54 research outputs found
Mammalian systems biotechnology: An integrative framework for combining in silico modeling and multi-Omics datasets in different CHO parental cell lines
The increasing availability of multi-omics data from Chinese hamster ovary (CHO) cell cultures entails both opportunity and challenges toward next generation cell culture engineering. Herein, we present a comprehensive and integrative framework to systematically combine trancriptome, proteome, metabolome and glycome datasets in conjunction with a genome-scale metabolic model of CHO cells. We then apply the framework to compare and contrast the metabolic characteristics of the three commonly used parental cell lines (CHO-K1, CHO-DUKXB11 and CHO-DG44) so that “global” attributes of the parental hosts (e.g. growth related characteristics, glycosylation patterns, etc.) could be highlighted (Figure 1).
The unique characteristics of the adherent against the suspension cell lines reveal that the latter are in an oxidative stress and that they differentially express genes/proteins associated with the lipid biosynthetic process. The unique transcriptomic and proteomic signatures of the different suspension cell lines, more relevant in an industrial context than the adherent, reflect the known historic divergence of the cell lines, i.e. the very different nature of the -DG44 cell line than the other two. Genes/proteins related with the purine nucleotide biosynthetic process (as expected, due to the Dhfr gene copy number differences), epigenetic regulation and programmed cell death present the major expression differences between the three parental cell lines. As far as the host N-glycome for each of the cell lines is concerned, it reveals similar profiles. Nevertheless, the cell lines present several differences in the expression of N-glycosylation related genes (e.g.Man2a1 and Fut8 are differentially expressed for -DG44 and Mgat4a for the -DXB11 cell line) and the pools of nucleotide sugar donors (-K1 presents higher UDP-Glc / UDP-Gal and CMP-sialic acid pools than -DG44; while -DG44 higher GDP-Fuc pools). Growth profiles of the various cell lines were also assessed and our results demonstrate that -K1 cells present significantly higher growth rate than the other two cell lines in suspension culture. Interestingly, adherent cells present a significantly faster growth profile than suspension cells that we attribute to the different media used for the two culture formats, i.e. to the presence of serum for adherent cells.
The integrative framework also involves the use of the genome-scale metabolic model as a scaffold to map the multiomics datasets. Such an analysis allows us to readily pinpoint the heterogeneity in cellular metabolism between the multiple conditions and/or cell lines tested, as well as their correlations. Moreover, the correlation analysis of transcriptome and proteome for a given cell line revealed the plausible regulatory intracellular events that can be targeted for genetic engineering to achieve the enhanced productivity and quality of recombinant proteins in the context of bioprocessing. Interestingly, we identified many differences in the reactions associated with the N-glycan processing pathways for the various parental cell lines analyzed, which may be associated with different glycosylation capacity. Further investigation at the glycomics level may validate our hypothesis that choice of CHO hosts should be product-specific. It is expected that our results can serve as the golden standard for the comprehensive comparison of the various CHO cell lines used worldwide
A synbiotic intervention modulates meta-omics signatures of gut redox potential and acidity in elective caesarean born infants.
Background
The compromised gut microbiome that results from C-section birth has been hypothesized as a risk factor for the development of non-communicable diseases (NCD). In a double-blind randomized controlled study, 153 infants born by elective C-section received an infant formula supplemented with either synbiotic, prebiotics, or unsupplemented from birth until 4 months old. Vaginally born infants were included as a reference group. Stool samples were collected from day 3 till week 22. Multi-omics were deployed to investigate the impact of mode of delivery and nutrition on the development of the infant gut microbiome, and uncover putative biological mechanisms underlying the role of a compromised microbiome as a risk factor for NCD.
