138 research outputs found

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    Biologically consistent annotation of CHO cell culture metabolomics data

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    Metabolomics represents the effort to understand the role of metabolites in a biological system. Unfortunately, unambiguous metabolite identification represents a major bottleneck in liquid chromatography-mass spectrometry (LC-MS) based untargeted metabolomics. A widely used approach is to search spectral (MS/MS) libraries in reference databases for matching metabolites; however, this approach is limited by incomplete coverage. An alternative approach is to match detected features to candidate chemical structures based on their mass and computationally predicted fragmentation pattern. Both approaches often return too many possible matches; moreover, the results from different annotation tools rarely agree. This presentation describes a novel annotation tool that combines search results from several MS/MS libraries and computational fragmentation tools, and evaluates these results based on the content of a metabolic model. This captures the relevant biological context to determine the most likely identity for a given LC-MS data feature. This workflow, termed Biologically Consistent Annotation (BioCAn), improves on other publicly available annotation tools, achieving superior accuracy and sensitivity, while reducing the false discovery rate. The utility of this tool for investigating metabolic inefficiencies in cell culture processes is demonstrated by identifying novel CHO cell metabolites associated with enhanced or reduced cell growth and monoclonal antibody production. The function of these metabolites was evaluated in shake flask and controlled bioreactor experiments

    Development of an N-1 perfusion process and optimized scale-down models for implementation in a platform CHO cell culture manufacturing process

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    The use of N-1 perfusion, coupled with high-inoculum fed batch in CHO cell culture manufacturing processes, has been shown to increase volumetric productivity and shorten the duration of the fed-batch production phase. Implementation of N-1 perfusion as part of a platform process requires the ability to screen multiple clones and to optimize media and process parameters in a high-throughput manner. We have developed an N-1 perfusion process, along with a series of scale-down models for N-1 perfusion using shake flasks, cell culture tubes, and deep-well plates. Process parameters for scale-down models were optimized to maximize comparability of growth profiles and cell culture performance relative to 5L N-1 perfusion bioreactors. Scale-down models were used to inoculate fed-batch experiments in Ambr15 micro-bioreactors at high seeding density, in order to compare growth and productivity profiles to those observed in 5L bench scale bioreactors. Multiple cell lines derived from different CHO hosts were evaluated in order to verify the robustness of the scale-down models. Results demonstrated that cell growth and viability in the optimized scale-down models were comparable to those observed in 5L N-1 perfusion bioreactors. Furthermore, growth, productivity, and product quality profiles from high-inoculum fed-batch experiments were comparable regardless of inoculum source. Optimized scale down models of N-1 perfusion, coupled with Ambr15 fed-batch production micro-bioreactors, have now been integrated into a high-throughput and robust workflow to enable DOE and screening experiments for clone selection, media development and parameter optimization in a platform N-1 perfusion process for monoclonal antibody manufacturing

    Applying genome scale metabolic models integrated with OMICs technologies for improvemwent of commercial CHO cell culture process

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    Although metabolic flux analysis has been established in microbial fermentation, their application in CHO cell culture is sparse. In general CHO cell culture process development is highly rely on empirical experience with limited cell and metabolite data without good mechanism understanding. The purpose of this research is to apply genome scale metabolic modeling for CHO cell culture process improvement. Recently we found that several medium components had significant impact on mAb production by BMSCHO1, a proprietary cell line (Fig. 1). Some of medium components at a low concentration, though within normal ranges for CHO cell culture, caused the BMSCHO1 crashed. Meanwhile some of the other medium components at a low concentration did not cause cell crash, but significantly decreased productivity. The preliminary genetic test results indicated no change in DNA copy number and southern blot integration profile under different medium conditions. Currently we are investigating both supernatant and cell pellets for metabolomics analysis using NMR and LCMS, and assessing epigenetic characteristics. In addition, transcriptomics data have been analyzed by RNA sequence and RT-PCR. Genome-scale modeling integrated with these OMICS datasets have been built and analyzed. In the presentation, we plan to share the investigation details of commercial cell-line and manufacturing process based on the application of genome scale modeling integrated with OMICS technology. Please click Additional Files below to see the full abstract

