scholarly journals Mechanical Stress Induces Ca2+-Dependent Signal Transduction in Erythroblasts and Modulates Erythropoiesis

2021 ◽  
Vol 22 (2) ◽  
pp. 955
Author(s):  
Francesca Aglialoro ◽  
Asena Abay ◽  
Nurcan Yagci ◽  
Minke A. E. Rab ◽  
Lars Kaestner ◽  
...  
Keyword(s):  
Mechanical Stress ◽  
Mammalian Cells ◽  
Large Scale ◽  
Culture Conditions ◽  
Signaling Cascades ◽  
Mechanical Forces ◽  
Orbital Shaking ◽  
Dependent Signal ◽  
Physiological Outcomes ◽  
Stirred Bioreactors

Bioreactors are increasingly implemented for large scale cultures of various mammalian cells, which requires optimization of culture conditions. Such upscaling is also required to produce red blood cells (RBC) for transfusion and therapy purposes. However, the physiological suitability of RBC cultures to be transferred to stirred bioreactors is not well understood. PIEZO1 is the most abundantly expressed known mechanosensor on erythroid cells. It is a cation channel that translates mechanical forces directly into a physiological response. We investigated signaling cascades downstream of PIEZO1 activated upon transitioning stationary cultures to orbital shaking associated with mechanical stress, and compared the results to direct activation of PIEZO1 by the chemical agonist Yoda1. Erythroblasts subjected to orbital shaking displayed decreased proliferation, comparable to incubation in the presence of a low dose of Yoda1. Epo (Erythropoietin)-dependent STAT5 phosphorylation, and Calcineurin-dependent NFAT dephosphorylation was enhanced. Phosphorylation of ERK was also induced by both orbital shaking and Yoda1 treatment. Activation of these pathways was inhibited by intracellular Ca2+ chelation (BAPTA-AM) in the orbital shaker. Our results suggest that PIEZO1 is functional and could be activated by the mechanical forces in a bioreactor setup, and results in the induction of Ca2+-dependent signaling cascades regulating various aspects of erythropoiesis. With this study, we showed that Yoda1 treatment and mechanical stress induced via orbital shaking results in comparable activation of some Ca2+-dependent pathways, exhibiting that there are direct physiological outcomes of mechanical stress on erythroblasts.

scholarly journals Automation of large scale transient protein expression in mammalian cells

2011 ◽  
Vol 175 (2) ◽  
pp. 209-215 ◽  
Author(s):  
Yuguang Zhao ◽  
Benjamin Bishop ◽  
Jordan E. Clay ◽  
Weixian Lu ◽  
Margaret Jones ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mammalian Cells ◽  
Large Scale ◽  
Transient Protein Expression ◽  
Expression In Mammalian Cells

An epidermal growth factor receptor/ret chimera generates mitogenic and transforming signals: evidence for a ret-specific signaling pathway

1994 ◽  
Vol 14 (1) ◽  
pp. 663-675
Author(s):  
M Santoro ◽  
W T Wong ◽  
P Aroca ◽  
E Santos ◽  
B Matoskova ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinases ◽  
Mammalian Cells ◽  
Egf Receptor ◽  
Biological Effects ◽  
Ectopic Expression ◽  
Growth Factor Receptor ◽  
Dependent Signal ◽  
Epidermal Growth

A chimeric expression vector which encoded for a molecule encompassing the extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) and the intracellular domain of the ret kinase (EGFR/ret chimera) was generated. Upon ectopic expression in mammalian cells, the EGFR/ret chimera was correctly synthesized and transported to the cell surface, where it was shown capable of binding EGF and transducing an EGF-dependent signal intracellularly. Thus, the EGFR/ret chimera allows us to study the biological effects and biochemical activities of the ret kinase under controlled conditions of activation. Comparative analysis of the growth-promoting activity of the EGFR/ret chimera expressed in fibroblastic or hematopoietic cells revealed a biological phenotype clearly distinguishable from that of the EGFR, indicating that the two kinases couple with mitogenic pathways which are different to some extent. Analysis of biochemical pathways implicated in the transduction of mitogenic signals also evidenced significant differences between the ret kinase and other receptor tyrosine kinases. Thus, the sum of our results indicates the existence of a ret-specific pathway of mitogenic signaling.

