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intact cell protein profiling

Veronika Dill, Bernd Hoffmann, Aline Zimmer, Martin Beer, Michael Eschbaumer
BACKGROUND: Suspension culture of BHK cells allows large-scale virus propagation and cost-efficient vaccine production, while the shift to animal-component-free cell culture media without serum is beneficial for the quality and downstream processing of the product. Foot-and-mouth disease virus is still endemic in many parts of the world and high-quality vaccines are essential for the eradication of this highly contagious and economically devastating disease. METHODS: Changes to the viral genome sequence during passaging in an adherent and a suspension cell culture system were compared and the impact of amino acid substitutions on receptor tropism, antigenicity and particle stability was examined...
March 16, 2018: Virology Journal
Yan Weng, Tetsuya Ishino, Annette Sievers, Saswata Talukdar, Jeffrey R Chabot, Amy Tam, Weili Duan, Kelvin Kerns, Eric Sousa, Tao He, Alison Logan, Darwin Lee, Dongmei Li, Yingjiang Zhou, Barbara Bernardo, Alison Joyce, Mania Kavosi, Denise M O'Hara, Tracey Clark, Jie Guo, Craig Giragossian, Mark Stahl, Roberto A Calle, Ron Kriz, Will Somers, Laura Lin
Pharmacological administration of FGF21 analogues has shown robust body weight reduction and lipid profile improvement in both dysmetabolic animal models and metabolic disease patients. Here we report the design, optimization, and characterization of a long acting glyco-variant of FGF21. Using a combination of N-glycan engineering for enhanced protease resistance and improved solubility, Fc fusion for further half-life extension, and a single point mutation for improving manufacturability in Chinese Hamster Ovary cells, we created a novel FGF21 analogue, Fc-FGF21[R19V][N171] or PF-06645849, with substantially improved solubility and stability profile that is compatible with subcutaneous (SC) administration...
March 9, 2018: Scientific Reports
Xiao-Lu Xie, Xue-Jiao Chang, Yan Gao, Dong-Li Li, Ling-Ke Liu, Man-Jun Liu, Ke-Jian Wang, Hai-Peng Liu
Influenza A virus non-structural-1A binding protein (named as Ns1abp) was originally identified as a host protein from human that bound to the viral NS-1 protein. In our previous study, the expression of an Ns1abp-like gene (denoted as CqNs1abp-like gene) was found to be up-regulated in a transcriptome library from the haematopoietic tissue (Hpt) cells of red claw crayfish Cherax quadricarinatus post white spot syndrome virus (WSSV) infection. To elucidate the role of CqNs1abp-like gene involved in WSSV infection, we cloned the CqNs1abp-like gene in which the open reading frame was 2232 bp, encoding 743 amino acids with two typical domains of one BTB (Broad-Complex, Tramtrack and Bric a brac) domain at N-terminal and six Kelch domains at C-terminal...
March 3, 2018: Developmental and Comparative Immunology
Kelei Guo, Xian Lin, Yongtao Li, Wei Qian, Zhong Zou, Huanchun Chen, Hongbo Zhou, Meilin Jin
Non-structural 1 (NS1) protein is a key virulence factor that regulates replication of influenza virus. A recombinant H5N1 virus lacking the eIF4GI-binding domain of NS1 (rNS1-SD30) exhibits significantly lower pathogenicity than H5N1 virus with an intact eIF4GI-binding domain (rNS1-wt). To further investigate this phenomenon, we performed comparative proteomics analyses to profile host proteins in chicken embryo fibroblasts (CEFs) infected with rNS1-wt and rNS1-SD30 viruses. In total, 81 differentially expressed (DE) proteins were identified at 12, 24, and 36 h post-infection...
