Multimodal treatment combining cold atmospheric plasma and acidic fibroblast growth factor for multi-tissue regeneration.

Cold atmospheric plasma (CAP) is an emerging technology for biomedical applications, exemplified by its antimicrobial and antineoplastic potentials. On the contrary, acidic fibroblast growth factor (aFGF) has been a long-standing potent mitogen for cells from various origins. In this study, we are the first to develop a multimodal treatment combining the aforementioned physicochemical and pharmacological treatments and investigated their individual and combined effects on wound healing, angiogenesis, neurogenesis, and osteogenesis. This work was performed at the tissue, cellular, protein, and gene levels, using histochemical staining, flow cytometry, ELISA, and PCR, respectively. Depending on the type of target tissue, various combinations of aforementioned methods were used. The results showed that the enhancement on would healing and angiogenesis by CAP and aFGF were synergistic. The former was manifested by increased murine fibroblast proliferation and reduced cutaneous tissue inflammation, whereas the latter by upregulated proangiogenic markers in vivo, for example, CD31, VEGF, and TGF-ß, and downregulated antiangiogenic proteins in vitro, for example, angiostatin and angiopoietin-2, respectively. In addition, aFGF outperformed CAP during neurogenesis, which was evidenced by superior neurite outgrowth, while CAP exceeded aFGF in osteogenesis which was demonstrated by more substantial bone nodule formation. These novel findings not only support the fact that CAP and aFGF are both multipotent agents during tissue regeneration, but also highlight the potential of our multimodal treatment combining the individual advantages of CAP and aFGF. The versatile administration route, that is, topical and/or systemic, might further broaden its applications.

Cold atmospheric plasma as an interface biotechnology for enhancing surgical implants.

Cold atmospheric plasma (CAP) has been intensively researched for direct treatment of living cells and tissues. Significant attention is now being given to its indirect applications in plasma medicine. Surgical implant is an exemplary conveyor to deliver the therapeutic effects of plasma to patients. There is a constant drive to enhance the clinical performance of surgical implants, targeting at the implant-tissue interface. As a versatile and potent tool, CAP is capable of ameliorating surgical implants using various strategies of interface biotechnology, such as surface modification, coating deposition, and drug delivery. Understanding the chemical, physical, mechanical, electrical, and pharmacological processes occurring at the implant-tissue interface is crucial to effective application of CAP as an interface biotechnology. This preclinical review focuses on the recent advances in CAP-assisted implant-based therapy for major surgical specialties. The ultimate goal here is to elicit unique opportunities and challenges for translating implant science to plasma medicine.

The relationship of metabolic burden to productivity levels in CHO cell lines.

The growing demand for recombinant therapeutics has driven biotechnologists to develop new production strategies. One such strategy for increasing the expression of heterologous proteins has focused on enhancing cell-specific productivity through environmental perturbations. In this work, the effects of hypothermia, hyperosmolarity, high shear stress, and sodium butyrate treatment on growth and productivity were studied using three (low, medium, and high producing) CHO cell lines that differed in their specific productivities of monoclonal antibody. In all three cell lines, the inhibitory effect of these parameters on proliferation was demonstrated. Additionally, compared to the control, specific productivity was enhanced under all conditions and exhibited a consistent cell line specific pattern, with maximum increases (50-290%) in the low producer, and minimum increases (7-20%) in the high producer. Thus, the high-producing cell line was less responsive to environmental perturbations than the low-producing cell line. We hypothesize that this difference is most likely due to the bottleneck associated with a higher metabolic burden caused by higher antibody expression. Increased recombinant mRNA levels and pyruvate carboxylase activities due to low temperature and hyperosmotic stress were found to be positively associated with the metabolic burden.

Understanding central carbon metabolism of rapidly proliferating mammalian cells based on analysis of key enzymatic activities in GS-CHO cell lines.

