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Kuo, C.-C.

Publications and source records attributed to Kuo, C.-C..

32 records · Page 2Linked to original sources

DNMT3A knockouts in human iPSCs prevent de novo DNA methylation and reveal growth advantage during hematopoietic differentiation

DNA methyltransferase 3A (DNMT3A) is a frequently mutated gene in many hematological malignancies, indicating that it may be essential for hematopoietic differentiation. Here, we addressed the functional relevance of DNMT3A for differentiation of human induced pluripotent stem cells (iPSCs) by knocking out exon 2, 19, or 23. Exon 19-/- and 23-/- lines revealed absence of almost the entire de novo DNA methylation during mesenchymal and hematopoietic differentiation. Yet, differentiation was only slightly reduced in exon 19-/- and increased in exon 23-/- lines, whereas there was no significant impact on gene expression in hematopoietic progenitors (iHPCs). Notably, DNMT3A-/- iHPCs recapitulate some DNA methylation differences of acute myeloid leukemia with DNMT3A mutations. Furthermore, multicolor genetic barcoding revealed competitive growth advantage of exon 23-/- iHPCs. Our results demonstrate that de novo DNA methylation during hematopoietic differentiation of iPSCs is almost entirely dependent on DNMT3A and exon 23-/- iHPCs even gained growth advantage.

cell biology↗

HIF1α-AS1 is a DNA:DNA:RNA triplex-forming lncRNA interacting with the HUSH complex

DNA:DNA:RNA triplexes that are formed through Hoogsteen base-pairing have been observed in vitro, but the extent to which these interactions occur in cells and how they impact cellular functions remains elusive. Using a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies, we set out to identify functionally important DNA:DNA:RNA triplex-forming long non-coding RNAs (lncRNA) in human endothelial cells. The lncRNA HIF1-AS1 was retrieved as a top hit. Endogenous HIF1-AS1 reduced the expression of numerous genes, including EPH Receptor A2 and Adrenomedullin through DNA:DNA:RNA triplex formation by acting as an adapter for the repressive human silencing hub complex (HUSH). Moreover, the oxygen-sensitive HIF1-AS1 was down-regulated in pulmonary hypertension and loss-of-function approaches not only resulted in gene de-repression but also enhanced angiogenic capacity. As exemplified here with HIF1-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control.

molecular biology↗

Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins

Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify bottlenecks limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant overexpression. Surprisingly, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. While most components of the secretory machinery showed comparable expression levels in both expression hosts, genes with significant expression variation were identified. Among these, ATF4, SRP9, JUN, PDIA3 and HSPA8 were validated as productivity boosters in CHO. Further, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components improving recombinant protein yield in HEK293 and CHO.

systems biology↗

Do pesticide residues have enduring negative effect on macroinvertebrates and vertebrates in fallow rice paddies?

Rice is one of the most important staple food in the world, with irrigated rice paddies largely converted from natural wetlands. The effectiveness of rice fields in help preserve species depends partially on management practices, including the usage of pesticides. However, related studies have focused predominately on the cultivation period, leaving the effects of soil pesticide residues on aquatic invertebrates during the fallow periods little explored; other animals, such as waterbirds, also rely on aquatic invertebrates in flooded fallow fields for their survival. We therefore investigated vertebrates and macroinvertebrates (terrestrial and aquatic) on rice stands and in flooded water during cultivation and fallow periods in organic and conventional rice fields in Taiwan. Association of environmental factors with terrestrial and aquatic organisms was also analyzed. In total, 32,880 individuals of 144 invertebrate families and 381 individuals of 15 vertebrate families were recorded after nine samplings each for six organic and six conventional rice fields. Family richness and abundance of all invertebrates (terrestrial and aquatic) were higher in organic than in conventional fields during the cultivation period, but were similar between the two agricultural practices during the fallow period. Richness and abundance of terrestrial invertebrates in both organic and conventional fields increased with the progression of rice cultivation, so did the differences between the two practices. Richness of aquatic invertebrates was mostly constant across the sampling period, while abundance increased but differences decreased during the fallow period. Richness and abundance of terrestrial invertebrates were positively associated with ambient temperature and height of rice stand. Abundance of aquatic invertebrates were positively associated with pH value and amount of dissolved oxygen but negatively associated with water temperature. Richness and abundance of all vertebrates and each of the constituting groups (fish, amphibian, reptile, bird, and migratory waterbird) were statistically similar between the two practices although abundance of migratory waterbirds in organic fields were two times those in conventional fields during the fallow period. Our study suggested accumulated effects of pesticides on suppressing terrestrial invertebrates during the cultivation period, but diminishing effects of pesticide residues on repressing aquatic invertebrates during the fallow period. This comprehensive study provided a holistic picture on macroinvertebrate and vertebrate fauna, as well as ramifications of pesticide usage, in a representative Southeast Asia rice paddy ecosystem. Further study should compare rice fields with natural wetlands to better assess how to capitalize on agroecosystems for biodiversity conservation.

