bluuebunny/crossref_metadata_2025_split huggingface dataset:
Green synthesis and physiochemical characterization of nickel oxide nanoparticles: Interaction studies with Calf thymus DNA
Authors: Sarkar Niloy , Sharma Radhey Shyam , Kaushik Mahima 
Published: October 2019

AbstractOne of the most promising applications of nanomaterials is that of nanobiosensors, using biomolecules such as nucleic acids as receptors. This study aimed to synthesize nickel oxide nanoparticles (NiO NPs) by an environmentally friendly green synthesis, using the extract of the herb Coriandrum sativum (coriander). The synthesized NPs were characterized using UV–Visible spectroscopy, Fourier transform infrared spectroscopy, X‐ray diffraction, X‐ray photon spectroscopy, field emission scanning electron microscopy coupled with energy dispersive spectroscopy, dynamic light scattering, zeta potential and transmission electron microscopy. All results confirmed the synthesis of pure, spherical, positively charged NiO NPs of around 95 nm in diameter with prominent hydroxyl groups attached to the surface. Furthermore, interaction studies of synthesized NiO NPs with calf thymus DNA (CT DNA) were performed using UV–Visible spectroscopy, UV–thermal melting, circular dichroism, and fluorescence spectroscopy. CT DNA served as a substitute for nucleic acid biosensors. All experimental studies indicated that the NiO NPs bound electrostatically with CT DNA. These studies may facilitate exploring the potential of NiO NP–nucleic acid conjugated materials to be used as nanobiosensors for various applications, especially in pharmacological, epidemiological, and environmental diagnostic applications, and in detection.
Unraveling what makes a monoclonal antibody difficult‐to‐express: From intracellular accumulation to incomplete folding and degradation via ERAD
Authors: Mathias Sven , Wippermann Anna , Raab Nadja , Zeh Nikolas , Handrick René , Gorr Ingo , Schulz Patrick , Fischer Simon , Gamer Martin , Otte Kerstin 
Published: October 2019

AbstractAlthough most therapeutic monoclonal antibodies (mAbs) can routinely be produced in the multigram per litre range, some mAb candidates turn out to be difficult‐to‐express (DTE). In addition, the class of more complex biological formats is permanently increasing and mammalian expression systems like Chinese hamster ovary (CHO) cell lines can show low performance. Hence, there is an urgent need to identify any rate limiting processing step during cellular synthesis. Therefore, we assessed the intracellular location of the DTE antibody mAb2 by fluorescence and electron microscopy (EM) and revealed an accumulation of the antibody, which led to an aberrant morphology of the endoplasmic reticulum (ER). Analysis of underlying cellular mechanisms revealed that neither aggregation nor antibody assembly, but folding represented the reason for hampered secretion. We identified that the disulfide bridge formation within the antibody light chain (LC) was impaired due to less recognition by protein disulfide isomerase (PDI). As a consequence, the DTE molecule was degraded intracellularly by the ubiquitin proteasome system via ER‐associated degradation (ERAD). This study revealed that with the continuous emergence of DTE therapeutic protein candidates, special attention needs to be drawn to optimization processes to ensure manufacturability.
Raman spectroscopy as a method to replace off‐line pH during mammalian cell culture processes
Authors: Rafferty Carl , O'Mahony Jim , Burgoyne Barbara , Rea Rosemary , Balss Karin M. , Latshaw David C. 
Published: October 2019

AbstractRaman spectroscopy is a robust, well‐established tool utilized for measuring important cell culture process variables for example, feed, metabolites, and biomass in real‐time. This study further expands the functionality of in‐line Raman spectroscopy coupled with partial least squares (PLS) regression modelling to develop a pH measurement tool. Cell line specific models were developed to enhance the robustness for processes with different pH setpoints, deadbands, and cellular metabolism. The modelling strategy further improved robustness by reducing the temporal complexity of pH shifts by splitting data sets into two time zones reflective of major changes in pH. In addition, models were developed to assess if lactate and partial pressure of carbon dioxide (pCO2) could be used in a PLS model for pH. Splitting the data sets into early and late for the process resulted in errors of 0.035 pH and 0.034 pH for the two respective Raman cell lines models which was within acceptance criteria. The lactate and pCO2 PLS model with values provided by Raman models had a further 0.001 pH error reduction. This study illustrates the potential to eliminate off‐line samples to correct for in‐line measurements of pH and further illustrates the capabilities of Raman to measure additional process variables.
Awakening dormant glycosyltransferases in CHO cells with CRISPRa
Authors: Karottki Karen Julie la Cour , Hefzi Hooman , Xiong Kai , Shamie Isaac , Hansen Anders Holmgaard , Li Songyuan , Pedersen Lasse Ebdrup , Li Shangzhong , Lee Jae Seong , Lee Gyun Min , Kildegaard Helene Faustrup , Lewis Nathan E. 
Published: October 2019