The central carbon metabolism (glycolysis, the pentose phosphate pathway [PPP], and the tricarboxylic acid [TCA] cycle) plays an essential role in the supply of biosynthetic precursors and energy. How the central carbon metabolism changes with the varying growth rates in the in vitro cultivation of rapidly proliferating mammalian cells, such as cancer cells and continuous cell lines for recombinant protein production, remains elusive. Based on relationships between the growth rate and the activity of seven key enzymes from six cell clones, this work reports finding an important metabolic characteristic in rapidly proliferating glutamine synthetase-Chinese hamster ovary cells. The key enzymatic activity involved in the TCA cycle that is responsible for the supply of energy became elevated as the growth rate exhibited increases, while the activity of key enzymes in metabolic pathways (glycolysis and the PPP), responsible for the supply of biosynthetic precursors, tended to decrease-suggesting that rapidly proliferating cells still depended predominantly on the TCA cycle rather than on aerobic glycolysis for their energetic demands. Meanwhile, the growth-limiting resource was most likely biosynthetic substrates rather than energy provision. In addition, the multifaceted role of glucose-6-phosphate isomerase (PGI) was confirmed, based on a significant correlation between PGI activity and the percentage of G2/M-phase cells.

The relationship between mTOR signalling pathway and recombinant antibody productivity in CHO cell lines.

BACKGROUND: High recombinant protein productivity in mammalian cell lines is often associated with phenotypic changes in protein content, energy metabolism, and cell growth, but the key determinants that regulate productivity are still not clearly understood. The mammalian target of rapamycin (mTOR) signalling pathway has emerged as a central regulator for many cellular processes including cell growth, apoptosis, metabolism, and protein synthesis. This role of this pathway changes in response to diverse environmental cues and allows the upstream proteins that respond directly to extracellular signals (such as nutrient availability, energy status, and physical stresses) to communicate with downstream effectors which, in turn, regulate various essential cellular processes. RESULTS: In this study, we have performed a transcriptomic analysis using a pathway-focused polymerase chain reaction (PCR) array to compare the expression of 84 target genes related to the mTOR signalling in two recombinant CHO cell lines with a 17.4-fold difference in specific monoclonal antibody productivity (qp). Eight differentially expressed genes that exhibited more than a 1.5-fold change were identified. Pik3cd (encoding the Class 1A catalytic subunit of phosphatidylinositol 3-kinase [PI3K]) was the most differentially expressed gene having a 71.3-fold higher level of expression in the high producer cell line than in the low producer. The difference in the gene's transcription levels was confirmed at the protein level by examining expression of p110δ. CONCLUSION: Expression of p110δ correlated with specific productivity (qp) across six different CHO cell lines, with a range of expression levels from 3 to 51 pg/cell/day, suggesting that p110δ may be a key factor in regulating productivity in recombinant cell lines.

Automated flow cytometry for monitoring CHO cell cultures.

Flow cytometry has been used to accurately monitor cell events that indicate the spatio-temporal state of a bioreactor culture. The introduction of process analytical technology (PAT) has led to process improvements using real-time or semi real-time monitoring systems. Integration of flow cytometry into an automated scheme for improved process monitoring can benefit PAT in bioreactor-based biopharmaceutical productions by establishing optimum process conditions and better quality protocols. Herein, we provide detailed protocols for establishing an automated flow cytometry system that can be used to investigate and monitor cell growth, viability, cell size, and cell cycle data. A method is described for the use of such a system primarily focused on CHO cell culture, although it is foreseen the information gathered from automated flow cytometry can be applied to a variety of cell lines to address both PAT requirements and gain further understanding of complex biological systems.

Application of statistical techniques for elucidating flow cytometric data of batch and fed-batch cultures.

The objective of this work is to develop structured, segregated stochastic models for bioprocesses using time-series flow cytometric (FC) data. To this end, mammalian CHO cells were grown in both batch and fed-batch cultures, and their viable cell numbers (VCDs), monoclonal antibody (MAb), cell cycle phases, mitochondria membrane potential/mitochondria mass, Golgi apparatus, and endoplasmic reticulum (ER) were analyzed. For the fed-batch mode, soy hydrolysate was introduced at 24-H intervals. The cytometric data were analyzed for early indicators of growth and productivity by multiple linear regression analysis, which involved taking into account multicollinearity diagnostics, Durbin-Watson statistics, and Houston tests to determine and refine statistically significant correlations between categorical variables (FC parameters) and response variables (yield parameters). The results indicate that the percentage of G1 cells and ER was significantly correlated with VCD and MAb in the case of batch culture, whereas for fed-batch culture, the percentage of G2 cells and ER was correlated significantly. There was a significant difference between cells in the batch and fed-batch cultures in their ER content, suggesting that the increase in protein synthesis as reflected by the ER content and consequent increase in growth rate and MAb productivity both can be monitored at the cellular level by FC analysis of ER content.

