scholarly journals Genetic and Process Engineering Strategies for Enhanced Recombinant N-glycoprotein Production in Bacteria

2020 ◽  
Author(s):  
Fenryco Pratama ◽  
Dennis Linton ◽  
Neil Dixon
Keyword(s):  
Protein Translocation ◽  
Scale Up ◽  
Strain Engineering ◽  
Process Engineering ◽  
Target Protein ◽  
Knock Out ◽  
Isomerase Activity ◽  
Secretion Signal ◽  
Secretion Pathways ◽  
The Impact

Abstract BackgroundThe production of N-linked glycoproteins in genetically amenable bacterial hosts offers great potential for reduced cost, faster/simpler bioprocesses, greater customisation and utility for distributed manufacturing of glycoconjugate vaccines and glycoprotein therapeutics. Efforts to optimize production hosts have included heterologous expression of glycosylation enzymes, metabolic engineering, use of alternative secretion pathways, and attenuation of gene expression. However, a major bottleneck to enhance glycosylation efficiency, which limits the utility of the other improvements is the impact of target protein sequon accessibility during glycosylation.ResultsHere, we explore a series genetic and process engineering strategies to increase recombinant N-linked glycosylation mediated by the Campylobacter-derived PglB oligosaccharyltransferase in Escherichia coli. Strategies include increasing membrane residency time of the target protein by modifying the cleavage site of its secretion signal, and modulating protein folding in the periplasm by use of oxygen limitation or strains with compromised oxidoreductases or disulphide-bond isomerase activity. These approaches could achieve up to 90% improvement in glycosylation efficiency. Furthermore, we also demonstrated that supplementation with the chemical oxidant cystine enhanced glycoprotein production and improved cell fitness in the oxidoreductase knock out strain.ConclusionsIn this study, we demonstrated that improved glycosylation in the heterologous host could be achieved by mimicking the coordination between protein translocation, folding and glycosylation observed in native such as Campylobacter jejuni and mammalian hosts. Furthermore, it provides insight into strain engineering and bioprocess strategy, to improve glycoprotein yield and to avoid physiological burden of unfolded protein stress to cell growth. The process and genetic strategies identified herein will inform further optimisation and scale-up of heterologous recombinant N-glycoprotein production

scholarly journals Genetic and process engineering strategies for enhanced recombinant N-glycoprotein production in bacteria

2020 ◽  
Author(s):  
Fenryco Pratama ◽  
Dennis Linton ◽  
Neil Dixon
Keyword(s):  
Protein Translocation ◽  
Scale Up ◽  
Strain Engineering ◽  
Process Engineering ◽  
Target Protein ◽  
Knock Out ◽  
Isomerase Activity ◽  
Secretion Signal ◽  
Secretion Pathways ◽  
The Impact

BackgroundThe production of N-linked glycoproteins in genetically amenable bacterial hosts offers great potential for reduced cost, faster/simpler bioprocesses, greater customisation and utility for distributed manufacturing of glycoconjugate vaccines and glycoprotein therapeutics. Efforts to optimize production hosts have included heterologous expression of glycosylation enzymes, metabolic engineering, use of alternative secretion pathways, and attenuation of gene expression. However, a major bottleneck to enhance glycosylation efficiency, which limits the utility of the other improvements is the impact of target protein sequon accessibility during glycosylation.ResultsHere, we explore a series genetic and process engineering strategies to increase recombinant N-linked glycosylation mediated by the Campylobacter-derived PglB oligosaccharyltransferase in Escherichia coli. Strategies include increasing membrane residency time of the target protein by modifying the cleavage site of its secretion signal, and modulating protein folding in the periplasm by use of oxygen limitation or strains with compromised oxidoreductases or disulphide-bond isomerase activity. These approaches could achieve up to 90% improvement in glycosylation efficiency. Furthermore, we also demonstrated that supplementation with the chemical oxidant cystine enhanced glycoprotein production and improved cell fitness in the oxidoreductase knock out strain.ConclusionsIn this study, we demonstrated that improved glycosylation in the heterologous host could be achieved by mimicking the coordination between protein translocation, folding and glycosylation observed in native such as Campylobacter jejuni and mammalian hosts. Furthermore, it provides insight into strain engineering and bioprocess strategy, to improve glycoprotein yield and to avoid physiological burden of unfolded protein stress to cell growth. The process and genetic strategies identified herein will inform further optimisation and scale-up of heterologous recombinant N-glycoprotein production

scholarly journals Genetic and process engineering strategies for enhanced recombinant N-glycoprotein production in bacteria