AbstractChinese hamster ovary (CHO) cells are the preferred workhorse for the biopharmaceutical industry, and CRISPR/Cas9 has proven powerful for generating targeted gene perturbations in CHO cells. Here, we expand the CRISPR engineering toolbox with CRISPR activation (CRISPRa) to increase transcription of endogenous genes. We successfully increased transcription of Mgat3 and St6gal1, and verified their activity on a functional level by subsequently detecting that the appropriate glycan structures were produced. This study demonstrates that CRISPRa can make targeted alterations of CHO cells for desired phenotypes.
Characterization and mutagenesis of Chinese hamster ovary cells endogenous retroviruses to inactivate viral particle release
Authors: Duroy Pierre‐Olivier , Bosshard Sandra , Schmid‐Siegert Emanuel , Neuenschwander Samuel , Arib Ghislaine , Lemercier Philippe , Masternak Jacqueline , Roesch Lucien , Buron Flavien , Girod Pierre‐Alain , Xenarios Ioannis , Mermod Nicolas 
Published: October 2019

AbstractThe Chinese hamster ovary (CHO) cells used to produce biopharmaceutical proteins are known to contain type‐C endogenous retrovirus (ERV) sequences in their genome and to release retroviral‐like particles. Although evidence for their infectivity is missing, this has raised safety concerns. As the genomic origin of these particles remained unclear, we characterized type‐C ERV elements at the genome, transcriptome, and viral particle RNA levels. We identified 173 type‐C ERV sequences clustering into three functionally conserved groups. Transcripts from one type‐C ERV group were full‐length, with intact open reading frames, and cognate viral genome RNA was loaded into retroviral‐like particles, suggesting that this ERV group may produce functional viruses. CRISPR‐Cas9 genome editing was used to disrupt the gag gene of the expressed type‐C ERV group. Comparison of CRISPR‐derived mutations at the DNA and RNA level led to the identification of a single ERV as the main source of the release of RNA‐loaded viral particles. Clones bearing a Gag loss‐of‐function mutation in this ERV showed a reduction of RNA‐containing viral particle release down to detection limits, without compromising cell growth or therapeutic protein production. Overall, our study provides a strategy to mitigate potential viral particle contaminations resulting from ERVs during biopharmaceutical manufacturing.
Xylose assimilation enhances the production of isobutanol in engineered Saccharomyces cerevisiae
Authors: Lane Stephan , Zhang Yanfei , Yun Eun Ju , Ziolkowski Leah , Zhang Guochang , Jin Yong‐Su , Avalos José L. 
Published: October 2019

AbstractBioconversion of xylose—the second most abundant sugar in nature—into high‐value fuels and chemicals by engineered Saccharomyces cerevisiae has been a long‐term goal of the metabolic engineering community. Although most efforts have heavily focused on the production of ethanol by engineered S. cerevisiae, yields and productivities of ethanol produced from xylose have remained inferior as compared with ethanol produced from glucose. However, this entrenched focus on ethanol has concealed the fact that many aspects of xylose metabolism favor the production of nonethanol products. Through reduced overall metabolic flux, a more respiratory nature of consumption, and evading glucose signaling pathways, the bioconversion of xylose can be more amenable to redirecting flux away from ethanol towards the desired target product. In this report, we show that coupling xylose consumption via the oxidoreductive pathway with a mitochondrially‐targeted isobutanol biosynthesis pathway leads to enhanced product yields and titers as compared to cultures utilizing glucose or galactose as a carbon source. Through the optimization of culture conditions, we achieve 2.6 g/L of isobutanol in the fed‐batch flask and bioreactor fermentations. These results suggest that there may be synergistic benefits of coupling xylose assimilation with the production of nonethanol value‐added products.
Development of a robust nitrilase by fragment swapping and semi‐rational design for efficient biosynthesis of pregabalin precursor
Authors: Zhang Qin , Lu Xia‐Feng , Zhang Yan , Tang Xiao‐Ling , Zheng Ren‐Chao , Zheng Yu‐Guo 
Published: October 2019