February 2, 2018: Oncotarget
Bhagyashree Nandakishor Swarge, Winfried Roseboom, Linli Zheng, Wishwas R Abhyankar, Stanley Brul, Chris G Koster, Leo J Koning
PURPOSE: Bacterial endospores, the transmissible forms of pathogenic bacilli and clostridia, are heterogeneous multilayered structures composed of proteins. These proteins protect the spores against variety of stresses, thus helping spore survival, and assist in germination, by interacting with the environment to form vegetative cells. Owing to the complexity, insolubility, and dynamic nature of spore proteins, it has been difficult to obtain their comprehensive protein profiles. EXPERIMENTAL DESIGN: The intact spores of Bacillus subtilis, Bacillus cereus, and Peptoclostridium difficile and their vegetative counterparts were disrupted by bead-beating in 6M urea under reductive conditions...
February 27, 2018: Proteomics. Clinical Applications
Kavita Reginald, Fook Tim Chew
Epitope mapping of Der p 2, a clinically important dust-mite allergen is the first step in designing immunotherapy hypoallergen vaccine candidates. Twenty-one single alanine mutants of Der p 2 were generated and their secondary structure was analysed using circular dichroism spectra. Only one mutant, K96A resulted in a misfolded protein. All mutants were tested for serum IgE reactivity using serum from dust mite allergic individuals by immuno dot-blots. Mutations to five residues, N10, E25, K77, K96 and E102 consistently showed reduced IgE reactions compared to wild-type Der p 2, and therefore these residues constitute the major IgE epitopes of Der p 2...
February 21, 2018: Scientific Reports
Laura Di Francesco, Annalisa Verrico, Italia Anna Asteriti, Paola Rovella, Pietro Cirigliano, Giulia Guarguaglini, Maria Eugenia Schininà, Patrizia Lavia
Karyopherin beta-1/Importin beta-1 is a conserved nuclear transport receptor, acting in protein nuclear import in interphase and as a global regulator of mitosis. These pleiotropic functions reflect its ability to interact with, and regulate, different pathways during the cell cycle, operating as a major effector of the GTPase RAN. Importin beta-1 is overexpressed in cancers characterized by high genetic instability, an observation that highlights the importance of identifying its partners in mitosis. Here we present the first comprehensive profile of importin beta-1 interactors from human mitotic cells...
January 30, 2018: Scientific Reports
Megan Garland, Christopher J Schulze, Ian T Foe, Wouter A van der Linden, Matthew A Child, Matthew Bogyo
Protein palmitoylation is a dynamic post-translational modification (PTM) important for cellular functions such as protein stability, trafficking, localization, and protein-protein interactions. S-palmitoylation occurs via the addition of palmitate to cysteine residues via a thioester linkage, catalyzed by palmitoyl acyl transferases (PATs), with removal of the palmitate catalyzed by acyl protein thioesterases (APTs) and palmitoyl-protein thioesterases (PPTs). Tools that target the regulators of palmitoylation-PATs, APTs and PPTs-will improve understanding of this essential PTM...
2018: PloS One
Tianhua Wang, Kong Meng Hoi, Henning Stöckmann, Corrine Wan, Lyn Chiin Sim, Noor Hayati Bte Kamari, Shi Jie Tay, Ce Huang Poo, Susanto Woen, Yuangsheng Yang, Peiqing Zhang, Pauline M Rudd
Robust plate based antibody glycan analysis platforms are urgently needed for biopharmaceutical development and manufacturing as well as for clinical biomarker research. A 96-well plate based workflow has been developed to analyze both intact IgG antibodies and released N-glycans using an Orbitrap Fusion Mass Spectrometer and an LC/MS method on the Waters UNIFI platform. Here we describe such a workflow including protein A purification, PNGaseF digestion, 2-AB labeling and SPE clean-up. The measured IgG glycan profile is consistent with that obtained from non-plate based method and commercial kit and has the advantage of less hands-on time...
