously regarded as a nonoccluded baculovirus, recently has been placed in the Nudivirus genus. This virus generates Hz NV-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 Hz NV-1 induces apoptosis through alternative caspase pathways.

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 ≤ l with mismatch tolerance ≥ 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.

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-α and -β (IFN-α/β) 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-α/β, 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-α/β responses diminished. Baculovirus transduction also triggered the expression of tumor necrosis factor-α and interleukin (IL)-6, but not IL-1β. 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. Copyright © 2009 John Wiley & Sons, Ltd.

This work presents a novel approach to acoustic and phoneme modeling in order to recognize ubiquitous mixed-language speech. The conventional approaches to perform multilingual speech recognition are the usage of a multilingual phone set. A confusion matrix combining acoustic between every two phonetic is built for phonetic unit clustering. In this work, we are interested in speaker independent voice command recognition. The IPA representation is adapted for phonetic unit modeling. The EAT is applied to construct speaker independent acoustic models. The experimental results show that the proposed method can perform 70-80% lexicon recognition accuracy.

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.

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. Copyright © 2006 John Wiley & Sons, Ltd.

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.

Intrusion Detection Systems (IDSs) are known as useful tools for identifying malicious attempts over the network. The most essential part to an IDS is the searching engine that inspects every packet through the network. To strictly defend the protectorate, an IDS must be able to inspect packets at line rate and also provide guaranteed performance even under heavy attacks. Therefore, in this paper we propose an efficient string matching algorithm (named ACM) with compact memory as well as high worst-case performance. Using a magic number heuristic based on the Chinese remainder theorem, the proposed ACM significantly reduces the memory requirement without bringing complex processes. Furthermore, the latency of off-chip memory references is drastically reduced. The proposed ACM can be easily implemented in hardware and software. As a result, ACM enables cost-effective and efficient IDSs.

In this paper, we present a robust text-independent speaker recognition system. The proposed system mainly includes an SNR-aware subspace-based enhancement technique and probabilistic support vector machines (SVMs). First, we construct a perceptual filterbank from psycho-acoustic model and incorporate it with the subspace-based enhancement approach. The prior SNR of each subband within the perceptual filterbank is taken to decide the estimator's gain to effectively suppress environmental background noises. Next, this study uses probabilistic SVMs to identify the speaker from the enhanced speech. The superiority of the proposed system has been demonstrated by twenty speaker recognition from AURORA-2 database with in-car noises.

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.

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.

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.

Expressed Sequence Tags (EST) are widely used for the discovery of new genes, particularly those involved in human disease processes. A subsequence in an EST dataset is unique if it appears only in one EST sequence of the dataset but does not appear in any other EST sequence. The unique subsequences can be regarded as signatures that distinguish an EST from all the others, and provide valuable information for many applications, such as PCR primer designs and microarray experiments. The discoveries of unique signatures on large-scale EST datasets are previously computational challenges. In this paper, we propose two efficient algorithms to extract the unique signatures from EST databases. The algorithms perform impressive discovery efficiencies in the experiments on real human ESTs.