Chew Kiat Heng

Metabolomics-driven approach for the improvement of Chinese hamster ovary cell growth: Overexpression of malate dehydrogenase II

We have established a liquid chromatography-mass spectrometry based metabolomics platform to identify extracellular metabolites in the medium of recombinant Chinese hamster ovary (CHO) fed-batch reactor cultures. Amongst the extracellular metabolites identified, malate accumulation was the most significant. The contributing factors to malate efflux were found to be the supply of aspartate from the medium, and an enzymatic bottleneck at malate dehydrogenase II (MDH II) in the tricarboxylic acid cycle. Subsequent metabolic engineering to overexpress MDH II in CHO resulted in increases in intracellular ATP and NADH, and up to 1.9-fold improvement in integral viable cell number.

Metabolomics profiling of extracellular metabolites in recombinant Chinese Hamster Ovary fed-batch culture.

A metabolomics-based approach was used to time profile extracellular metabolites in duplicate fed-batch bioreactor cultures of recombinant Chinese Hamster Ovary (CHO) cells producing monoclonal IgG antibody. Culture medium was collected and analysed using a high-performance liquid chromatography (HPLC) system in tandem with an LTQ-Orbitrap mass spectrometer. An in-house software was developed to pre-process the LC/MS data in terms of filtering and peak detection. This was followed by principal component analysis (PCA) to assess variance amongst the samples, and hierarchical clustering to categorize mass peaks by their time profiles. Finally, LC/MS2 experiments using the LTQ-Orbitrap (where standard was available) and SYNAPT HDMS (where standard was unavailable) were performed to confirm the identities of the metabolites. Two groups of identified metabolites were of particular interest; the first consisted of metabolites that began to accumulate when the culture entered stationary phase. The majority of them were amino acid derivatives and they were likely to be derived from the amino acids in the feed media. Examples included acetylphenylalanine and dimethylarginine which are known to be detrimental to cell growth. The second group of metabolites showed a downward trend as the culture progressed. Two of them were medium components--tryptophan and choline, and these became depleted midway into the culture despite the addition of feed media. The findings demonstrated the potential of utilizing metabolomics to guide medium design for fed-batch culture to potentially improve cell growth and product titer.

Metabolomics-based identification of apoptosis-inducing metabolites in recombinant fed-batch CHO culture media.

A liquid chromatography-mass spectrometry (LC-MS) based metabolomics platform was previously established to identify and profile extracellular metabolites in culture media of mammalian cells. This presented an opportunity to isolate novel apoptosis-inducing metabolites accumulating in the media of antibody-producing Chinese hamster ovary (CHO mAb) fed-batch bioreactor cultures. Media from triplicate cultures were collected daily for the metabolomics analysis. Concurrently, cell pellets were obtained for determination of intracellular caspase activity. Metabolite profiles from the LC-MS data were subsequently examined for their degree of correlation with the caspase activity. A panel of extracellular metabolites, the majority of which were nucleotides/nucleosides and amino acid derivatives, exhibited good (R² > 0.8) and reproducible correlation. Some of these metabolites, such as oxidized glutathione, AMP and GMP, were later shown to induce apoptosis when introduced to fresh CHO mAb cultures. Finally, metabolic engineering targets were proposed to potentially counter the harmful effects of these metabolites.

A genome-wide association study of n-3 and n-6 plasma fatty acids in a Singaporean Chinese population