January 17, 2018: Biotechnology Journal
Seong-Ryong Kim, Hye-Jin Yi, Yu Na Lee, Ji Yoon Park, Robert M Hoffman, Teruo Okano, In Kyong Shim, Song Cheol Kim
Post-operative pancreatic fistula (POPF) following pancreatic resection is a life-threatening surgical complication. Cell sheets were prepared and harvested using temperature-responsive culture dishes and transplanted as patches to seal POPF. Two different mesenchymal stem cell (MSC) sheets were compared in terms of the preventative ability for pancreatic leakage in a rat model. Both rat adipose-derived stem cell (rADSC) and bone marrow-derived stem cell (rBMSC) sheets were transplanted. Those rADSC and rBMSC sheets are created without enzymes and thus maintained their cell-cell junctions and adhesion proteins with intact fibronectin on the basal side, as well as characteristics of MSCs...
January 10, 2018: Scientific Reports
Benjamin-Florian Hempel, Maik Damm, Bayram Göçmen, Mert Karis, Mehmet Anıl Oguz, Ayse Nalbantsoy, Roderich D Süssmuth
The Nose-horned Viper (Vipera ammodytes) is one of the most widespread and venomous snakes in Europe, which causes high frequent snakebite accidents. The first comprehensive venom characterization of the regional endemic Transcaucasian Nose-horned Viper (Vipera ammodytes transcaucasiana) and the Transdanubian Sand Viper (Vipera ammodytes montandoni) is reported employing a combination of intact mass profiling and bottom-up proteomics. The bottom-up analysis of both subspecies identified the major snake protein families of viper venoms...
January 1, 2018: Toxins
Ivana Jarić, Jasmina Živanović, Marko Miler, Vladimir Ajdžanović, Duško Blagojević, Nataša Ristić, Verica Milošević, Nataša Nestorović
This study aimed to investigate the effects of soy isoflavones, genistein (GEN) and daidzein, (DAI) on the uterine function in ovary-intact middle-aged rats. GEN and DAI (35mg/kg) were subcutaneously administrated to acyclic (12-month-old) Wistar females, daily, for 4weeks. Control group received either vehicle (olive oil and ethanol, 9:1) or remained intact. We found that GEN and DAI differently affect uterine morphophysiology. GEN significantly increased the uterine wet weight which was associated with hyperplastic changes, revealed by stereological and histomorphometrical analyses...
January 15, 2018: Toxicology and Applied Pharmacology
Nasit Igci, Parisa Sharafi, Duygu Ozel Demiralp, Cemil Ozerk Demiralp, Aysel Yuce, Serap Dokmeci Emre
BACKGROUND: Gaucher disease (GD) is defined as an autosomal recessive disorder resulting from the deficiency of glucocerebrosidase (E.C. Glucocerebrosidase is responsible for the degradation of glucosylceramide into ceramide and glucose. The deficiency of this enzyme results in the accumulation of undegraded glucosylceramide, almost exclusively in macrophages. With Fourier transform infrared (FTIR) spectroscopy, the complete molecular diversity of the samples can be studied comparatively and the amount of the particular materials can be determined...
October 2017: Advances in Clinical and Experimental Medicine: Official Organ Wroclaw Medical University
Masaki Takahashi
High affinity binders targeting specific cell surface proteins are vital for development of basic and applied biosciences. However, despite sustained efforts to generate such binders by chemicals and antibodies, there are still many cell surface proteins that lack high affinity binders. Nucleic acid aptamers have potential as binding molecules for cell surface proteins, because they form distinct structures that have high affinity and specificity for a wide range of targets. Aptamers are isolated from large combinatorial libraries using a unique iterative selection-amplification process known as systematic evolution of ligands by exponential enrichment (SELEX)...
February 2018: Biochimie
Jung Hwa Lee, Xiang Hou, Evangel Kummari, Abdolsamad Borazjani, Mariola J Edelmann, Matthew K Ross
The endocannabinoids (eCBs) are endogenous arachidonoyl-containing lipid mediators with important roles in host defense. Macrophages are first-line defenders of the innate immune system and biosynthesize large amounts of eCBs when activated. The cellular levels of eCBs are controlled by the activities of their biosynthetic enzymes and catabolic enzymes, which include members of the serine hydrolase (SH) superfamily. The physiologic activity of SHs can be assessed in a class-specific way using chemoproteomic activity-based protein profiling (ABPP) methods...