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Fenryco Pratama ◽  
Dennis Linton ◽  
Neil Dixon
Keyword(s):  
Mammalian Cells ◽  
Protein Translocation ◽  
Scale Up ◽  
Strain Engineering ◽  
Process Engineering ◽  
Protein Glycosylation ◽  
Target Protein ◽  
Isomerase Activity ◽  
Secretion Signal ◽  
The Impact

Abstract Background The production of N-linked glycoproteins in genetically amenable bacterial hosts offers great potential for reduced cost, faster/simpler bioprocesses, greater customisation, and utility for distributed manufacturing of glycoconjugate vaccines and glycoprotein therapeutics. Efforts to optimize production hosts have included heterologous expression of glycosylation enzymes, metabolic engineering, use of alternative secretion pathways, and attenuation of gene expression. However, a major bottleneck to enhance glycosylation efficiency, which limits the utility of the other improvements, is the impact of target protein sequon accessibility during glycosylation. Results Here, we explore a series of genetic and process engineering strategies to increase recombinant N-linked glycosylation, mediated by the Campylobacter-derived PglB oligosaccharyltransferase in Escherichia coli. Strategies include increasing membrane residency time of the target protein by modifying the cleavage site of its secretion signal, and modulating protein folding in the periplasm by use of oxygen limitation or strains with compromised oxidoreductase or disulphide-bond isomerase activity. These approaches achieve up to twofold improvement in glycosylation efficiency. Furthermore, we also demonstrate that supplementation with the chemical oxidant cystine enhances the titre of glycoprotein in an oxidoreductase knockout strain by improving total protein production and cell fitness, while at the same time maintaining higher levels of glycosylation efficiency. Conclusions In this study, we demonstrate that improved protein glycosylation in the heterologous host could be achieved by mimicking the coordination between protein translocation, folding and glycosylation observed in native host such as Campylobacter jejuni and mammalian cells. Furthermore, it provides insight into strain engineering and bioprocess strategies, to improve glycoprotein yield and titre, and to avoid physiological burden of unfolded protein stress upon cell growth. The process and genetic strategies identified herein will inform further optimisation and scale-up of heterologous recombinant N-glycoprotein production.

128 Development of an M1-polarized, non-viral chimeric antigen receptor macrophage (CAR-M) platform for cancer immunotherapy

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A141-A141
Author(s):  
Yumi Ohtani ◽  
Kayleigh Ross ◽  
Aditya Dandekar ◽  
Rashid Gabbasov ◽  
Michael Klichinsky
Keyword(s):  
Cancer Immunotherapy ◽  
Chimeric Antigen Receptor ◽  
Scale Up ◽  
Antigen Receptor ◽  
Innate Immune ◽  
List Type ◽  
M1 Phenotype ◽  
The Impact ◽  
Anti Tumor Activity ◽  
Immunosuppressive Factors