    Acidochromic organic photovoltaic integrated device

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    Tremendous efforts have been devoted to boosting the power conversion efficiency (PCE) of organic solar cells (OSCs) via the introduction of cathode interlayers (CILs). However, CIL materials have limited diversity and the development of multifunctional devices is largely neglected. Herein, an acidochromic organic photovoltaic integrated device is firstly proposed by introducing an acid-sensitive stimulating-reaction organic molecule as both the CIL of OSCs and the sensor of monitoring environmental acidity. The oxazolidine unit of acidochromic molecule can form a ring-opening structure after acid treatment, resulting in the remarkable color change with the direct reflection of pH value of ecological environment. The additive-free PM6:Y6 OSCs using the acidochromic molecule as the CIL achieve an excellent PCE of above 15.29 %, which is 47 % higher than that of the control device. The PCE can even maintain above 92 % after treating CIL with various strong acids (pH = 1). Moreover, the color of acidified films and the degraded performance of acidified OSCs can be easily restored by alkaline treatment. The successful application of CIL in other highly efficient photovoltaic systems proves its good universality. This work triggers the promising application of acidochromic molecules in solar cells as CIL with the additional function of recognition of acid environment

    Two C-terminal Sequence Variations Determine Differential Neurotoxicity Between Human and Mouse α-synuclein

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    BACKGROUND: α-Synuclein (aSyn) aggregation is thought to play a central role in neurodegenerative disorders termed synucleinopathies, including Parkinson\u27s disease (PD). Mouse aSyn contains a threonine residue at position 53 that mimics the human familial PD substitution A53T, yet in contrast to A53T patients, mice show no evidence of aSyn neuropathology even after aging. Here, we studied the neurotoxicity of human A53T, mouse aSyn, and various human-mouse chimeras in cellular and in vivo models, as well as their biochemical properties relevant to aSyn pathobiology. METHODS: Primary midbrain cultures transduced with aSyn-encoding adenoviruses were analyzed immunocytochemically to determine relative dopaminergic neuron viability. Brain sections prepared from rats injected intranigrally with aSyn-encoding adeno-associated viruses were analyzed immunohistochemically to determine nigral dopaminergic neuron viability and striatal dopaminergic terminal density. Recombinant aSyn variants were characterized in terms of fibrillization rates by measuring thioflavin T fluorescence, fibril morphologies via electron microscopy and atomic force microscopy, and protein-lipid interactions by monitoring membrane-induced aSyn aggregation and aSyn-mediated vesicle disruption. Statistical tests consisted of ANOVA followed by Tukey\u27s multiple comparisons post hoc test and the Kruskal-Wallis test followed by a Dunn\u27s multiple comparisons test or a two-tailed Mann-Whitney test. RESULTS: Mouse aSyn was less neurotoxic than human aSyn A53T in cell culture and in rat midbrain, and data obtained for the chimeric variants indicated that the human-to-mouse substitutions D121G and N122S were at least partially responsible for this decrease in neurotoxicity. Human aSyn A53T and a chimeric variant with the human residues D and N at positions 121 and 122 (respectively) showed a greater propensity to undergo membrane-induced aggregation and to elicit vesicle disruption. Differences in neurotoxicity among the human, mouse, and chimeric aSyn variants correlated weakly with differences in fibrillization rate or fibril morphology. CONCLUSIONS: Mouse aSyn is less neurotoxic than the human A53T variant as a result of inhibitory effects of two C-terminal amino acid substitutions on membrane-induced aSyn aggregation and aSyn-mediated vesicle permeabilization. Our findings highlight the importance of membrane-induced self-assembly in aSyn neurotoxicity and suggest that inhibiting this process by targeting the C-terminal domain could slow neurodegeneration in PD and other synucleinopathy disorders

    Antifungal mechanisms of the antagonistic bacterium Bacillus mojavensis UTF-33 and its potential as a new biopesticide