December 1, 2017: Molecular and Cellular Biochemistry
Mya M Hlaing, Bayden R Wood, Don McNaughton, Julian I Rood, Edward M Fox, Mary Ann Augustin
The ability of bacteria to tolerate acid stress plays an important role in their growth and survival. In particular, aciduric bacteria have several survival systems that prevent cell damage from acid stress. In this study, the effect of the bacterial stress induced by pre-adaptation at different pH values on the cellular macromolecules of Lactobacillus plantarum was investigated using Raman spectroscopy and Fourier transform infrared spectroscopy. The expression of key genes was also quantified to provide understanding of the transcriptional response of the cells to lethal acid stress conditions...
October 29, 2017: Applied Microbiology and Biotechnology
Virginie Sabado, Ludovic Vienne, Emi Nagoshi
Circadian behavioral rhythms offer an excellent model to study intricate interactions between the molecular and neuronal mechanisms of behavior. In mammals, pacemaker neurons in the suprachiasmatic nucleus (SCN) generate rhythms cell-autonomously, which are synchronized by the network interactions within the circadian circuit to drive behavioral rhythms. However, whether this principle is universal to circadian systems in animals remains unanswered. Here, we examined the autonomy of the Drosophila circadian clock by monitoring transcriptional and post-transcriptional rhythms of individual clock neurons in dispersed culture with time-lapse microscopy...
2017: Frontiers in Cellular Neuroscience
Ana Karina Morao, Erwann Caillieux, Vincent Colot, François Roudier
Progression of a cell along a differentiation path is characterized by changes in gene expression profiles. Alterations of these transcriptional programs result from cell type-specific transcription factors that act in a dynamic chromatin environment. Understanding the precise contribution of these molecular factors during the differentiation process requires accessing specific cell types within a developing organ. This chapter describes a streamlined and alternative version of INTACT, a method enabling the isolation of specific cell populations by affinity-purification of tagged nuclei and the subsequent analysis of gene expression, transcription factor binding profiles, as well as chromatin state at a genome-wide scale...
2018: Methods in Molecular Biology
George R Young, Sandra N Terry, Lara Manganaro, Alvaro Cuesta-Dominguez, Gintaras Deikus, Dabeiba Bernal-Rubio, Laura Campisi, Ana Fernandez-Sesma, Robert Sebra, Viviana Simon, Lubbertus C F Mulder
Endogenous retroviruses (ERVs) occupy extensive regions of the human genome. Although many of these retroviral elements have lost their ability to replicate, those whose insertion took place more recently, such as the HML-2 group of HERV-K elements, still retain intact open reading frames and the capacity to produce certain viral RNA and/or proteins. Transcription of these ERVs is, however, tightly regulated by dedicated epigenetic control mechanisms. Nonetheless, it has been reported that some pathologic states, such as viral infections and certain cancers, coincide with ERV expression suggesting transcriptional reawakening is possible...
October 18, 2017: Journal of Virology
Kanchan Vaswani, Yong Qin Koh, Fatema B Almughlliq, Hassendrini N Peiris, Murray D Mitchell
Exosomes are nanovesicles that play important roles in intercellular communication as they carry information to target cells. Isolation of high purity exosomes will aid in studying the exosomal cargo and quantity as well as how cell-specific messages are carried. We describe a new method incorporating size exclusion chromatography (SEC) to enrich milk-derived exosomes from extracellular vesicles (EVs). This involved the initial isolation of EVs from bovine milk via milk processing and ultracentrifugation; followed by a new method to enrich exosomes using SEC...
December 2017: Reproductive Biology
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