scholarly journals Transgenic goats producing an improved version of cetuximab in milk

2020 ◽  
Author(s):  
Götz Laible ◽  
Sally Cole ◽  
Brigid Brophy ◽  
Paul Maclean ◽  
Li How Chen ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Large Scale ◽  
Growth Factor Receptor ◽  
Cost Effective ◽  
Scale Production ◽  
Cancer Treatments ◽  
Large Scale Production ◽  
Immunogenic Epitope ◽  
Dependent Cell ◽  
Transgenic Goats

ABSTRACTTherapeutic monoclonal antibodies (mAbs) represent one of the most important classes of pharmaceutical proteins to treat human diseases. Most are produced in cultured mammalian cells which is expensive, limiting their availability. Goats, striking a good balance between a relatively short generation time and copious milk yield, present an alternative platform for the cost-effective, flexible, large-scale production of therapeutic mAbs. Here, we focused on cetuximab, a mAb against epidermal growth factor receptor, that is commercially produced under the brand name Erbitux and approved for anti-cancer treatments. We generated several transgenic goat lines that produce cetuximab in their milk. Two lines were selected for detailed characterization. Both showed stable genotypes and cetuximab production levels of up to 10g/L. The mAb could be readily purified and showed improved characteristics compared to Erbitux. The goat-produced cetuximab (gCetuximab) lacked a highly immunogenic epitope that is part of Erbitux. Moreover, it showed enhanced binding to CD16 and increased antibody-dependent cell-dependent cytotoxicity compared to Erbitux. This indicates that these goats produce an improved cetuximab version with the potential for enhanced effectiveness and better safety profile compared to treatments with Erbitux. In addition, our study validates transgenic goats as an excellent platform for large-scale production of therapeutic mAbs.

scholarly journals Infection with intracellular parasite Amoeboaphelidium protococcarum induces shifts in associated bacterial communities in microalgae cultures

2021 ◽  
Vol 33 (5) ◽  
pp. 2863-2873
Author(s):  
Anna-Lena Höger ◽  
Carola Griehl ◽  
Matthias Noll
Keyword(s):  
Bacterial Communities ◽  
Large Scale ◽  
Algal Growth ◽  
Dry Weight ◽  
Illumina Miseq ◽  
Culture Conditions ◽  
Rrna Gene ◽  
Scale Production ◽  
Market Demand ◽  
Freshwater Microalgae

AbstractIn recent years microalgae products have developed increasing market demand, but sustainable industrial production is still challenged by biological stability of large-scale production plants. Yet the relationships between algal hosts, associated microbiomes, and contaminants in photobioreactors remains widely understudied. The aim of this study was to investigate the temporal development of microbiomes of four freshwater microalgae species Scenedesmus vacuolatus, Desmodesmus quadricauda, Chlorella sorokiniana, and Botryococcus braunii, in presence and absence of the zoosporic parasite Amoeboaphelidium protococcarum. To compare the effects of sterile and nonsterile culture conditions, infection experiments were performed in sterile laboratory (sterile) and simulated industrial conditions (open). Algal growth (dry weight, optical density, and nutrient consumption) was observed for 21 days, and samples of the associated microbiome were collected for bacterial 16S rRNA gene Illumina MiSeq sequencing. Infection patterns of A. protococcarum were algae species-specific, irrespectively of culture conditions. Bacterial community analysis demonstrated distinct and stable bacterial communities for each algae species, which were mostly dominated by α- and γ-Proteobacteria. Upon aphelid parasitosis, bacterial diversity increased, and community compositions diverged algae-specific over time. Moreover, bacterial functional traits shifted to detoxification, degradation, and cellulolysis once algae were infected. This study provides a first insight into the close connection between algae, associated bacterial microbiomes and appearing contaminants in photobioreactor systems.

