Cryopreservation of Neurospheres Derived from Human Glioblastoma Multiforme

Cancer stem cells have been shown to initiate and sustain tumor growth. In many instances, clinical material is limited, compounded by a lack of methods to preserve such cells at convenient time points. Although brain tumor-initiating cells grown in a spheroid manner have been shown to maintain their integrity through serial transplantation in immune-compromised animals, practically, it is not always possible to have access to animals of suitable ages to continuously maintain these cells. We therefore explored vitrification as a cryopreservation technique for brain tumor-initiating cells. Tumor neurospheres were derived from five patients with glioblastoma multiforme (GBM). Cryopreservation in 90% serum and 10% dimethyl sulfoxide yielded greatest viability and could be explored in future studies. Vitrification yielded cells that maintained self-renewal and multipotentiality properties. Karyotypic analyses confirmed the presence of GBM hallmarks. Upon implantation into NOD/SCID mice, our vitrified cells reformed glioma masses that could be serially transplanted. Transcriptome analysis showed that the vitrified and nonvitrified samples in either the stem-like or differentiated states clustered together, providing evidence that vitrification does not change the genotype of frozen cells. Upon induction of differentiation, the transcriptomes of vitrified cells associated with the original primary tumors, indicating that tumor stem-like cells are a genetically distinct population from the differentiated mass, underscoring the importance of working with the relevant tumor-initiating population. Our results demonstrate that vitrification of brain tumor-initiating cells preserves the biological phenotype and genetic profiles of the cells. This should facilitate the establishment of a repository of tumor-initiating cells for subsequent experimental designs

Engineering biofilm matrix for efficient biofilm-mediated contaminant removal

The naturally sorptive nature of biofilm matrix is an attractive feature to be exploited for environmental application. The objective of this thesis work is to engineer biofilm matrix into an enhanced biosorbent for efficient biofilm-mediated contaminant removal. Shewanella oneidensis was used as an environmentally relevant model organism for biofilm matrix modification through manipulating an abundant matrix-associated protein, BpfA. Arsenic (As) was the contaminant in focus to demonstrate the aimed enhanced sorption by the engineered biofilm. To understand the role of BpfA in the interaction between S. oneidensis and As, comparisons were performed on the global transcriptomics from the RNA-sequencing of the wild-type (WT) and the BpfA-lacking mutant bpfA. When treated with As, both the WT and the bpfA were shown to upregulate the ars-detoxification system for As tolerance, and the master regulator of metabolic processes, TyrR. Energy-consuming metabolic enzymes and low energy efficient metabolic pathways were downregulated in both. The lack of BpfA resulted in expression loss of genes that are primarily important for managing oxidative stress, protein synthesis and their quality assessment, amino acid and phospholipid biosynthesis, uptake of sulfate and exports of xenobiotics. These losses were observed to be compensated with the expression of megaplasmid-borne genes promoting cellular preservation by regulating the cellular metal content and defense, genetic information, and flux of exogenous genetic materials. With the aim to display As-binding domains in the S. oneidensis biofilm matrix, the chromosomal bpfA was genetically fused to arsR from E. coli, which encodes for a well-characterized As binding protein. The recombinant exhibited poor aggregation under rapid agitation but showed competency in biofilm formation under hydrodynamic conditions. Sorption of As by the engineered biofilms was quantified using packed-bed biofilm reactors and the results showed ~2-fold higher sorption by the recombinant biofilms than the WT biofilms. To overcome the limitation of expressing BpfA-ArsR through the only 1 copy chromosomal bpfA in S. oneidensis, an expression of a higher copy number of bpfA was attempted using vector systems. Due to its high repeats content and to accommodate its insertion into a vector system, the bpfA gene was truncated to encode domains deemed necessary for biofilm formation. Biofilm formation assay revealed the necessity of the RTX domain. Constitutive and high induction of BpfA truncates expression resulted in low amounts of biofilm formation. In contrast, substantial biofilm formed when the BpfA truncates were expressed in an IPTG-inducible vector system. This then allowed a further engineering of it to co-express ArsR, As-specific binding module, at its C-terminal end to be displayed as a component in the biofilm matrix for As sorption. When compared to WT, the As sorption by the biofilms of the vector-expressed BpfA-ArsR recombinants exhibited 3.6- fold more, while the biofilms of the recombinant expressing BpfA-ArsR in the chromosome exhibited 2.4-fold more. Therefore, implicating that vector-expressed BpfA-ArsR can increase As sorption through higher expression of BpfA-ArsR. Taken together, this thesis work reports the practicality of engineering biofilm matrix into a sustainable biological tool for environmental applications. Modifying an extracellular polymeric substance in biofilms can enhance its sorptive nature as shown in this thesis work and may also serve as a strategy to create other functional biofilms.Doctor of Philosoph