ecology↗

Commensal rodent habitat expansion enhances arthropod disease vectors on a tropical volcanic island

Release from predators and competitors on volcanic islands can lead to increased body size and population density, as well as expanded habitat usage of introduced animals relative to their mainland counterparts. Such alterations might help the spread of diseases on islands when these exotic animals also carry pathogenic agents, but is rarely investigated. The commensal Asian house rat (Rattus tanezumi) is confined to human residential surroundings in Taiwan mainland but can be observed in the primary forests of the nearby Orchid Island, a tropical volcanic island. Orchid Island is also a hotspot of scrub typhus, a lethal febrile disease transmitted by larval trombiculid mites (chiggers) infective of the rickettsia Orientia tsutsugamushi (OT). We predicted an increase in chigger abundance when rodents, the primary host of chiggers, invade forests from human settlements as soils are largely devoid of in the latter habitat but is necessary for the survival of nymphal and adult mites. A trimonthly rodent survey in ten sites of three habitats (human resident, grassland, and forest) found only R. tanezumi and showed more R. tanezumi and chiggers in forest than in human residential sites. There was a positive association between rodent and chigger abundance, as well between rodent body weight and load of chiggers. Lastly, >95% of chiggers were Leptotrombidium deliense and their OT infection rates were similar among the habitats. Our study demonstrated potentially elevated risks of scrub typhus when the commensal rat is allowed to invade natural habitats on islands. In addition, while the success of invasive species can be ascribed to their parasites being left behind, island invaders might instead attain more parasites when the parasite requires only a single host (e.g. trombiculid mite), is a host generalist (L. deliense), and is transferred from unsuitable to suitable habitats (human settlement on mainland to forest on island).

ecology↗

Restoration of deficient DNA Repair Genes Mitigates Genome Instability and Increases Productivity of Chinese Hamster Ovary Cells

Chinese Hamster Ovary (CHO) cells are the primary host used for manufacturing of therapeutic proteins. However, production instability of high-titer cell lines is a major problem and is associated with genome instability, as chromosomal aberrations reduce transgene copy number and decrease protein titer. We analyzed whole-genome sequencing data from 11 CHO cell lines and found deleterious single-nucleotide polymorphisms (SNPs) in DNA repair genes. Comparison with other mammalian cells confirmed DNA repair is compromised in CHO. Restoration of key DNA repair genes by SNP reversal or expression of intact cDNAs improved DNA repair and genome stability. Moreover, the restoration of LIG4 and XRCC6 in a CHO cell line expressing secreted alkaline phosphatase mitigated transgene copy loss and improved protein titer retention. These results show for the first time that correction of key DNA repair genes yields considerable improvements in stability and protein expression in CHO, and provide new opportunities for cell line development and a more efficient and sustainable production of therapeutic proteins.

cell biology↗

Dysregulation of the secretory pathway connects Alzheimer's disease genetics to aggregate formation

A hallmark of amyloid disorders, such as Alzheimers disease, is aggregation of secreted proteins. However, it is largely unclear how the hundreds of secretory pathway proteins contribute to amyloid formation. We developed a systems biology framework that integrates expression data with protein-protein interaction networks to successfully estimate a tissues fitness for producing specific secreted proteins. Using this framework, we analyzed the fitness of the secretory pathway of various brain regions and cell types for synthesizing the Alzheimers disease-associated amyloid-precursor protein (APP). While none of the key amyloidogenic pathway components were differentially expressed in AD brain, we found the deposition of A{beta} is associated with repressed expression of the secretory pathway components proximal to APP. Concurrently, we detected systemic up-regulation of the secretory pathway components proximal to {beta}- and {gamma}-secretases in AD brains. Our analyses suggest that perturbations from 3 high confidence AD risk genes cascade through the secretory machinery support network for APP and into the endocytosis pathway. Thus, we present a model where amyloidogenesis is associated with dysregulation of dozens of secretory pathway components supporting APP, which could yield novel therapeutic targets for the treatment of AD.