Results
As early as day 3, infants born vaginally presented a hypoxic and acidic gut environment characterized by an enrichment of strict anaerobes (Bifidobacteriaceae). Infants born by C-section presented the hallmark of a compromised microbiome driven by an enrichment of Enterobacteriaceae. This was associated with meta-omics signatures characteristic of a microbiome adapted to a more oxygen-rich gut environment, enriched with genes associated with reactive oxygen species metabolism and lipopolysaccharide biosynthesis, and depleted in genes involved in the metabolism of milk carbohydrates. The synbiotic formula modulated expression of microbial genes involved in (oligo)saccharide metabolism, which emulates the eco-physiological gut environment observed in vaginally born infants. The resulting hypoxic and acidic milieu prevented the establishment of a compromised microbiome.
Conclusions
This study deciphers the putative functional hallmarks of a compromised microbiome acquired during C-section birth, and the impact of nutrition that may counteract disturbed microbiome development.
Trial registration
The study was registered in the Dutch Trial Register (Number: 2838 ) on 4th April 2011
A new gigantopterid genus from the late Permian of the Daha Coalfield, Tibetan Plateau and its implication on plant-insect interactions
An Internal Ribosome Entry Site (IRES) Mutant Library for Tuning Expression Level of Multiple Genes in Mammalian Cells
International audienceA set of mutated Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) elements with varying strengths is generated by mutating the translation initiation codons of 10 th , 11 th , and 12 th AUG to non-AUG triplets. They are able to control the relative expression of multiple genes over a wide range in mammalian cells in both transient and stable transfections. The relative strength of each IRES mutant remains similar in different mammalian cell lines and is not gene specific. The expressed proteins have correct molecular weights. Optimization of light chain over heavy chain expression by these IRES mutants enhances monoclonal antibody expression level and quality in stable transfections. Uses of this set of IRES mutants can be extended to other applications such as synthetic biology, investigating interactions between proteins and its complexes, cell engineering, multi-subunit protein production, gene therapy, and reprogramming of somatic cells into stem cells
An RpoN-dependent PEP-CTERM gene is involved in floc formation of an Aquincola tertiaricarbonis strain
Abstract Background The floc is a characteristic of microbial aggregate growth, displaying cloudy suspensions in water. Floc formation has been demonstrated in a series of bacteria and the floc-forming bacteria play a crucial role in activated sludge (AS) process widely used for municipal sewage and industrial wastewater treatment over a century. It has been demonstrated that some exopolysaccharide biosynthesis genes and the sigma factor (sigma54 or rpoN) were required for floc forming in some bacteria. However, the mechanism underlying the floc formation stills need to be elucidated. Results In this study, we demonstrate that a TPR (Tetratricopeptide repeats) protein-encoding gene prsT is required for floc formation of Aquincola tertiaricarbonis RN12 and an upstream PEP-CTERM gene (designated pepA), regulated by RpoN1, is involved in its floc formation but not swarming motility and biofilm formation. Overexpression of PepA could rescue the floc-forming phenotype of the rpoN1 mutant by decreasing the released soluble exopolysaccharides and increasing the bound polymers. Conclusion Our results indicate that the wide-spread PEP-CTERM proteins play an important role in the self-flocculation of bacterial cells and may be a component of extracellular polymeric substances required for floc-formation
Cottonseed Meal Protein Isolate as a New Source of Alternative Proteins: A Proteomics Perspective