BackgroundWe have previously developed CAR-M as a novel cell therapy approach for the treatment of solid tumors.1 CAR-M have the potential to overcome key challenges that cell therapies face in the solid tumor setting – tumor infiltration, immunosuppression, lymphocyte exclusion – and can induce epitope spreading to overcome target antigen heterogeneity. While macrophages transduced with the adenoviral vector Ad5f35 (Ad CAR-M) traffic to tumors, provide robust anti-tumor activity, and recruit and activate T cells, we sought to identify a robust non-viral method of macrophage engineering in order to reduce the cost of goods, manufacturing complexity, and potential immunogenicity associated with viral vectors.MethodsAs innate immune cells, macrophages detect exogenous nucleic acids and respond with inflammatory and apoptotic programs. Thus, we sought to identify a means of mRNA delivery that avoids recognition by innate immune sensors. We screened a broad panel of mRNA encoding an anti-HER2 CAR comprising multiplexed 5’Cap and base modifications using an optimized and scalable electroporation approach and evaluated the impact of interferon-β priming on CAR-M phenotype and function.ResultsWe identified the optimal multiplexed mRNA modifications that led to maximal macrophage viability, transfection efficiency, intensity of CAR expression, and duration of expression. Non-viral HER2 CAR-M phagocytosed and killed human HER2+ tumor cells. Unlike Ad CAR-M, mRNA CAR-M were not skewed toward an M1 state by mRNA electroporation. Priming non-viral CAR-M with IFN-β induced a durable M1 phenotype, as shown by stable upregulation of numerous M1 markers and pathways. IFN-β priming significantly enhanced the anti-tumor activity of CAR but not control macrophages. IFN-β primed mRNA CAR-M were resistant to M2 conversion, maintaining an M1 phenotype despite challenge with various immunosuppressive factors, and converted bystander M2 macrophages toward M1. Interestingly, priming mRNA CAR-M with IFN-β significantly enhanced the persistence of CAR expression, overcoming the known issue of rapid mRNA turnover. RNA-seq analysis revealed that IFN-β priming affected pathways involved in increasing translation and decreasing RNA degradation in human macrophages.ConclusionsWe have established a novel, optimized non-viral CAR-M platform based on chemically modified mRNA and IFN-β priming. IFN-β priming induced a durable M1 phenotype, improved CAR expression, improved CAR persistence, led to enhanced anti-tumor function, and rendered resistance to immunosuppressive factors. This novel platform is amenable to scale-up, GMP manufacturing, and represents an advance in the development of CAR-M.ReferenceKlichinsky M, Ruella M, Shestova O, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol 2020;38(8):947–953.

scholarly journals Developmental up-regulation of NMDA receptors in the prefrontal cortex and hippocampus of mGlu5 receptor knock-out mice

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Tiziana Imbriglio ◽  
Remy Verhaeghe ◽  
Nico Antenucci ◽  
Stefania Maccari ◽  
Giuseppe Battaglia ◽  
...  
Keyword(s):  
Prefrontal Cortex ◽  
Nmda Receptor ◽  
Metabotropic Glutamate Receptors ◽  
Adult Life ◽  
Developmental Time ◽  
Postnatal Life ◽  
Developmental Studies ◽  
Knock Out ◽  
Nmda Receptor Subunits ◽  
The Impact

AbstractmGlu5 metabotropic glutamate receptors are highly expressed and functional in the early postnatal life, and are known to positively modulate NMDA receptor function. Here, we examined the expression of NMDA receptor subunits and interneuron-related genes in the prefrontal cortex and hippocampus of mGlu5−/− mice and wild-type littermates at three developmental time points (PND9, − 21, and − 75). We were surprised to find that expression of all NMDA receptor subunits was greatly enhanced in mGlu5−/− mice at PND21. In contrast, at PND9, expression of the GluN2B subunit was enhanced, whereas expression of GluN2A and GluN2D subunits was reduced in both regions. These modifications were transient and disappeared in the adult life (PND75). Changes in the transcripts of interneuron-related genes (encoding parvalbumin, somatostatin, vasoactive intestinal peptide, reelin, and the two isoforms of glutamate decarboxylase) were also observed in mGlu5−/− mice across postnatal development. For example, the transcript encoding parvalbumin was up-regulated in the prefrontal cortex of mGlu5−/− mice at PND9 and PND21, whereas it was significantly reduced at PND75. These findings suggest that in mGlu5−/− mice a transient overexpression of NMDA receptor subunits may compensate for the lack of the NMDA receptor partner, mGlu5. Interestingly, in mGlu5−/− mice the behavioral response to the NMDA channel blocker, MK-801, was significantly increased at PND21, and largely reduced at PND75. The impact of adaptive changes in the expression of NMDA receptor subunits should be taken into account when mGlu5−/− mice are used for developmental studies.

scholarly journals Stigma, gay men and biomedical prevention: the challenges and opportunities of a rapidly changing HIV prevention landscape