Large–Scale Production of Proteins from Mammalian Cells

1988 ◽  
Vol 6 (5) ◽  
pp. 518-523 ◽  
Author(s):  
Malcolm Rhodes ◽  
John Birch
Keyword(s):  
Mammalian Cells ◽  
Large Scale ◽  
Scale Production ◽  
Large Scale Production

Sustainability and income opportunities of farming milkfish (Chanos chanos) to local communities in Kenya: assessment of initial trials of earthen ponds.

2020 ◽  
Author(s):  
David Oersted Mirera ◽  
Charles C Ngugi
Keyword(s):  
Large Scale ◽  
Production Capacity ◽  
Maximum Growth ◽  
Culture Conditions ◽  
Chicken Manure ◽  
Scylla Serrata ◽  
Good Growth ◽  
Chanos Chanos ◽  
Training Centre ◽  
Seed Collection

Abstract This case study documents the development of earthen pond mariculture from the late 1980s to 2009 in Kenya and prospects of community milkfish (Chanos chanos) culture using satellite farmers along the coast of Kenya, as well as challenges to this kind of farming and approaches towards sustainability. Pond mariculture was started in the 1980s by the Kenyan Fisheries Department through the onset of prawn farming (Penaeus indicus and P. monodon) in large scale demonstration ponds at Ngomeni under funding from FAO. The total culture area was estimated at 60 ha and milkfish occurred as a by-product of the prawn culture operations with indications of good growth. Similar observations were made for the milkfish in the artisanal Kwetu prawn culture pond (0.8 ha) that was started in the late 1990s, and one individual middle-scale farmer in Mtwapa creek (Wampare) who had interest in prawn culture at the time and constructed ponds covering an area of 3 ha. The initial recommendations for the potential to culture milkfish in Kenya were made through a research project (Mwaluma, 2002) whose objectives were to assess the potential of mud crab (Scylla serrata) pen culture in natural mangrove channels at Mtwapa creek. Intense participatory research into potential for milkfish culture coupled with extension was initiated at the Kwetu Training Centre along the Kenya coast with satellite groups in 2004. Consequently, improvements have since been achieved with wild seed collection in mangrove pools, use of chicken manure as organic pond fertilizer, and dependency of tidal water exchange, which has led to a maximum growth rate of 1.2 g/day. The demand for milkfish production has increased greatly among the coastal poor (Mirera, 2009b) and presently (Sept 2009) five communities have constructed more than 25 milkfish production ponds with a maximum production capacity of more than 70 kg per crop. These farmers have appreciated the contribution of milkfish culture to achieving food security and income generation for their families. The first harvest of milkfish in the community ponds were bought by the research/extension officer, and were fried and eaten together by all members of the community (including none group members) to demonstrate that milkfish can grow under local culture conditions. In subsequent harvests, the whole community is informed a day before the harvest, whereby they come to the farms during harvesting and buy the fish in quotas at two dollars per kg.

scholarly journals Outdoor Large-Scale Cultivation of the Acidophilic Microalga Coccomyxa onubensis in a Vertical Close Photobioreactor for Lutein Production

Processes ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 324 ◽  
Author(s):  
Juan-Luis Fuentes ◽  
Zaida Montero ◽  
María Cuaresma ◽  
Mari-Carmen Ruiz-Domínguez ◽  
Benito Mogedas ◽  
...  
Keyword(s):  
Biomass Production ◽  
Large Scale ◽  
Culture Conditions ◽  
High Concentration ◽  
Ph Values ◽  
Lutein Content ◽  
Acidic Waters ◽  
Large Scale Cultivation ◽  
Rate Limiting ◽  
Lutein Production

The large-scale biomass production is an essential step in the biotechnological applications of microalgae. Coccomyxa onubensis is an acidophilic microalga isolated from the highly acidic waters of Río Tinto (province of Huelva, Spain) and has been shown to accumulate a high concentration of lutein (9.7 mg g−1dw), a valuable antioxidant, when grown at laboratory-scale. A productivity of 0.14 g L−1 d−1 was obtained by growing the microalga under outdoor conditions in an 800 L tubular photobioreactor. The results show a stable biomass production for at least one month and with a lutein content of 10 mg g−1dw, at pH values in the range 2.5–3.0 and temperature in the range 10–25 °C. Culture density, temperature, and CO2 availability in highly acidic medium are rate-limiting conditions for the microalgal growth. These aspects are discussed in this paper in order to improve the outdoor culture conditions for competitive applications of C. onubensis.