neuroscience↗

In situ detection of protein interactions for recombinant therapeutic enzymes

Despite their therapeutic potential, many protein drugs remain inaccessible to patients since they are difficult to secrete. Each recombinant protein has unique physicochemical properties and requires different machinery for proper folding, assembly, and post-translational modifications (PTMs). Here we aimed to identify the machinery supporting recombinant protein secretion by measuring the protein-protein interaction (PPI) networks of four different recombinant proteins (SERPINA1, SERPINC1, SERPING1 and SeAP) with various PTMs and structural motifs using the proximity-dependent biotin identification (BioID) method. We identified PPIs associated with specific features of the secreted proteins using a Bayesian statistical model, and found proteins involved in protein folding, disulfide bond formation and N-glycosylation were positively correlated with the corresponding features of the four model proteins. Among others, oxidative folding enzymes showed the strongest association with disulfide bond formation, supporting their critical roles in proper folding and maintaining the ER stability. Knockdown of disulfide-isomerase PDIA4, a measured interactor with significance for SERPINC1 but not SERPINA1, led to the decreased secretion of SERPINC1, which relies on its extensive disulfide bonds, compared to SERPINA1, which has no disulfide bonds. Proximity-dependent labeling successfully identified the transient interactions supporting synthesis of secreted recombinant proteins and refined our understanding of key molecular mechanisms of the secretory pathway during recombinant protein production.

synthetic biology↗

New Targeted Approaches for Epigenetic Age Predictions

Aging causes epigenetic modifications, which are utilized as a biomarker for the aging process. While genome-wide DNA methylation profiles enable robust age-predictors by integration of many age-associated CG dinucleotides (CpGs), there are various alternative approaches for targeted measurements at specific CpGs that better support standardized and cost-effective high-throughput analysis. In this study, we utilized 4,650 Illumina BeadChip datasets of blood to select the best suited CpG sites for targeted analysis. DNA methylation analysis at these sites with either pyrosequencing or droplet digital PCR (ddPCR) revealed a high correlation with chronological age. In comparison, bisulfite barcoded amplicon sequencing (BBA-seq) gave slightly lower precision at individual CpGs. However, BBA-seq data revealed that the correlation of methylation levels with age at neighboring CpG sites follows a bell-shaped curve, often accompanied by a CTCF binding site at the peak. We demonstrate that within individual BBA-seq reads the DNA methylation at neighboring CpGs is not coherently modified but reveals a stochastic pattern. Based on this, we have developed an alternative model for epigenetic age predictions based on the binary sequel of methylated and non-methylated sites in individual reads, which reflects heterogeneity in epigenetic aging within a sample. Thus, the stochastic evolution of age-associated DNA methylation patterns, which seems to resemble epigenetic drift, enables epigenetic clocks for individual DNA strands.

genetics↗

Invasive plants facilitated by socioeconomic change shelter vectors of scrub typhus and spotted fever

BackgroundEcological determinants of most emerging vector-borne diseases are little studied, particularly for neglected tropical disease; meanwhile, although socioeconomic change can have significant downstream effect on human risks to vector-borne diseases via a change in land cover, particularly facilitating the invasion of exotic plants, related studies remain very scarce. Scrub typhus and spotted fever are neglected diseases emerging around the globe and are transmitted by chigger mites and ticks, respectively, with small mammals as the primary hosts of both vectors.\n\nMethodology/Principal findingsWe investigated how invasion of Leucaena leucocephala plant after extensive abandonment of farmlands driven by industrialization in Penghu Islands of Taiwan affected abundance of chiggers and ticks by trapping small mammals in three types of habitats (invasion site, agricultural field, human residence) every two months for a year. Invasion sites sheltered more chiggers and ticks than the other two habitats; moreover, both vectors maintained higher abundance in early winter and populations of chiggers were more stable across seasons in invasion sites, suggesting that the invasive sites could be a temporary refuge for both vectors and might help mitigate the negative influence of unfavorable climate. Infective rates of etiologic agents in chiggers and ticks were also higher in invasion sites. Top soil temperature and relative humility were similar across the three habitats, but invasion sites harbored more Rattus losea rat, on which infested chiggers and ticks were more well fed than those from the most commonly trapped species (Suncus murinus shrew), implicating that abundance of superior hosts instead of microclimate, might determine the abundance of both vectors.\n\nConclusions/SignificanceThis study highlights an important but largely neglected issue that socioeconomic change can have unexpected consequence for human health mediated particularly through invasive plants, which could become a hotspot for emerging infectious diseases but usually are very hard to be eradicated. In the future, a more holistic perspective that integrates socioeconomy, land use, exotic species, and human health should be considered to fully understand potential emergence of vector-borne diseases.\n\nAuthor summaryUnderstanding how environmental factors, such as land use change, affect risks to vector-borne diseases helps control and prevent human diseases, but ecological preference of vectors of most neglected diseases remain little investigated. In this study, we found that vectors of scrub typhus (chigger mites) and spotted fever (hard ticks), two emerging neglected diseases, were much more abundant in sites invaded by exotic plants than the other major land cover types in a small island of Taiwan; moreover, populations of chigger mite in invasion sites were more stable across seasons, suggesting that plant invasion sites could be a refuge for disease vectors under unfavorable climate. Higher abundance of chigger mites and ticks was related to higher abundance of a superior rodent host instead of a difference in soil micro-climate. More significantly, these invasive plants are facilitated by extensive abandonment of farmlands driven by industrialization and rural to urban migration, thus demonstrating an important but largely neglected issue that socioeconomic change, when mediated through a change in land cover, can have unexpected downstream effect on emerging neglected tropical diseases.