Growth, metabolic activity, and productivity of immobilized and freely suspended CHO cells in perfusion culture.

Chinese hamster ovary (CHO) cells producing ß-galactosidase (ß-gal) were successfully cultured on silicone-based porous microcarriers (ImmobaSil FS) in a 1 L stirred-tank perfusion bioreactor. We studied the growth, metabolism, and productivity of free and immobilized cells to understand cellular activity in immobilized conditions. CHO cells attached to ImmobaSil FS significantly better than to other microcarriers. Scanning electron microscope images showed that the CHO cells thoroughly colonized the porous surfaces of the ImmobaSil FS, exhibiting a spherical morphology with microvilli that extended to anchorage cells on the silicone surface. In perfusion culture, the concentration of the attached cells reached 8 × 10(8) cells/mL of carrier, whereas those that remained freely suspended reached 2 × 10(7) cells/mL medium. The ß-gal concentration reached more than 5 unit/mL in perfusion culture, more than fivefold that of batch culture. The maximum concentration per microcarrier was proportional to the initial cell density. The specific growth rate, the specific ß-gal production rate, the percentage of S phase, and the oxygen uptake rate were all relatively lower for immobilized cells than freely suspended cells in the same bioreactor, indicating that not only do cells survive and grow to a greater extent in a free suspension state, but they are also metabolically more active than viable cells inside the pores of the microcarriers.

Analysis of an artificially selected GS-NS0 variant with increased resistance to apoptosis.

Heterogeneity in gene expression and phenotypic traits is an inherent feature within the "clonal" cell lines used for biopharmaceutical production. This feature can allow the selection of cell lines with improved phenotypes and adaptation to growth under preferential conditions to improve productivity or provide a platform to study the molecular basis of improved characteristics. A repeated process of extended batch culture of a recombinant antibody producing GS-NS0 myeloma cell line generated a stable variant cell line displaying increased resistance to both environmental stresses and chemical apoptosis inducers, and resulted in extended culture viability and increased antibody production. An interesting feature of the variant cell line was an altered metabolic state with consumption of lactate as the culture progressed. The variant cell line also showed altered expression of proteins associated with autophagy suggesting a role for this process in extending cell survival in culture. Targeted transcriptomic analysis was carried out on the parental and variant cell lines using a qRT-PCR array containing a panel of apoptosis-associated genes to elucidate both the predominant apoptotic pathways in the NS0 cell line with batch culture progression, and the altered gene expression contributing to increased survival in the variant line. Results indicated a balance between pro- and anti-apoptotic signaling is triggered with the onset of cell death in the NS0 cell line. Pro-survival pathways such as NFκB signaling and the unfolded protein response were implicated along with death receptor, endoplasmic reticulum stress and p53 mediated apoptotic pathways. The identification of altered gene expression in the variant cell line also provides several potential targets for cell engineering strategies to create improved cell lines for production.

Osteoconductivity and growth factor production by MG63 osteoblastic cells on bioglass-coated orthopedic implants.

We have produced Bioglass coatings for Orthopedic implants by using a novel coating technique, CoBlast. The two resultant surfaces, designated BG and hydroxyapatite (HA)/BG, were compared with their HA counterpart, OsteoZip in terms of osteoblastic cell attachment, adhesion, proliferation, differentiation, and growth factor production. BG and HA/BG were demonstrated by goniometry to be more hydrophilic than OsteoZip. This corresponded to enhanced protein adsorption, cell attachment, and cell adhesion documented by both quantitative and qualitative assessments. BG and HA/BG surfaces had a significant initial release of Si and Ca ions, and this was consistent with elevated cell proliferation and basic fibroblast growth factor levels. However, OsteoZip, being similar to HA/BG, exhibited better osteogenic differentiation than BG did, shown by augmented differentiation marker activity at both protein and mRNA levels. Sandwich ELISA was used to quantify angiopoietin and inducible nitric oxide synthase which are involved in peri-prosthetic angiogenesis and aseptic loosening of total hip replacement, respectively. Both Bioglass-derived coatings provide superior initial osteoconductivity to OsteoZip, and HA/Bioglass composite coating outruns in long-term osteogenic differentiation and prognostic bioprocesses. The novel coatings discovered in this study have significant potential in providing both orthopedic and therapeutic functions.