scholarly journals Insect Cecropins, Antimicrobial Peptides with Potential Therapeutic Applications

2019 ◽  
Vol 20 (23) ◽  
pp. 5862 ◽  
Author(s):  
Daniel Brady ◽  
Alessandro Grapputo ◽  
Ottavia Romoli ◽  
Federica Sandrelli
Keyword(s):  
Infectious Diseases ◽  
Antimicrobial Peptides ◽  
Mammalian Cells ◽  
Large Scale ◽  
Scale Production ◽  
Gram Negative ◽  
Social Burden ◽  
Large Scale Production ◽  
Conventional Antibiotics ◽  
The Cost

The alarming escalation of infectious diseases resistant to conventional antibiotics requires urgent global actions, including the development of new therapeutics. Antimicrobial peptides (AMPs) represent potential alternatives in the treatment of multi-drug resistant (MDR) infections. Here, we focus on Cecropins (Cecs), a group of naturally occurring AMPs in insects, and on synthetic Cec-analogs. We describe their action mechanisms and antimicrobial activity against MDR bacteria and other pathogens. We report several data suggesting that Cec and Cec-analog peptides are promising antibacterial therapeutic candidates, including their low toxicity against mammalian cells, and anti-inflammatory activity. We highlight limitations linked to the use of peptides as therapeutics and discuss methods overcoming these constraints, particularly regarding the introduction of nanotechnologies. New formulations based on natural Cecs would allow the development of drugs active against Gram-negative bacteria, and those based on Cec-analogs would give rise to therapeutics effective against both Gram-positive and Gram-negative pathogens. Cecs and Cec-analogs might be also employed to coat biomaterials for medical devices as an approach to prevent biomaterial-associated infections. The cost of large-scale production is discussed in comparison with the economic and social burden resulting from the progressive diffusion of MDR infectious diseases.

scholarly journals Interrogation of kinase genetic interactions provides a global view of PAK1-mediated signal transduction pathways

2020 ◽  
Vol 295 (50) ◽  
pp. 16906-16919
Author(s):  
Jae-Hong Kim ◽  
Yeojin Seo ◽  
Myungjin Jo ◽  
Hyejin Jeon ◽  
Young-Seop Kim ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cell Migration ◽  
Intracellular Signaling ◽  
Mammalian Cells ◽  
Drug Targets ◽  
Large Scale ◽  
Genetic Interaction ◽  
Genetic Interactions ◽  
Signal Transduction Pathways ◽  
Global View

Kinases are critical components of intracellular signaling pathways and have been extensively investigated with regard to their roles in cancer. p21-activated kinase-1 (PAK1) is a serine/threonine kinase that has been previously implicated in numerous biological processes, such as cell migration, cell cycle progression, cell motility, invasion, and angiogenesis, in glioma and other cancers. However, the signaling network linked to PAK1 is not fully defined. We previously reported a large-scale yeast genetic interaction screen using toxicity as a readout to identify candidate PAK1 genetic interactions. En masse transformation of the PAK1 gene into 4,653 homozygous diploid Saccharomyces cerevisiae yeast deletion mutants identified ∼400 candidates that suppressed yeast toxicity. Here we selected 19 candidate PAK1 genetic interactions that had human orthologs and were expressed in glioma for further examination in mammalian cells, brain slice cultures, and orthotopic glioma models. RNAi and pharmacological inhibition of potential PAK1 interactors confirmed that DPP4, KIF11, mTOR, PKM2, SGPP1, TTK, and YWHAE regulate PAK1-induced cell migration and revealed the importance of genes related to the mitotic spindle, proteolysis, autophagy, and metabolism in PAK1-mediated glioma cell migration, drug resistance, and proliferation. AKT1 was further identified as a downstream mediator of the PAK1-TTK genetic interaction. Taken together, these data provide a global view of PAK1-mediated signal transduction pathways and point to potential new drug targets for glioma therapy.

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