AbstractPolyunsaturated fatty acids (PUFAs) have a major impact on human health. Recent genome-wide association studies (GWAS) have identified several genetic loci that are associated with plasma levels of n-3 and n-6 PUFAs in primarily subjects of European ancestry. However, the relevance of these findings has not been evaluated extensively in other ethnic groups. nThe primary aim of this study was to evaluate for genetic loci associated with n-3 and n-6 PUFAs and to validate the role of recently identified index loci using data from a Singaporean Chinese population. Using a GWAS approach, we evaluated associations with plasma concentrations of three n-3 PUFAs [alphalinolenic acid (ALA), eicosapentaenoic acid and docosahexaenoic acid], four n-6 PUFAs [linoleic acid (LA), gammalinolenic acid, dihomogammalinolenic acid (DGLA) and arachidonic acid], and estimates of delta-5 desaturase and delta-6 desaturase activities among the participants (Nxa0=xa01361) of the Singaporean Chinese Health Study. Our results reveal robust genome-wide associations (p value <5xa0×xa010−8) with ALA, all four n-6 PUFAs, and delta-6 desaturase activity at the FADS1/FADS2 locus. We further replicated the associations between common index variants at the NTAN1/PDXDC1 locus and n-6 PUFAs LA and DGLA, and between the JMJD1C locus and n-6 PUFA LA (p value between 0.0490 and 9.88xa0×xa010−4). These associations were independent of dietary intake of PUFAs. In aggregate, we show that genetic loci that influence plasma concentrations of n-3 and n-6 PUFAs are shared across different ethnic groups.

Carbon nanotube-based labels for highly sensitive colorimetric and aggregation-based visual detection of nucleic acids

A novel carbon nanotube (CNT) derived label capable of dramatic signal amplification of nucleic acid detection and direct visual detection of target hybridization has been developed. Highly sensitive colorimetric detection of human acute lymphocytic leukemia (ALL) related oncogene sequences amplified by the novel CNT-based label was demonstrated. Atomic force microscope (AFM) images confirmed that a monolayer of horseradish peroxidase and detection probe molecules was immobilized along the carboxylated CNT carrier. The resulting CNT labels significantly enhanced the nucleic acid assay sensitivity by at least 1000 times compared to that of conventional labels used in enzyme-linked oligosorbent assay (ELOSA). An excellent detection limit of 1 × 10−12xa0M (60 × 10−18xa0mol in 60xa0µl) and a four-order wide dynamic range of target concentration were achieved. Hybridizations using these labels were coupled to a concentration-dependent formation of visible dark aggregates. Targets can thus be detected simply with visual inspection, eliminating the need for expensive and sophisticated detection systems. The approach holds promise for ultrasensitive and low cost visual inspection and colorimetric nucleic acid detection in point-of-care and early disease diagnostic application.

Visfatin and its genetic variants are associated with obesity‐related morbidities and cardiometabolic risk in severely obese children

Visfatin is an adipokine, associated with obesity and possibly glucose regulation.

Electrochemical detection of leukemia oncogenes using enzyme-loaded carbon nanotube labels

We describe an ultrasensitive electrochemical nucleic acid assay amplified by carbon nanotubes (CNTs)-based labels for the detection of human acute lymphocytic leukemia (ALL)-related p185 BCR-ABL fusion transcript. The carboxylated CNTs were functionalized with horseradish peroxidase (HRP) molecules and target-specific detection probes (DP) via diimide-activated amidation and used to label and amplify the target hybridization signal. The activity of captured HRP was monitored by square-wave voltammetry measuring the electroactive enzymatic product in the presence of 2-aminophenol and hydrogen peroxide substrate solution. The signal-amplified assay achieved a detection limit of 83 fM (5 × 10(-18) mol in 60 μL) targets oligonucleotides and has a 4-order-wide dynamic range of target concentration. The resulting assay allowed robust discrimination between the perfect match and a three-base mismatch sequence. When exposed to the full-length (491 bp) DNA oncogene, the approach demonstrated a detection limit of 1 × 10(-16) mol in 60 μL, corresponding to approximately 33 pg of the target gene. The high sensitivity and specificity of the assay enabled a PCR-free detection of target transcripts in as little as 65 ng of mRNA extracted from positive ALL cell lines SUP-B15 in comparison to those obtained from negative cell line HL-60. The approach enables a simple, low-cost and ultrasensitive electrochemical nucleic acid detection in portable devices, point-of-care and early disease diagnostic applications.

Differences in AMY1 Gene Copy Numbers Derived from Blood, Buccal Cells and Saliva Using Quantitative and Droplet Digital PCR Methods: Flagging the Pitfall.

