Victor Vai Tak Wong

A novel normalization method for effective removal of systematic variation in microarray data

Normalization of cDNA and oligonucleotide microarray data has become a standard procedure to offset non-biological differences between two samples for accurate identification of differentially expressed genes. Although there are many normalization techniques available, their ability to accurately remove systematic variation has not been sufficiently evaluated. In this study, we performed experimental validation of various normalization methods in order to assess their ability to accurately offset non-biological differences (systematic variation). The limitations of many existing normalization methods become apparent when there are unbalanced shifts in transcript levels. To overcome this limitation, we have proposed a novel normalization method that uses a matching algorithm for the distribution peaks of the expression log ratio. The robustness and effectiveness of this method was evaluated using both experimental and simulated data.

Sonochemistry and sonoluminescence in microfluidics

One way to focus the diffuse energy of a sound field in a liquid is by acoustically driving bubbles into nonlinear oscillation. A rapid and nearly adiabatic bubble collapse heats up the bubble interior and produces intense concentration of energy that is able to emit light (sonoluminescence) and to trigger chemical reactions (sonochemistry). Such phenomena have been extensively studied in bulk liquid. We present here a realization of sonoluminescence and sonochemistry created from bubbles confined within a narrow channel of polydimethylsiloxane-based microfluidic devices. In the microfluidics channels, the bubbles form a planar/pancake shape. During bubble collapse we find the formation of OH radicals and the emission of light. The chemical reactions are closely confined to gas–liquid interfaces that allow for spatial control of sonochemical reactions in lab-on-a-chip devices. The decay time of the light emitted from the sonochemical reaction is several orders faster than that in the bulk liquid. Multibubble sonoluminescence emission in contrast vanishes immediately as the sound field is stopped.

Creation of cavitation activity in a microfluidic device through acoustically driven capillary waves

We present a study on achieving intense acoustic cavitation generated by ultrasonic vibrations in polydimethylsiloxane (PDMS) based microfluidic devices. The substrate to which the PDMS is bonded was forced into oscillation with a simple piezoelectric transducer attached at 5 mm from the device to a microscopic glass slide. The transducer was operated at 100 kHz with driving voltages ranging between 20 V and 230 V. Close to the glass surface, pressure and vibration amplitudes of up to 20 bar and 400 nm were measured respectively. It is found that this strong forcing leads to the excitation of nonlinear surface waves when gas-liquid interfaces are present in the microfluidic channels. Also, it is observed that nuclei leading to intense inertial cavitation are generated by the entrapment of gas pockets at those interfaces. Subsequently, cavitation bubble clusters with void fractions of more than 50% are recorded with high-speed photography at up to 250,000 frames/s. The cavitation clusters can be sustained through the continuous injection of gas using a T-junction in the microfluidic device.

Elucidation of metabolism in hybridoma cells grown in fed‐batch culture by genome‐scale modeling

Genome‐scale modeling of mouse hybridoma cells producing monoclonal antibodies (mAb) was performed to elucidate their physiological and metabolic states during fed‐batch cell culture. Initially, feed media nutrients were monitored to identify key components among carbon sources and amino acids with significant impact on the desired outcome, for example, cell growth and antibody production. The monitored profiles indicated rapid assimilation of glucose and glutamine during the exponential growth phase. Significant increase in mAb concentration was also observed when glutamine concentration was controlled at 0.5 mM as a feeding strategy. Based on the reconstructed genome‐scale metabolic network of mouse hybridoma cells and fed‐batch profiles, flux analysis was then implemented to investigate the cellular behavior and changes in internal fluxes during the cell culture. The simulated profile of the cell growth was consistent with experimentally measured specific growth rate. The in silico simulation results indicated (i) predominant utilization of glycolytic pathway for ATP production, (ii) importance of pyruvate node in metabolic shifting, and (iii) characteristic pattern in lactate to glucose ratio during the exponential phase. In future, experimental and in silico analyses can serve as a promising approach to identifying optimal feeding strategies and potential cell engineering targets as well as facilitate media optimization for the enhanced production of mAb or recombinant proteins in mammalian cells. Biotechnol. Bioeng. 2009;102: 1494–1504.