Chemostat-based transcriptional analysis of growth rate change and BCL-2 over-expression in NS0 cells.

We investigated the transcriptional response of NS0 cells undergoing controlled nutrient growth change in continuous chemostat culture using mouse microarrays. A 50% reduction in growth rate resulted in detectable alterations in the expression of 29 genes in NS0 cells. Notably, expression of genes in three major biological processes, namely transcriptional, translational, and protein processing functions, were modified. To further elucidate the advantage of the chemostat environment for establishment of "omic" data sets, an expression profile of the over-expressed gene bcl-2 in NS0 cells was probed. Functional analysis revealed that the underlying altered molecular mechanism was particularly associated with G1 cell cycle progression, protein synthesis, and apoptosis. Importantly, these findings agreed with the physical function of the cells. Despite an increase in survival rate, bcl-2 over-expression resulted in a decrease of specific productivity, glucose consumption, oxygen uptake rate and intracellular protein content, indicating a lower energy generating metabolism. Further, a prolongation of G1 cell cycle phase was evident on lowering the growth rate. Overall, the application of microarray analysis to chemostat-grown cultures offers an excellent combination for the interpretation of transcriptomic profiles to elucidate the molecular mechanisms during nutrient growth change and bcl-2 over-expression.

Defining viability in mammalian cell cultures.

A large number of assays are available to monitor viability in mammalian cell cultures with most defining loss of viability as a loss of plasma membrane integrity, a characteristic of necrotic cell death. However, the majority of cultured cells die by apoptosis and early apoptotic cells, although non-viable, maintain an intact plasma membrane and are thus ignored. Here we measure the viability of cultures of a number of common mammalian cell lines by assays that measure membrane integrity (a measure of necrotic cell death) and assays that measure apoptotic cells, and show that discrepancies in the measurement of culture viability have a significant impact on the calculation of cell culture parameters and lead to skewed experimental data.

A multifunctional dexamethasone-delivery implant fabricated using atmospheric plasma and its effects on apoptosis, osteogenesis and inflammation.

Implant-based local drug delivery is a unique surgical therapy with many clinical advantages. Atmospheric pressure plasma is a novel non-thermal surface biotechnology that has only recently been applied in enhancing a surgical implant. We are the first to use this technology to successfully create a dexamethasone-delivery metallic implant. Irrespective of the loaded medication, the surface of this novel implant possesses advantageous material features including homogeneity, hydrophilicity, and optimal roughness. UV-vis spectroscopy revealed much more sustainable drug release compared to the implants produced using simple drug attachment. In addition, our drug-releasing implant was found to have multiple biological benefits. As proven by the ELISA data, this multi-layer drug complex provides differential regulation on the cell apoptosis, as well as pro-osteogenic and anti-inflammatory effects on the peri-implant tissue. Furthermore, using the pathway-specific PCR array, our study discovered 28 and 26 upregulated and downregulated genes during osteogenesis and inflammation on our newly fabricated drug-delivery implant, respectively. The medication-induced change in molecular profile serves as a promising clue for designing future implant-based therapy. Collectively, we present atmospheric pressure plasma as a potent tool for creating a surgical implant-based drug-delivery system, which renders multiple therapeutic potentials. Graphical abstract Schematic of the APP-facilitated Dex-delivery implant. This layer-by-layer drug-releasing complex consisted of bottom plasma activation layer, middle medication layer, and top absorbable polymer layer.

A proteomic study of cMyc improvement of CHO culture.

BACKGROUND: The biopharmaceutical industry requires cell lines to have an optimal proliferation rate and a high integral viable cell number resulting in a maximum volumetric recombinant protein product titre. Nutrient feeding has been shown to boost cell number and productivity in fed-batch culture, but cell line engineering is another route one may take to increase these parameters in the bioreactor. The use of CHO-K1 cells with a c-myc plasmid allowing for over-expressing c-Myc (designated cMycCHO) gives a higher integral viable cell number. In this study the differential protein expression in cMycCHO is investigated using two-dimensional gel electrophoresis (2-DE) followed by image analysis to determine the extent of the effect c-Myc has on the cell and the proteins involved to give the new phenotype. RESULTS: Over 100 proteins that were differentially expressed in cMycCHO cells were detected with high statistical confidence, of which 41 were subsequently identified by tandem mass spectrometry (LC-MS/MS). Further analysis revealed proteins involved in a variety of pathways. Some examples of changes in protein expression include: an increase in nucleolin, involved in proliferation and known to aid in stabilising anti-apoptotic protein mRNA levels, the cytoskeleton and mitochondrial morphology (vimentin), protein biosynthesis (eIF6) and energy metabolism (ATP synthetase), and a decreased regulation of all proteins, identified, involved in matrix and cell to cell adhesion. CONCLUSION: These results indicate several proteins involved in proliferation and adhesion that could be useful for future approaches to improve proliferation and decrease adhesion of CHO cell lines which are difficult to adapt to suspension culture.