ecology↗

The human secretome - the proteins secreted from human cells

The proteins secreted by human tissues (the secretome) are important for the basic understanding of human biology, but also for identification of potential targets for future diagnosis and therapy. Here, we present an annotation of all predicted secreted proteins (n=2,623) with information about their spatial distribution in the human body. A high-throughput mammalian cell factory was established to create a resource of recombinant full-length proteins. This resource was used for phenotypic assays involving {beta}-cell dedifferentiation and for development of targeted proteomics assays. A comparison between host cells, including omics analysis, shows that many of the proteins that failed to be generated in CHO cells could be rescued in human HEK293 cells. In conclusion, the human secretome has been mapped and characterized to facilitate further exploration of the human secretome.

molecular biology↗

Combating viral contaminants in CHO cells by engineering STAT1 mediated innate immunity

Viral contamination in biopharmaceutical manufacturing can lead to shortages in the supply of critical therapeutics. To facilitate the protection of bioprocesses, we explored the basis for the susceptibility of CHO cells, the most commonly used cell line in biomanufacturing, to RNA virus infection. Upon infection with certain ssRNA and dsRNA viruses, CHO cells fail to generate a significant interferon (IFN) response. Nonetheless, the downstream machinery for generating IFN responses and its antiviral activity is intact in these cells: treatment of cells with exogenously-added type I IFN or poly I:C prior to infection limited the cytopathic effect from Vesicular stomatitis virus (VSV), Encephalomyocarditis virus (EMCV), and Reovirus-3 virus (Reo-3) in a STAT1-dependent manner. To harness the intrinsic antiviral mechanism, we used RNA-Seq to identify two upstream repressors of STAT1: Gfi1 and Trim24. By knocking out these genes, the engineered CHO cells exhibited increased resistance to the prototype RNA viruses tested. Thus, omics-guided engineering of mammalian cell culture can be deployed to increase safety in biotherapeutic protein production among many other biomedical applications.

bioengineering↗

Increasing consensus of context-specific metabolic models by integrating data-inferred cell functions

Genome-scale metabolic models provide a valuable context for analyzing data from diverse high-throughput experimental techniques. Models can quantify the activities of diverse pathways and cellular functions. Since some metabolic reactions are only catalyzed in specific environments, several algorithms exist that build context-specific models. However, these methods make differing assumptions that influence the content and associated predictive capacity of resulting models, such that model content varies more due to methods used than cell types. Here we overcome this problem with a novel framework for inferring the metabolic functions of a cell before model construction. For this, we curated a list of metabolic tasks and developed a framework to infer the activity of these functionalities from transcriptomic data. We protected the data-inferred tasks during the implementation of diverse context-specific model extraction algorithms for 44 cancer cell lines. We show that the protection of data-inferred metabolic tasks decreases the variability of models across extraction methods. Furthermore, resulting models better capture the actual biological variability across cell lines. This study highlights the potential of using biological knowledge, inferred from omics data, to obtain a better consensus between existing extraction algorithms. It further provides guidelines for the development of the next-generation of data contextualization methods.

systems biology↗

Variants of DNMT3A cause transcript-specific DNA methylation patterns and affect hematopoietic differentiation

The de novo DNA methyltransferase 3A (DNMT3A) plays pivotal roles in hematopoietic differentiation. In this study, we followed the hypothesis that alternative splicing of DNMT3A has characteristic epigenetic and functional sequels. Specific DNMT3A transcripts were either downregulated or overexpressed in human hematopoietic stem and progenitor cells and this resulted in complementary and transcript-specific DNA methylation and gene expression changes. Functional analysis indicated that particularly transcript 2 (coding for DNMT3A2) activates proliferation and induces loss of a primitive immunophenotype, whereas transcript 4 interferes with colony formation of the erythroid lineage. Notably, in acute myeloid leukemia (AML) expression of transcript 2 correlates with its in vitro DNA methylation and gene expression signatures and is associated with overall survival, indicating that DNMT3A variants impact also on malignancies. Our results demonstrate that specific DNMT3A variants have distinct epigenetic and functional impact. Particularly DNMT3A2 triggers hematopoietic differentiation and the corresponding signatures are reflected in AML.

cell biology↗