Introduction The human salivary (AMY1) gene, encoding salivary α-amylase, has variable copy number variants (CNVs) in the human genome. We aimed to determine if real-time quantitative polymerase chain reaction (qPCR) and the more recently available Droplet Digital PCR (ddPCR) can provide a precise quantification of the AMY1 gene copy number in blood, buccal cells and saliva samples derived from the same individual. Methods Seven participants were recruited and DNA was extracted from the blood, buccal cells and saliva samples provided by each participant. Taqman assay real-time qPCR and ddPCR were conducted to quantify AMY1 gene copy numbers. Statistical analysis was carried out to determine the difference in AMY1 gene copy number between the different biological specimens and different assay methods. Results We found significant within-individual difference (p<0.01) in AMY1 gene copy number between different biological samples as determined by qPCR. However, there was no significant within-individual difference in AMY1 gene copy number between different biological samples as determined by ddPCR. We also found that AMY1 gene copy number of blood samples were comparable between qPCR and ddPCR, while there is a significant difference (p<0.01) between AMY1 gene copy numbers measured by qPCR and ddPCR for both buccal swab and saliva samples. Conclusions Despite buccal cells and saliva samples being possible sources of DNA, it is pertinent that ddPCR or a single biological sample, preferably blood sample, be used for determining highly polymorphic gene copy numbers like AMY1, due to the large within-individual variability between different biological samples if real time qPCR is employed.

Functional Characterization of Variants in MC4R Gene Promoter Region Found in Obese Children

CONTEXTnMutations in the MC4R gene are the most common cause of monogenic obesity, and there are few studies on mutations in the promoter region.nnnOBJECTIVEnThe objective of the study was to sequence the promoter region of the MC4R gene in a cohort of obese children to identify rare variants.nnnDESIGN, SETTING, AND PATIENTSnA region 1500 bp upstream of the MC4R gene was sequenced in 267 unrelated local children younger than 10 years, with body weight of at least 150% of ideal. An 891-bp upstream region of the MC4R gene was cloned into a luciferase reporter vector for reporter gene assays.nnnINTERVENTIONSnThere were no interventions.nnnMAIN OUTCOME MEASURESnThe basal transcriptional activity of the MC4R promoter was analyzed in human embryonic kidney 293 cells using reporter gene assays.nnnRESULTSnThree rare variants were detected: c.-803A>G, c.-105C>G, and c.-216C>T. The novel c.-803A>G variant was found in a 9-year-old severely obese Malay boy. This variant was not found in his severely obese mother but was present in his overweight father, who had type 2 diabetes, and also in his normal-weight brother. The novel c.-105C>G variant was found in an obese 9-year-old Malay boy. The c.-216C>T variant was found in an obese Chinese girl with Downs syndrome. The transcriptional activities of the c.-803A>G and c.-105C>G promoters were significantly reduced compared with the wild type but not the c.-216C>T promoter.nnnCONCLUSIONSnWe have described, for the first time, two novel human MC4R gene promoter variants found in obese children that resulted in a decrease in basal transcriptional activity.

Microfluidic chips for viral RNA extraction & detection

Sensing biomolecules at minute quantities demands laborious and skill-laden laboratory protocols for sample preparation and amplification in order to improve signal-to-noise ratio. Nucleic-acid-based detection of viral particles in whole blood requires separation of viral particles from blood cells followed by extraction, amplification, and detection of viral nucleic acids. Here, three microfluidic chips have been independently shown to be capable of performing each critical step. Separation of viral particles involves a flow-through, shear-type microfilter chip that can handle large volume of blood. The remaining chips, although developed for genomic DNA, have been adopted for extraction and amplification of viral RNA. In the extraction chip, protein coatings around viral particles are chemically broken to liberate viral RNA which can reversibly bind to the chip surface under high-salt conditions. Viral RNA can be eluted out with a low-salt buffer after removal of unwanted debris. In the amplification chip, viral RNA is first transcribed into cDNA and then multiplied exponentially in copies by a continuous, isothermal, enzyme-based technique known as Nucleic Acid Sequence-Based Amplification (NASBA). The amplicons are detected on the same chip using DNA probes conjugated with horse radish peroxide (HRP) for colorimetry