Recent advances in the implant-based drug delivery in otorhinolaryngology.

The surgical implant is an interdisciplinary therapeutic modality that offers unique advantages in the daily practice of otorhinolaryngology. Some well-known examples include cochlear implants, bone-anchored hearing aids, sinus stents, and tracheostomy tubes. Neuroprotective, osteogenic, anti-inflammatory, and antimicrobial effects are among their established or pursued functions. Implant-based drug delivery affords an efficient and potent approach to enhancing these therapeutic functions. Recent innovations have infiltrated all four elements of a drug-eluting implant. The purpose of this pre-clinical, biotechnology-oriented review is to discuss these developments in terms of the implant biomaterial, loaded medication, delivery pattern, and system fabrication. Cell-mediated neurotrophin release, fabrication of a hydroxyapatite-supported system, biodegradable polymer-based implants, and multiclass and multidrug delivery are some representative advancements. The ultimate goal here is to bridge the gap between biotechnology advances and clinical needs. The review is concluded with a perspective regarding the future opportunities and challenges in this popular and rapidly developing subject of research. STATEMENT OF SIGNIFICANCE: Surgical implants and local drug delivery are representative modern modalities of surgical treatment and medical treatment, respectively. Their synergy offers unique therapeutic advantages, such as minimal systemic side effects, proximity-related high efficiency, and potential absorbability. The applications of implant-based drug delivery have infiltrated otorhinolaryngology and head & neck surgery, which is well known for its related tissue diversity and surgical complexity. Examples discussed here include cochlear implants, bone-anchored hearing aids, sinus stents, and airway tubes. This timely review focuses primarily on the four fundamental components of an implant-based drug delivery system, namely implant biomaterial, loaded medication, delivery pattern, and system fabrication. A particular emphasis is placed upon the in vitro cellular and in vivo animal studies that demonstrate pre-clinical potentials.

Controlling stem cell fate using cold atmospheric plasma.

The stem cell is the foundation of regenerative medicine and tissue engineering. Regulating specific stem cell fate, such as cell attachment, proliferation, differentiation, and even death, undergoes continuous development. Cold atmospheric plasma (CAP), the core technology of plasma medicine, is attracting tremendous attention due to its ability and versatility to manipulate various types of cells, including stem cells. Specifically, the direct and indirect applications of CAP in controlling cell fate are best exemplified by upfront irradiation of the stem cells and modification of the stem cell niche, respectively. This review will describe the recent advances in various CAP strategies, both direct and indirect, and their influence on the fate of healthy and cancer stem cells. Particular emphasis will be placed on the mechanism of connecting the physical and chemical cues carried by the plasma and biological changes presented by the cells, especially at the transcriptomic level. The ultimate goal is to exploit CAP's potential in regenerative medicine.

cMyc increases cell number through uncoupling of cell division from cell size in CHO cells.

BACKGROUND: Over the past decades, the increase in maximal cell numbers for the production of mammalian derived biologics has been in a large part due to the development of optimal feeding strategies. Engineering of the cell line is one of probable approaches for increasing cell numbers in bioreactor. RESULTS: We have demonstrated that the over-expression of the c-myc gene in immortalised CHO cells can increase proliferation rate and maximal cell density in batch culture compared to the control. The changes were attributed to a rapid transition into S-phase from a shortened duration of G1 phase and to the uncoupling of cell size from cell proliferation. To achieve the >70% increase in maximal cell density without additional supply of nutrients the cells underwent an overall reduction of 14% in size as well as a significant decrease in glucose and amino acid consumption rate. Consequently, the total biomass accumulation did not show a significant change from the control. The amount of hSEAP-hFc activity of the over expressing c-myc cell line was found to be within 0.7% of the control. CONCLUSION: It is shown that the manipulation of cell cycle kinetics and indirectly cell metabolism gives higher cell densities in CHO batch cultures. The unaltered apoptotic rate supported the proposition that the increase in cell number was a result of enhance cell cycle kinetics and cellular metabolism rather than increasing viability. Production of hSEAP-hFc from a constitutive c-myc over-expressing cell line did not increase with the increase in cell number.