Evaluation of insulin-mimetic trace metals as insulin replacements in mammalian cell cultures

Insulin is involved in a number of cellular functions, including the stimulation of cell growth, cell cycle progression and glucose uptake and is a common protein supplement in serum-free mammalian cell culture media. However, several trace metals have previously been reported to exhibit insulin-like effects on specific cell types. As a step towards developing chemically-defined, protein-free media for mammalian cells, we tested the effectiveness of five trace metals (cadmium, nickel, lithium, vanadium and zinc) as a replacement for insulin. Four cell lines of biotechnological relevance were used, including the hybridoma CRL1606, the myeloma NS0, and the Chinese hamster ovary cell lines CHO-IFN and CHO-K1. Zinc was found to be an effective insulin replacement for the hybridoma, myeloma and CHO-K1 cells. Cell growth, cell cycle progression and antibody production was not affected by the substitution. Furthermore, no adaptation procedure was required.

Co-expression of Skp and FkpA chaperones improves cell viability and alters the global expression of stress response genes during scFvD1.3 production

BackgroundThe overexpression of scFv antibody fragments in the periplasmic space of Escherichia coli frequently results in extensive protein misfolding and loss of cell viability. Although protein folding factors such as Skp and FkpA are often exploited to restore the solubility and functionality of recombinant protein products, their exact impact on cellular metabolism during periplasmic antibody fragment expression is not clearly understood. In this study, we expressed the scFvD1.3 antibody fragment in E. coli BL21 and evaluated the overall physiological and global gene expression changes upon Skp or FkpA co-expression.ResultsThe periplasmic expression of scFvD1.3 led to a rapid accumulation of insoluble scFvD1.3 proteins and a decrease in cell viability. The co-expression of Skp and FkpA improved scFvD1.3 solubility and cell viability in a dosage-dependent manner. Through mutagenesis experiments, it was found that only the chaperone activity of FkpA, not the peptidyl-prolyl isomerase (PPIase) activity, is required for the improvement in cell viability. Global gene expression analysis of the scFvD1.3 cells over the chaperone-expressing cells showed a clear up-regulation of genes involved in heat-shock and misfolded protein stress responses. These included genes of the major HSP70 DnaK chaperone family and key proteases belonging to the Clp and Lon protease systems. Other metabolic gene expression trends include: (1) the differential regulation of several energy metabolic genes, (2) down-regulation of the central metabolic TCA cycle and transport genes, and (3) up-regulation of ribosomal genes.ConclusionsThe simultaneous activation of multiple stress related and other metabolic genes may constitute the stress response to protein misfolding in the scFvD1.3 cells. These gene expression information could prove to be valuable for the selection and construction of reporter contructs to monitor the misfolded protein stress response during antibody fragment production.

Cytotoxic antibody fragments for eliminating undifferentiated human embryonic stem cells

Human embryonic stem cells (hESC) possess great potential for applications in regenerative medicine due to their ability to differentiate into any cell type in the body. However, it is crucial to remove residual undifferentiated hESC from the differentiated population to avoid teratoma formation in vivo. The monoclonal antibody, mAb 84, has been shown to bind and kill undifferentiated hESC and is very useful for the elimination of contaminating undifferentiated hESC prior to transplantation. As mAb 84 is an IgM, its large size may impede penetration into embryoid bodies (EB) or cell clumps. To improve penetration, four antibody fragment formats of mAb 84 were engineered and expressed in Escherichia coli: Fab 84, scFv 84, scFv 84-diabody and scFv 84-HTH. All 4 fragments bound specifically to hESC, but only scFv 84-HTH, a single chain variable fragment with a dimerizing helix-turn-helix motif, could recapitulate the cytotoxicity of mAb 84 on multiple hESC lines. The results suggest that multivalency and flexibility between the antigen-binding sites may be essential features required for killing of hESC by mAb 84 and its derivatives. Imaging of EB treated with scFv 84-HTH or mAb 84 showed an even distribution of scFv 84-HTH throughout the EB whereas mAb 84 was localized more to the periphery.