AbstractProtein engineering is a powerful tool for improving the properties of enzymes. However, large changes in enzyme properties are still challenging for traditional evolution strategies because they usually require multiple amino acid substitutions. In this study, a feasible evolution approach by a combination of fragment swapping and semi‐rational design was developed for the engineering of nitrilase. A chimera BaNIT harboring 12 amino acid substitutions was obtained using nitrilase from Arabis alpine (AaNIT) and Brassica rapa (BrNIT) as parent enzymes, which exhibited higher enantioselectivity and activity toward isobutylsuccinonitrile for the biosynthesis of pregabalin precursor. The semi‐rational design was executed on BaNIT to further generate variant BaNIT/L223Q/H263D/Q279E with the concurrent improvement of activity, enantioselectivity, and solubility. The robust nitrilase displayed a 5.4‐fold increase in whole‐cell activity and the enantiomeric ratio (E) increased from 180 to higher than 300. Molecular dynamics simulation and molecular docking demonstrated that the substitution of residues on the A and C surface contributed to the conformation alteration of nitrilase, leading to the simultaneous enhancement of enzyme properties. The results obtained not only successfully engineered the nitrilase with great industrial potential for the production of pregabalin precursor, but also provided a new perspective for the development of novel industrially important enzymes.
Engineered Fn3 protein has targeted therapeutic effect on mesothelin‐expressing cancer cells and increases tumor cell sensitivity to chemotherapy
Authors: Sirois Allison R. , Deny Daniela A. , Li Yanxuan , Fall Yacine D. , Moore Sarah J. 
Published: October 2019

AbstractMesothelin is a protein expressed at high levels on the cell surface in a variety of cancers, with limited expression in healthy tissues. The presence of mesothelin on tumor tissue correlates with increased invasion and metastasis, and resistance to traditional chemotherapies, through mechanisms that remain poorly understood. Molecules that specifically recognize mesothelin and interrupt its contribution to tumor progression have significant potential for targeted therapy and targeted drug delivery applications. A number of mesothelin‐targeting therapies are in preclinical and clinical development, although none are currently approved for routine clinical use. In this work, we report the development of a mesothelin‐targeting protein based on the fibronectin type‐III non‐antibody protein scaffold, which offers opportunities for applications where antibodies have limitations. We engineered protein variants that bind mesothelin with high affinity and selectively initiate apoptosis in tumor cells expressing mesothelin. Interestingly, apoptosis does not occur through a caspase‐mediated pathway and does not require downregulation of cell‐surface mesothelin, suggesting a currently unknown pathway through which mesothelin contributes to cancer progression. Importantly, simultaneous treatment with mesothelin‐binding protein and chemotherapeutic mitomycin C had a greater cytotoxic effect on mesothelin‐positive cells compared to either molecule alone, underscoring the potential for combination therapy including biologics targeting mesothelin.
Automatic real‐time calibration, assessment, and maintenance of generic Raman models for online monitoring of cell culture processes
Authors: Tulsyan Aditya , Wang Tony , Schorner Gregg , Khodabandehlou Hamid , Coufal Myra , Undey Cenk 
Published: October 2019

AbstractRaman spectroscopy is a multipurpose analytical technology that has found great utility in real‐time monitoring and control of critical performance parameters of cell culture processes. As a process analytical technology (PAT) tool, the performance of Raman spectroscopy relies on chemometric models that correlate Raman signals to the parameters of interest. The current calibration techniques yield highly specific models that are reliable only on the operating conditions they are calibrated in. Furthermore, once models are calibrated, it is typical for the model performance to degrade over time due to various recipe changes, raw material variability, and process drifts. Maintaining the performance of industrial Raman models is further complicated due to the lack of a systematic approach to assessing the performance of Raman models. In this article, we propose a real‐time just‐in‐time learning (RT‐JITL) framework for automatic calibration, assessment, and maintenance of industrial Raman models. Unlike traditional models, RT‐JITL calibrates generic models that can be reliably deployed in cell culture experiments involving different modalities, cell lines, media compositions, and operating conditions. RT‐JITL is a first fully integrated and fully autonomous platform offering a self‐learning approach for calibrating and maintaining industrial Raman models. The efficacy of RT‐JITL is demonstrated on experimental studies involving real‐time predictions of various cell culture performance parameters, such as metabolite concentrations, viability, and viable cell density. RT‐JITL framework introduces a paradigm shift in the way industrial Raman models are calibrated, assessed, and maintained, which to the best of authors' knowledge, have not been done before.
Massive computational identification of somatic variants in exonic splicing enhancers using The Cancer Genome Atlas
Authors: Tanimoto Kousuke , Muramatsu Tomoki , Inazawa Johji 
Published: October 2019