Identification of AHR and hypoxia co-regulated immune target genes as potential factors of inflammation-driven oncogenesis

The transcription factors AHR and HIFs dimerize with the common co-activator ARNT during adaptive responses to xenobiotic exposure and oxygen tension. This cross-talk may subsequently modulate inflammation and/or physiological adaptation via immune cellular survival and maturation. Dysregulation of AHR and HIF pathways can cause chronic inflammation leading to oncogenesis, thus we hypothesized that AHR and hypoxia signaling co-operate in potentiating inflammation-driven oncogenesis. To address this, we analyzed AHR-mediated transcriptional responses in three frequently xenobiotic-exposed study models (murine liver, human keratinocytes, human intestinal epithelial cells), especially in the intestine, where AHR and hypoxia signaling constantly co-exist. Although AHR signaling is evolutionary conserved, we find that the AHR mediates diverse responses in a cell type- and species-dependent manner. Concurrent activation with hypoxia signaling in intestinal cells indicate a promotion of chronic inflammation through down-regulation of numerous anti-inflammatory target genes, and up-regulation of pro-inflammatory genes such as PTGS2, GZMB, and MMP1.​Master of Scienc

The Core- and Pan-Genomic Analyses of the Genus Comamonas: From Environmental Adaptation to Potential Virulence

Comamonas is often reported to be one of the major members of microbial communities in various natural and engineered environments. Versatile catabolic capabilities of Comamonas have been studied extensively in the last decade. In contrast, little is known about the ecological roles and adaptation of Comamonas to different environments as well as the virulence of potentially pathogenic Comamonas strains. In this study, we provide genomic insights into the potential ecological roles and virulence of Comamonas by analysing the entire gene set (pangenome) and the genes present in all genomes (core genome) using 34 genomes of 11 different Comamonas species. The analyses revealed that the metabolic pathways enabling Comamonas to acquire energy from various nutrient sources are well conserved. Genes for denitrification and ammonification are abundant in Comamonas, suggesting that Comamonas plays an important role in the nitrogen biogeochemical cycle. They also encode sophisticated redox sensory systems and diverse c-di-GMP controlling systems, allowing them to be able to effectively adjust their biofilm lifestyle to changing environments. The virulence factors in Comamonas were found to be highly species-specific. The conserved strategies used by potentially pathogenic Comamonas for surface adherence, motility control, nutrient acquisition and stress tolerance were also revealed.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore)Published versio

Establishment of plastic-associated microbial community from superworm gut microbiome

Gut microbial communities of plastic-munching worms provide novel insights for the development of plastic-processing biotechnologies. Considering the complexity of worm maintenance and the gut microbial communities, it is challenging to apply the worms directly in plastic processing. Harnessing the power of microbial communities derived from the worm gut microbiomes in vitro may enable a promising bioprocess for plastic degradation. Here, we established stable and reproducible plastic-associated biofilm communities derived from the gut microbiome of a superworm, Zophobas atratus, through a two-stage enrichment process: feeding with plastics (HDPE, PP, and PS) and in vitro incubation of gut microbiomes obtained from the plastic-fed worms. Plastic feeding exhibited marginal influence on bacterial diversity but substantially changed the relative abundance of different bacterial groups, and intriguingly, enriched potential plastic degraders. More prominent shifts of microbial communities were observed during the in vitro incubation of the gut microbiomes. Taxa containing plastic-degrading strains were further enriched, while other taxa represented by lactic acid bacteria were depleted. Additionally, the plastic characterization confirmed the degradation of the incubated plastics by the plastic-associated microbial communities. Community functional inference for both gene abundance and community phenotype suggested that the in vitro incubation enhanced plastic-degrading potential. Deterministic ecological effects, in particular, selection processes, were identified as the main driving force of the observed community shifts. Our findings provide novel insights into plastic-munching-worm-inspired bioprocessing of plastic wastes

Responses of exogenous bacteria to soluble extracellular polymeric substances in wastewater : a mechanistic study and implications on bioaugmentation