Cottonseed meal (CSM) is a good source of dietary proteins but is unsuitable for human consumption due to its gossypol content. To unlock its potential, we developed a protein extraction process with a gossypol removal treatment to generate CSM protein isolate (CSMPI) with ultra-low gossypol content. This process successfully reduced the free and total gossypol content to 4.8 ppm and 147.2 ppm, respectively, far below the US FDA limit. In addition, the functional characterisation of CSMPI revealed a better oil absorption capacity and water solubility than pea protein isolate. Proteome profiling showed that the treatment improved protein identification, while SDS-PAGE analysis indicated that the treatment did not induce protein degradation. Amino acid analysis revealed that post-treated CSMPI was rich in branched-chain amino acids (BCAAs). Mass spectrometry analysis of various protein fractions obtained from an in vitro digestibility assay helped to establish the digestibility profile of CSM proteins. Several potential allergens in CSMPI were also found using allergenic prediction software, but further evaluation based on their digestibility profiles and literature reviews suggests that the likelihood of CSMPI allergenicity remains low. Overall, our results help to navigate and direct the application of CSMPIs as alternative proteins toward nutritive human food application
RING1B O-GlcNAcylation regulates gene targeting of polycomb repressive complex 1 in human embryonic stem cells
International audienceO-linked-N-acetylglucosamine (O-GlcNAc) post-translationally modifies and regulates thousands of proteins involved in various cellular mechanisms. Recently, O-GlcNAc has been linked to human embryonic stem cells (hESC) differentiation, however the identity and function of O-GlcNAc proteins regulating hESC remain unknown. Here, we firstly identified O-GlcNAc modified human stem cell regulators such as hnRNP K, HP1Îł, and especially RING1B/RNF2. Thereafter, we focused our work on RING1B which is the catalytic subunit of the polycomb repressive complex 1 (PRC1) a major epigenetic repressor essential for pluripotency maintenance and differentiation. By point-mutation, we show that T 250 /S 251 and S 278 RING1B residues are bearing O-GlcNAc, and that T 250 /S 251 O-GlcNAcylation decreases during differentiation. O-GlcNAc seems to regulate RING1B-DNA binding as suggested by our ChIP-sequencing results. Non-O-GlcNAcylated RING1B is found to be enriched near cell cycle genes whereas O-GlcNAcylated RING1B seems preferentially enriched near neuronal genes. Our data suggest that during hESC differentiation, the decrease of RING1B O-GlcNAcylation might enable PRC1 to switch its target to induce neuro
Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells
Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing.ASTAR (Agency for Sci., Tech. and Research, S’pore)Accepted versio
Multiple Reaction Monitoring Mass Spectrometry for the Discovery and Quantification of O‑GlcNAc-Modified Proteins
O-linked <i>N</i>-acetylglucosamine
(O-GlcNAc) is a post-translational
modification regulating proteins involved in a variety of cellular
processes and diseases. Unfortunately, O-GlcNAc remains challenging
to detect and quantify by shotgun mass spectrometry (MS) where it
is time-consuming and tedious. Here, we investigate the potential
of Multiple Reaction Monitoring Mass Spectrometry (MRM-MS), a targeted
MS method, to detect and quantify native O-GlcNAc modified peptides
without extensive labeling and enrichment. We report the ability of
MRM-MS to detect a standard O-GlcNAcylated peptide and show that the
method is robust to quantify the amount of O-GlcNAcylated peptide
with a method detection limit of 3 fmol. In addition, when diluted
by 100-fold in a trypsin-digested whole cell lysate, the O-GlcNAcylated
peptide remains detectable. Next, we apply this strategy to study
glycogen synthase kinase-3 beta (GSK-3β), a kinase able to compete
with O-GlcNAc transferase and modify identical site on proteins. We
demonstrate that GSK-3β is itself modified by O-GlcNAc in human
embryonic stem cells (hESC). Indeed, by only using gel electrophoresis
to grossly enrich GSK-3β from whole cell lysate, we discover
by MRM-MS a novel O-GlcNAcylated GSK-3β peptide, bearing 3 potential
O-GlcNAcylation sites. We confirm our finding by quantifying the increase
of O-GlcNAcylation, following hESC treatment with an O-GlcNAc hydrolase
inhibitor. This novel O-GlcNAcylation could potentially be involved
in an autoinhibition mechanism. To the best of our knowledge, this
is the first report utilizing MRM-MS to detect native O-GlcNAc modified
peptides. This could potentially facilitate rapid discovery and quantification
of new O-GlcNAcylated peptides/proteins
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