AbstractOwing to the development of next‐generation sequencing (NGS) technologies, a large number of somatic variants have been identified in various types of cancer. However, the functional significance of most somatic variants remains unknown. Somatic variants that occur in exonic splicing enhancer (ESE) regions are thought to prevent serine and arginine‐rich (SR) proteins from binding to ESE sequence motifs, which leads to exon skipping. We computationally identified somatic variants in ESEs by compiling numerous open‐access datasets from The Cancer Genome Atlas (TCGA). Using somatic variants and RNA‐seq data from 9635 patients across 32 TCGA projects, we identified 646 ESE‐disrupting variants. The false positive rate of our method, estimated using a permutation test, was approximately 1%. Of these ESE‐disrupting variants, approximately 71% were located in the binding motifs of four classical SR proteins. ESE‐disrupting variants occurred in proportion to the number of somatic variants, but not necessarily in the specific genes associated with the biological processes of cancer. Existing bioinformatics tools could not predict the pathogenicity of ESE‐disrupting variants identified in this study, although these variants could cause exon skipping. We demonstrated that ESE‐disrupting nonsense variants tended to escape nonsense‐mediated decay surveillance. Using integrated analyses of open access data, we could specifically identify ESE‐disrupting variants. We have generated a powerful tool, which can handle datasets without normal samples or raw data, and thus contribute to reducing variants of uncertain significance because our statistical approach only uses the exon‐junction read counts from the tumor samples.
Strategies for the Development of Metallic‐Nanoparticle‐Based Label‐Free Biosensors and Their Biomedical Applications
Authors: Kaushal Sandeep , Nanda Sitansu Sekhar , Samal Shashadhar , Yi Dong Kee 
Published: October 2019

AbstractLabel‐free biosensors offer accurate sensing capabilities due to the reliable quantification of biological and biochemical processes. These devices function by establishing a dynamic interaction of analyte and receptor molecules and convert this interaction into a measurable signal through a transducer. In recent decades, label‐free biosensors have attracted attention in biomedical applications due to the ease of linking nanomaterials with bioreceptor molecules. In this review, recent advances in sensitivity, specificity, and sensing mechanism related to label‐free biosensors of metallic nanoparticles of gold, silver, aluminium, copper, and zinc oxide are presented. Selected sensing methods based on fluorescence, surface plasmon resonance, surface‐enhanced Raman scattering, metal‐enhanced fluorescence, and electrochemical sensors are discussed. New measurement techniques and rapid progress of label‐free biosensors are going to play a vital role in the real‐time detection of biomarkers in clinical samples, such as blood plasma, serum, and urine, as well as in targeted drug delivery. Future trends of these label‐free biosensing mechanisms and their development are also discussed.
Cellular senescence and EMT crosstalk in bleomycin‐induced pathogenesis of pulmonary fibrosis—an in vitro analysis
Authors: Muthuramalingam Karthika , Cho Moonjae , Kim Youngmee 
Published: October 2019

AbstractWith poor prognosis and aberrant lung remodeling, pulmonary fibrosis exhibits worldwide prevalence accompanied by an increase in burden in terms of hospitalization and death. Apart from genetic and non‐genetic factors, fibrosis occurs as a side effect of bleomycin antineoplastic activity. Elucidating the cellular and molecular mechanism could help in the development of effective anti‐fibrotic treatment strategies. In the present study, we investigated the underlying mechanism behind bleomycin‐induced fibrosis using human alveolar epithelial cells (A549 cells). On the basis of the experimental observation, it was demonstrated that with transforming growth factor‐β (TGF‐β) as a central mediator of fibrosis progression, a cross‐talk between epithelial–mesenchymal transition (EMT) and senescence upon bleomycin treatment occurs. This results in the advancement of this serious fibrotic condition. Fibrosis was initiated through integrin activation and imbalance in the redox state (NOX expression) of the cell. It progressed along the TGF‐β‐mediated non‐canonical pathway (via ERK phosphorylation) followed by the upregulation of α‐smooth muscle actin and collagen synthesis. Additionally, in this process, the loss of the epithelial marker E‐cadherin was observed. Furthermore, the expressions of senescence markers, such as p21 and p53, were upregulated upon bleomycin treatment, thereby intensifying the fibrotic condition. Accordingly, the molecular pathway mediating the bleomycin‐induced fibrosis was explored in the current study.
Sex‐related difference in the use of percutaneous left ventricular assist device in patients undergoing complex high‐risk percutaneous coronary intervention: Insight from the cVAD registry
Authors: Alraies M Chadi , Kaki Amir , Kajy Marvin , Blank Nimrod , Hasan Reema , Htun Wah Wah , Glazier James J. , Elder Mahir , O'Neill William W. , Grines Cindy L. , Schreiber Theodore 
Published: October 2019