Glutamine or Glucose Starvation in Hybridoma Cultures Induces Death Receptor and Mitochondrial Apoptotic Pathways

Glutamine and glucose are often controlled at low levels in fed-batch strategies to limit ammonia and lactate accumulation and improve productivity of mammalian cell cultures. However, this risks triggering apoptosis if cells are depleted of glutamine or glucose. To examine the apoptosis cascade during glutamine or glucose limitation, the transcriptional profile of FAS, FASL, FADD, FLIP, BAX, p53 and PEG3 in CRL 1606 hybridoma culture was investigated using quantitative real-time PCR. Activities of caspases 2, 3, 8 and 9 were also analyzed. Increase in the activities of the caspases was observed with up-regulation in the expression of FAS (6-8–fold) and PEG3 (2.5-fold), suggesting that the cells experienced apoptotic cell death via both the death receptor and mitochondrial pathways.

Integrative analysis workflow for the structural and functional classification of C-type lectins

BackgroundIt is important to understand the roles of C-type lectins in the immune system due to their ubiquity and diverse range of functions in animal cells. It has been observed that currently confirmed C-type lectins share a highly conserved domain known as the C-type carbohydrate recognition domain (CRD). Using the sequence profile of the CRD, an increasing number of putative C-type lectins have been identified. Hence, it is highly needed to develop a systematic framework that enables us to elucidate their carbohydrate (glycan) recognition function, and discover their physiological and pathological roles.ResultsPresented herein is an integrated workflow for characterizing the sequence and structural features of novel C-type lectins. Our workflow utilizes web-based queries and available software suites to annotate features that can be found on the C-type lectin, given its amino acid sequence. At the same time, it incorporates modeling and analysis of glycans - a major class of ligands that interact with C-type lectins. Thereafter, the results are analyzed together with context-specific knowledge to filter off unlikely predictions. This allows researchers to design their subsequent experiments to confirm the functions of the C-type lectins in a systematic manner.ConclusionsThe efficacy and usefulness of our proposed immunoinformatics workflow was demonstrated by applying our integrated workflow to a novel C-type lectin -CLEC17A - and we report some of its possible functions that warrants further validation through wet-lab experiments.

Production of Functional Soluble Dectin-1 Glycoprotein Using an IRES-Linked Destabilized-Dihydrofolate Reductase Expression Vector

Dectin-1 (CLEC7A) is a C-type lectin receptor that binds to β-glucans found in fungal cell walls to act as a major pattern recognition receptor (PRR). Since β-glucans epitope is not present in human cells, we are of the opinion that Dectin-1 can have therapeutic functions against fungal infections. We thus set out to produce a soluble extracellular domain of murine Dectin-1 (called sDectin-1) in sufficient titers to facilitate such studies in mouse models. Since sDectin-1 has previously been shown to be glycosylated, we chose to produce this protein using Chinese Hamster Ovary (CHO) cells, a mammalian host cell line suitable for the high-titer production of recombinant glycoproteins. To ensure a high titer production of sDectin-1 and minimize the effects of gene fragmentation, we constructed a mammalian expression vector with a PEST-destabilized dhfr amplifiable marker downstream of an attenuated IRES element, which was in turn downstream of the sDectin-1 gene and a CMV IE promoter. Stably transfected and MTX-amplified cell pools were generated using this vector, and maximum sDectin-1 titers of 246 mg/l and 598 mg/l were obtained in shake flask batch culture and bioreactor fed-batch culture respectively. The purified recombinant sDectin-1 was shown to be glycosylated. Protein functionality was also demonstrated by its ability to bind to zymosan particles and to the cell wall of Saccharomyces cerevisiae. We describe for the first time the use of an attenuated IRES-linked PEST-destabilized dhfr amplifiable marker for the production of recombinant proteins with stably amplified cell pools. With our process, we reached the highest reported titer for producing recombinant proteins smaller than 50 kDa in cell pools. sDectin-1 protein produced is glycosylated and functional. This vector design can thus be used efficiently for the high-titer production of functional recombinant proteins.