Sexual Health ◽  
2017 ◽  
Vol 14 (1) ◽  
pp. 111 ◽  
Author(s):  
Graham Brown ◽  
William Leonard ◽  
Anthony Lyons ◽  
Jennifer Power ◽  
Dirk Sander ◽  
...  
Keyword(s):  
Hiv Prevention ◽  
Gay Men ◽  
Sexual Minorities ◽  
Research Policy ◽  
Scale Up ◽  
People Living With Hiv ◽  
Structural Level ◽  
Biomedical Technologies ◽  
Living With Hiv ◽  
The Impact

Improvements in biomedical technologies, combined with changing social attitudes to sexual minorities, provide new opportunities for HIV prevention among gay and other men who have sex with men (GMSM). The potential of these new biomedical technologies (biotechnologies) to reduce HIV transmission and the impact of HIV among GMSM will depend, in part, on the degree to which they challenge prejudicial attitudes, practices and stigma directed against gay men and people living with HIV (PLHIV). At the structural level, stigma regarding gay men and HIV can influence the scale-up of new biotechnologies and negatively affect GMSM’s access to and use of these technologies. At the personal level, stigma can affect individual gay men’s sense of value and confidence as they negotiate serodiscordant relationships or access services. This paper argues that maximising the benefits of new biomedical technologies depends on reducing stigma directed at sexual minorities and people living with HIV and promoting positive social changes towards and within GMSM communities. HIV research, policy and programs will need to invest in: (1) responding to structural and institutional stigma; (2) health promotion and health services that recognise and work to address the impact of stigma on GMSM’s incorporation of new HIV prevention biotechnologies; (3) enhanced mobilisation and participation of GMSM and PLHIV in new approaches to HIV prevention; and (4) expanded approaches to research and evaluation in stigma reduction and its relationship with HIV prevention. The HIV response must become bolder in resourcing, designing and evaluating programs that interact with and influence stigma at multiple levels, including structural-level stigma.

scholarly journals Farmer Field Schools (FFSs): A Tool Empowering Sustainability and Food Security in Peasant Farming Systems in the Nicaraguan Highlands

2018 ◽  
Vol 10 (9) ◽  
pp. 3020 ◽  
Author(s):  
Esperanza Arnés ◽  
Carlos G. H. Díaz-Ambrona ◽  
Omar Marín-González ◽  
Marta Astier
Keyword(s):  
Food Security ◽  
Scale Up ◽  
Farming Systems ◽  
Farming System ◽  
Production Costs ◽  
Farmer Field Schools ◽  
Basic Services ◽  
Long Term Impact ◽  
Field Schools ◽  
The Impact

Farmer field schools (FFSs) emerged in response to the gap left by the worldwide decline in agricultural extension services. With time, this methodology has been adapted to specific rural contexts to solve problems related to the sustainability of peasant-farming systems. In this study we draw upon empirical data regarding the peasant-farming system in the Nicaraguan highlands to evaluate whether FFSs have helped communities improve the sustainability of their systems and the food security of their residents using socioeconomic, environmental, and food and nutrition security (FNS) indicators. In order to appreciate the long-term impact, we studied three communities where FFSs were implemented eight, five, and three years ago, respectively, and we included participants and nonparticipants from each community. We found that FFSs have a gradual impact, as there are significant differences between participants and nonparticipants, and it is the community that first implemented FFSs that scores highest. The impact of FFSs is broad and long lasting for indicators related to participation, access to basic services, and conservation of natural resources. Finally, this paper provides evidence that FFSs have the potential to empower farmers; however, more attention needs to be paid to critical indicators like production costs and the use of external inputs in order to scale up their potential in the future.

scholarly journals Energy Production from Biogas: Competitiveness and Support Instruments in Latvia

2016 ◽  
Vol 53 (5) ◽  
pp. 43-53
Author(s):  
G. Klāvs ◽  
A. Kundziņa ◽  
I. Kudrenickis
Keyword(s):  
New Technologies ◽  
Local Economic Development ◽  
Renewable Energy Sources ◽  
Distribution Network ◽  
Scale Up ◽  
Electricity Production ◽  
Small Scale ◽  
Gas Distribution ◽  
Ex Post ◽  
The Impact