AbstractObjectiveTo assess the in‐hospital and short‐term outcome differences between males and females who underwent high‐risk PCI with mechanical circulatory support (MCS).BackgroundSex differences have been noted in several percutaneous coronary intervention (PCI) series with females less likely to be referred for PCI due increased risk of adverse events. However, data on sex differences in utilization and outcomes of high‐risk PCI with MCS is scarce.MethodsUsing the cVAD Registry, we identified 1,053 high‐risk patients who underwent PCI with MCS using Impella 2.5 or Impella CP. Patients with cardiogenic shock were excluded. A total of 792 (75.21%) males and 261 (24.79%) females were included in the analysis with median follow‐up of 81.5 days.ResultsFemales were more likely to be African American, older (72.05 ± 11.66 vs. 68.87 ± 11.17, p < .001), have a higher prevalence of diabetes (59.30 vs. 49.04%, p = .005), renal insufficiency (35.41 vs. 27.39%, p = .018), and peripheral vascular disease (31.89 vs. 25.39%, p of .05). Women had a higher mean STS score (8.21 ± 8.21 vs. 5.04 ± 5.97, p < .001) and lower cardiac output on presentation (3.64 ± 1.30 vs. 4.63 ± 1.49, p < .001). Although women had more comorbidities, there was no difference in in‐hospital mortality, stroke, MI or need for recurrent revascularization compared to males. Females were more likely to have multivessel revascularization than males. Ejection fraction improved in both males and females at the time of discharge (26.59 to 31.40% and 30.75 to 36.05%, respectively, p < .0001). However, females had higher rate of bleeding requiring transfusion compared with males (9.58 vs. 5.30%, p = .019).ConclusionFemale patients undergoing high PCI were older and had more comorbidities but had similar outcomes compared to males.
Enhanced High‐Temperature Electrochemical Performance of Layered Nickel‐Rich Cathodes for Lithium‐Ion Batteries after LiF Surface Modification
Authors: Huang Jinlong , Du Ke , Peng Zhongdong , Cao Yanbing , Xue Zhichen , Duan Jianguo , Wang Fei , Liu Yong , Hu Guorong 
Published: October 2019

AbstractLayered nickel (Ni)‐rich materials have been widely studied as attractive cathode materials for lithium‐ion batteries, owing to their high theoretical specific capacity and low cost; however, most Ni‐rich materials have poor cycle performance, especially at high temperatures. This study reports a simple sol‐gel method for developing lithium fluoride (LiF)‐coated LiNi0.90Co0.08Al0.02O2 (NCA@LiF) by immersing NCA powder in a 1‐butyl‐2,3‐dimethylimidazolium tetrafluoroborate (BdmimBF4) solution. The residual Li compound on the surface of NCA can be converted into a uniform LiF coating layer through the in situ hydrolysis of BdmimBF4. The Li‐conducting LiF coating layer inhibits side reactions (LiPF6→PF5+LiF, PF5+H2O→POF3+HF, POF3+Li2O→LixPOFy+LiF, Li2O/LiOH+HF→H2O+2LiF) with the electrolyte and protects the electrode from HF corrosion. The NCA@LiF sample shows a high capacity retention rate of 79.9 % after 200 cycles at 1 C and an excellent capacity of 155.7 mAh g−1 at 10 C at room temperature. Additionally, the capacity retention rate of the NCA@LiF sample at high temperatures exhibited a significant improvement in comparison with the NCA sample, reaching 75.7 % after 100 cycles at 60 °C at 1 C. This simple and effective method can be used for improving the electrochemistry stability and high‐temperature performance of other Ni‐based cathode materials.