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    Biological control has gradually become the dominant means of controlling fungal disease over recent years. In this study, an endophytic strain of UTF-33 was isolated from acid mold (Rumex acetosa L.) leaves. Based on 16S rDNA gene sequence comparison, and biochemical and physiological characteristics, this strain was formally identified as Bacillus mojavensis. Bacillus mojavensis UTF-33 was sensitive to most of the antibiotics tested except neomycin. Moreover, the filtrate fermentation solution of Bacillus mojavensis UTF-33 had a significant inhibitory effect on the growth of rice blast and was used in field evaluation tests, which reduced the infestation of rice blast effectively. Rice treated with filtrate fermentation broth exhibited multiple defense mechanisms in response, including the enhanced expression of disease process-related genes and transcription factor genes, and significantly upregulated the gene expression of titin, salicylic acid pathway-related genes, and H2O2 accumulation, in plants; this may directly or indirectly act as an antagonist to pathogenic infestation. Further analysis revealed that the n-butanol crude extract of Bacillus mojavensis UTF-33 could retard or even inhibit conidial germination and prevent the formation of adherent cells both in vitro and in vivo. In addition, the amplification of functional genes for biocontrol using specific primers showed that Bacillus mojavensis UTF-33 expresses genes that can direct the synthesis of bioA, bmyB, fenB, ituD, srfAA and other substances; this information can help us to determine the extraction direction and purification method for inhibitory substances at a later stage. In conclusion, this is the first study to identify Bacillus mojavensis as a potential agent for the control of rice diseases; this strain, and its bioactive substances, have the potential to be developed as biopesticides

    Probing Specific Interaction Forces Between Human IgG and Rat Anti-Human IgG by Self-Assembled Monolayer and Atomic Force Microscopy

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    Interaction forces between biological molecules such as antigen and antibody play important roles in many biological processes, but probing these forces remains technically challenging. Here, we investigated the specific interaction and unbinding forces between human IgG and rat anti-human IgG using self assembled monolayer (SAM) method for sample preparation and atomic force microscopy (AFM) for interaction force measurement. The specific interaction force between human IgG and rat anti-human IgG was found to be 0.6–1.0 nN, and the force required for unbinding a single pair of human IgG and rat anti-human IgG was calculated to be 144 ± 11 pN. The results are consistent with those reported in the literatures. Therefore, SAM for sample preparation combined with AFM for interaction measurement is a relatively simple, sensitive and reliable technique to probe specific interactions between biological molecules such as antigen and antibody

    Preparation and Characterization of Covalently Binding of Rat Anti-human IgG Monolayer on Thiol-Modified Gold Surface

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    The 16-mercaptohexadecanoic acid (MHA) film and rat anti-human IgG protein monolayer were fabricated on gold substrates using self-assembled monolayer (SAM) method. The surface properties of the bare gold substrate, the MHA film and the protein monolayer were characterized by contact angle measurements, atomic force microscopy (AFM), grazing incidence X-ray diffraction (GIXRD) method and X-ray photoelectron spectroscopy, respectively. The contact angles of the MHA film and the protein monolayer were 18° and 12°, respectively, all being hydrophilic. AFM images show dissimilar topographic nanostructures between different surfaces, and the thickness of the MHA film and the protein monolayer was estimated to be 1.51 and 5.53 nm, respectively. The GIXRD 2θ degrees of the MHA film and the protein monolayer ranged from 0° to 15°, significantly smaller than that of the bare gold surface, but the MHA film and the protein monolayer displayed very different profiles and distributions of their diffraction peaks. Moreover, the spectra of binding energy measured from these different surfaces could be well fitted with either Au4f, S2p or N1s, respectively. Taken together, these results indicate that MHA film and protein monolayer were successfully formed with homogeneous surfaces, and thus demonstrate that the SAM method is a reliable technique for fabricating protein monolayer

    Mathematical statistics algorithm in the bending performance test of corroded reinforced concrete beams under fatigue load

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    Harsh environments such as alternating wet and dry conditions and cyclic loading cause erosion in steel bars, leading to severe damage to structures. Steel bars are also prone to bending under long-term fatigue loads. This paper establishes the bending performance test of corroded reinforced concrete (RC) beam under fatigue load based on the mathematical–statistical algorithm. It investigates the influence of heavy load on the fatigue performance of damaged RC beams, the influence of corrosive environment on the fatigue performance of RC beams, the degree of corrosion of steel bars in concrete beams under fatigue loading, and the distribution of chloride ions. The study results found that the stress ratio has a significant effect on the maximum crack width of the specimen beam. The greater is the stress ratio, the longer the fatigue life. This directly affects the performance of the specimen beam under fatigue loading. For this reason, we should pay special attention to the impact of corrosion on the fatigue performance of a structure during the design of steel bars
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