Compared with the chemically defined synthetic wastewater (SynWW), real wastewater has been reported to exhibit distinct effects on microbial community development. Whether and how soluble microbial products in real wastewater contribute to different effects of synthetic and real wastewater on the fate of exogenous bacteria remains elusive. In this study, using a model wastewater bacterium Comamonas testosteroni, we first examined the influences of microfiltration filter-sterilized real wastewater (MF-WW) and SynWW on the retention of C. testosteroni in established wastewater flocs during bioaugmentation. In bioreactors fed with MF-WW, augmentation of C. testosteroni to wastewater flocs resulted in a substantially higher abundance of the augmented bacterial cells than those fed with SynWW. To identify the soluble microbial products in MF-WW contributing to the observed differences between bioaugmentation reactors fed with MF-WW and SynWW, we examined the effect of MF-WW and SynWW on the growth, floc formation, and biofilm development of C. testosteroni. When C. testosteroni grew in MF-WW, visible flocs formed within 2 h, which is in contrast to cell growth in SynWW where floc formation was not observed. We further demonstrated that the observed differences were mainly attributed to the high molecular weight fraction of the soluble extracellular polymeric substances (EPS) in MF-WW, in particular, proteins and extracellular DNA. The DLVO analysis suggested that, in the presence of soluble EPS, the bacterial cell surface exhibits an increased hydrophobicity and a diminished energy barrier, leading to irreversible attachment of planktonic cells and floc formation. The RNA-seq based transcriptional analysis revealed that, in the presence of soluble EPS, genes involved in nonessential metabolisms were downregulated while genes coding for Cco (cbb3-type) and Cox (aa3-type) oxidases with different oxygen affinities were upregulated, facilitating bacterial survival in flocs. Taken together, this study reveals the mechanisms underlying the contribution of soluble EPS in real wastewater to the recruitment of exogenous bacteria by microbial aggregates and provides implications to bioaugmentation.Ministry of Education (MOE)National Research Foundation (NRF)We thank Professor Nico Boon for providing strain I2::rfp. We also thank Chee Wai Liew for wastewater sampling, Dr. Daphne Ng for critically reading the manuscript, and Wu Siang Chia for assistance in some experiments. Dr. Yichao Wu thanks the National Natural Science Foundation of China (41807024). Dr. Xin Liu is grateful for the support of the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2017ZT07Z479). This research was supported by the National Research Foundation and MOE Singapore under its Research Centre of Excellence Programme, Singapore Centre for Environmental Life Sciences Engineering (SCELSE) (M4330005.C70), Nanyang Technological University, Singapore

Microbial recycling of lithium-ion batteries: challenges and outlook

The progression of green technologies has driven higher future demands for valuable metals such as lithium, cobalt, nickel, and manganese, hence necessitating the recycling of lithium-containing energy storage systems. Restrategizing conventional metal recycling technologies with sustainable biological approaches can explore the potential to curtail expensive process costs and emissions of hazardous by-products. This article advocates the benefits and persuasion for future studies and applications to exploit current concepts of microbial-based metal recycling technologies as environmentally cleaner options.National Environmental Agency (NEA)National Research Foundation (NRF)Submitted/Accepted versionThis research/project is supported by the National Research Foundation, Singapore, and National Environment Agency, Singapore, under its Closing the Waste Loop Funding Initiative (award no. USSIF- 2018-4)

Id1, inhibitor of differentiation, is a key protein mediating anti-tumor responses of gamma-tocotrienol in breast cancer cells

Gamma-tocotrienol has demonstrated anti-proliferative effect on breast cancer (BCa) cells, but mechanisms involved are largely unknown. This study aimed at deciphering the molecular pathways responsible for its activity. Our results showed that treatment of BCa cells with gamma-tocotrienol resulted in induction of apoptosis as evidenced by activation of pro-caspases, accumulation of sub-G1 cells and DNA fragmentations. Examination of the pro-survival genes revealed that the gamma-tocotrienol-induced cell death was associated with suppression of Id1 and NF-κB through modulation of their upstream regulators (Src, Smad1/5/8, Fak and LOX). Meanwhile, gamma-tocotrienol treatment also resulted in the induction of JNK signaling pathway and inhibition of JNK activity by specific inhibitor partially blocked the effect of gamma-tocotrienol. Furthermore, synergistic effect was observed when cells were co-treated with gamma-tocotrienol and Docetaxel. Interestingly, in cells that treated with gamma-tocotrienol, alpha-tocopherol or β-aminoproprionitrile were found to partially restore Id1 expression. Meanwhile, this restoration of Id1 was found to protect the cells from gamma-tocotrienol induced apoptosis. Consistent outcome was observed in cells ectopically transfected with the Id-1 gene. Our results suggested that the anti-proliferative and chemosensitization effect of gamma-tocotrienol on BCa cells may be mediated through downregulation of Id1 protein. © 2009 Elsevier Ireland Ltd. All rights reserved.link_to_subscribed_fulltex

Relative sensitivity of parkin and other cysteine-containing enzymes to stress-induced solubility alterations

10.1074/jbc.M609466200Journal of Biological Chemistry2821612310-12318JBCH