Metabolic characterization of a hyper-productive state in an antibody producing NS0 myeloma cell line.

Metabolic profiling or metabolomics is the analysis of a larger number of small metabolic compounds within cells. While this technique has been utilized to study microbial and yeast strains under different physiochemical conditions, very little has been reported regarding its application in mammalian cell culture. Here, the physiological and metabolic changes observed during the proliferation arrest of an antibody producing GS-NS0 mouse myeloma cell line were studied using conventional biochemical analysis and one-dimensional nuclear magnetic resonance (NMR)-based metabolic profiling. Proliferation-arrested cells had increased antibody productivity, enhanced normalized mitochondrial membrane potential, and showed changes in the consumption of several amino acids. Further investigation into these physiological changes was carried out by (1)H NMR profiling followed by principle component analysis (PCA). The resulting data showed a clear separation of the arrested and control spectra that related to the altered metabolic state of the arrested culture. Metabolites associated with phosphatidylcholine homeostasis, lipid and fatty acid metabolism, and ascorbate formation were found to be present in significant amount in these cultures. Taken together, the results suggested that there was a link between the metabolic alterations and the hyper-productive state, possibly relating to vesicle recycling and secretory functions, and mechanism to counteract against the generation of reactive oxygen species. While the use of metabolic profiling is still in its infancy, its potential to enhance the understanding of physiological processes in mammalian cell lines used for antibody production is certain.

The isolation and identification of a secreted biomarker associated with cell stress in serum-free CHO cell culture.

One approach to improving mammalian culture productivity has been to reduce cell stress and cell death in the bioreactor, thus enhancing productivity through a longer phase of viability. Here we describe the isolation and identification of a biomarker for stress and viability loss in CHO culture. Using SELDI-TOF mass spectrometry to profile the protein component of supernatant culture media we have identified a peak at 7.7 kDa that was associated with loss of viability toward the end of the culture and simulated stress from both toxic metabolite accumulation and nutrient depletion. The relative intensity (signal/noise ratio) of the peak increased rapidly at the onset of dropping viability toward the end of the growth phase. Also, the peak height was seen to increase significantly when cells were grown under conditions emulating ammonia accumulation and glutamine deprivation. The species has been identified as a fragment of Galectin-1 (Gal-1) via MS/MS fingerprinting. We propose that this peak could be utilized as a marker for early onset of stress in cell culture. This work demonstrates the efficacy of SELDI technology to identify biomarkers in mammalian cell culture and highlights its value as a tool for the monitoring and improvement of culture processes.

The effect of Bcl-2, YAMA, and XIAP over-expression on apoptosis and adenovirus production in HEK293 cell line.

Many viruses induce cell death and lysis as part of their replication and dissemination strategy, and in many cases features of apoptosis are observed. Attempts have been made to further increase productivity by prolonging cell survival via the over-expression of anti-apoptotic genes. Here, we extend the study to investigate the association between virus replication and apoptosis, pertinent to large-scale vector production for gene therapy. Infection of an HEK293 cell line with a replication defective type-5-adenovirus expressing a GFP reporter (Ad5GFP) resulted in rapid decline in viability associated with increased virus titer. The over-expression of bcl-2 resulted in improved cell resistance to apoptosis and prolonged culture duration, but reduced virus specific and total productivity. In contrast, the over-expression of pro-caspase-3 (Yama/CPP32/apopain) resulted in reduced cell survival but increased virus productivity. The treatment of infected cells with caspase inhibitors support the preposition that caspase-3 dependent apoptosis, and to a lesser degree caspase-9 dependent apoptosis, represent important steps in virus production, thus implicating the intrinsic apoptosis pathway in the production of adenovirus from HEK293 cells. The suppression of apoptosis by the over-expression of XIAP (inhibitors of caspase family cell death proteases) further shows that caspase-mediated activation plays an important role in virus infection and maturation.