Abstract Use of renewable energy sources (RES) might be one of the key factors for the triple win-win: improving energy supply security, promoting local economic development, and reducing greenhouse gas emissions. The authors ex-post evaluate the impact of two main support instruments applied in 2010-2014 – the investment support (IS) and the feed-in tariff (FIT) – on the economic viability of small scale (up to 2MWel) biogas unit. The results indicate that the electricity production cost in biogas utility roughly corresponds to the historical FIT regarding electricity production using RES. However, if in addition to the FIT the IS is provided, the analysis shows that the practice of combining both the above-mentioned instruments is not optimal because too high total support (overcompensation) is provided for a biogas utility developer. In a long-term perspective, the latter gives wrong signals for investments in new technologies and also creates unequal competition in the RES electricity market. To provide optimal biogas utilisation, it is necessary to consider several options. Both on-site production of electricity and upgrading to biomethane for use in a low pressure gas distribution network are simulated by the cost estimation model. The authors’ estimates show that upgrading for use in a gas distribution network should be particularly considered taking into account the already existing infrastructure and technologies. This option requires lower support compared to support for electricity production in small-scale biogas utilities.

scholarly journals Signal sequence insufficiency contributes to neurodegeneration caused by transmembrane prion protein

2010 ◽  
Vol 188 (4) ◽  
pp. 515-526 ◽  
Author(s):  
Neena S. Rane ◽  
Oishee Chakrabarti ◽  
Lionel Feigenbaum ◽  
Ramanujan S. Hegde
Keyword(s):  
Prion Protein ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Sequences ◽  
Hydrophobic Domain ◽  
Sequence Variations ◽  
Sufficient Magnitude ◽  
The Impact

Protein translocation into the endoplasmic reticulum is mediated by signal sequences that vary widely in primary structure. In vitro studies suggest that such signal sequence variations may correspond to subtly different functional properties. Whether comparable functional differences exist in vivo and are of sufficient magnitude to impact organism physiology is unknown. Here, we investigate this issue by analyzing in transgenic mice the impact of signal sequence efficiency for mammalian prion protein (PrP). We find that replacement of the average efficiency signal sequence of PrP with more efficient signals rescues mice from neurodegeneration caused by otherwise pathogenic PrP mutants in a downstream hydrophobic domain (HD). This effect is explained by the demonstration that efficient signal sequence function precludes generation of a cytosolically exposed, disease-causing transmembrane form of PrP mediated by the HD mutants. Thus, signal sequences are functionally nonequivalent in vivo, with intrinsic inefficiency of the native PrP signal being required for pathogenesis of a subset of disease-causing PrP mutations.

Strain Engineering of Thermal Conductivity of Two-Dimensional MoS2 and h-BN

MRS Advances ◽  
2016 ◽  
Vol 1 (32) ◽  
pp. 2297-2302 ◽  
Author(s):  
Xiaonan Wang ◽  
Alireza Tabarraei
Keyword(s):  
Thermal Conductivity ◽  
Tensile Strain ◽  
Phonon Dispersion ◽  
Hexagonal Boron Nitride ◽  
Three Dimensional ◽  
Strain Engineering ◽  
Normal Strain ◽  
Dynamics Modeling ◽  
Molecular Dynamics Modeling ◽  
The Impact

ABSTRACTWe have used reverse nonequlibrium molecular dynamics modeling to study the impact of uniaxial stretching on the thermal conductivity of monolayer molybdenum disulfide (MoS2) and hexagonal boron nitride (h-BN). Our results predict an anomalous response of the thermal conductivity of these materials to normal strain. Thermal conductivity of h-BN increases under a tensile strain whereas thermal conductivity of MoS2 remains fairly constant. These are in striking contrast to the impact of tensile strain on the thermal conductivity of three dimensional materials whose thermal conductivity decreases under tensile strain. We investigate the mechanism responsible for this unexpected behavior by studying the impact of tensile strain on the phonon dispersion curves and group velocities of these materials.

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