Established bioprocess monitoring is based on quick and reliable methods, including cell count and viability measurement, extracellular metabolite measurement, and the measurement of physicochemical ...qualities of the cultivation medium. These methods are sufficient for monitoring of process performance, but rarely give insight into the actual physiological states of the cell culture. However, understanding of the latter is essential for optimization of bioprocess development. Our study used LC‐MS metabolomics as a tool for additional resolution of bioprocess monitoring and was designed at three bioreactors scales (10 L, 100 L, and 1,000 L) to gain insight into the basal metabolic states of the Chinese hamster ovary (CHO) cell culture during fed‐batch. Metabolites characteristics of the four growth stages (early and late exponential phase, stationary phase, and the phase of decline) were identified by multivariate analysis. Enriched metabolic pathways were then established for each growth phase using the CHO metabolic network model. Biomass generation and nucleotide synthesis were enriched in early exponential phase, followed by increased protein production and imbalanced glutathione metabolism in late exponential phase. Glycolysis became downregulated in stationary phase and amino‐acid metabolism increased. Phase of culture decline resulted in rise of oxidized glutathione and fatty acid concentrations. Intracellular metabolic profiles of the CHO fed‐batch culture were also shown to be consistent with scale and thus demonstrate metabolomic profiling as an informative method to gain physiological insight into the cell culture states during bioprocess regardless of scale.
Established bioprocess monitoring is based on quick and reliable methods, including cell count and viability measurement, extracellular metabolite measurement and the measurement of physicochemical qualities of the cultivation medium.
Varroa destructor is one of the most common parasites of honey bee colonies and is considered as a possible co-factor for honey bee decline. At the same time, the use of pesticides in intensive ...agriculture is still the most effective method of pest control. There is limited information about the effects of pesticide exposure on parasitized honey bees. Larval ingestion of certain pesticides could have effects on honey bee immune defense mechanisms, development and metabolic pathways. Europe and America face the disturbing phenomenon of the disappearance of honey bee colonies, termed Colony Collapse Disorder (CCD). One reason discussed is the possible suppression of honey bee immune system as a consequence of prolonged exposure to chemicals. In this study, the effects of the neonicotinoid thiamethoxam on honey bee, Apis mellifera carnica, pupae infested with Varroa destructor mites were analyzed at the molecular level. Varroa-infested and non-infested honey bee colonies received protein cakes with or without thiamethoxam. Nurse bees used these cakes as a feed for developing larvae. Samples of white-eyed and brown-eyed pupae were collected. Expression of 17 immune-related genes was analyzed by real-time PCR. Relative gene expression in samples exposed only to Varroa or to thiamethoxam or simultaneously to both Varroa and thiamethoxam was compared. The impact from the consumption of thiamethoxam during the larval stage on honey bee immune related gene expression in Varroa-infested white-eyed pupae was reflected as down-regulation of spaetzle, AMPs abaecin and defensin-1 and up-regulation of lysozyme-2. In brown-eyed pupae up-regulation of PPOact, spaetzle, hopscotch and basket genes was detected. Moreover, we observed a major difference in immune response to Varroa infestation between white-eyed pupae and brown-eyed pupae. The majority of tested immune-related genes were upregulated only in brown-eyed pupae, while in white-eyed pupae they were downregulated.
Toll-like receptor 10 (TLR10) is the only member of the TLR family whose function and ligand have not been clearly described. Literature reports on its function are contradictory and suggest a ...possible immunomodulatory role that depends on the cell type, the pathogen, and the level of TLR10 expression. To investigate the regulatory role of TLR10 in A549 lung epithelial cells, we overexpressed
using CRISPRa technology and examined the differential expression of various genes involved in TLR signaling activated by different TLR ligands, namely dsRNA, LPS, and Pam3Cys. The expression of proinflammatory cytokines, such as
,
,
,
,
, and
decreased in the challenged cells overexpressing
, whereas the expression of the anti-inflammatory cytokine
and the antimicrobial peptide
increased. For several of the regulated inflammatory markers, we were able to show the change in gene expression was translated to the protein level. It appears that TLR10 can function as an anti-inflammatory in A549 cells, depending on its expression level and that the mode of action may be virulence factor-specific. The potential suppression of inflammation by regulating expression of
in lung epithelial cells may allow the development of new approaches to balance an inflammatory response and prevent extensive tissue damage in respiratory diseases.
Among numerous factors that contribute to honey bee colony losses and problems in beekeeping, pesticides and
have been often reported. In contrast to insecticides, whose effects on bees have been ...widely studied, fungicides did not attract considerable attention. Prochloraz, an imidazole fungicide widely used in agriculture, was detected in honey and pollen stored inside hives and has been already proven to alter immune gene expression of honey bees at different developmental stages. The aim of this study was to simulate the realistic conditions of migratory beekeeping, where colonies, both uninfected and infected with
, are frequently transported to the vicinity of crop fields treated with prochloraz. We investigated the combined effect of prochloraz and
on honey bees that faced fungicide during the larval stage through food consumption and microsporidium infection afterwards. The most pronounced changes in gene expression were observed in newly emerged
-free bees originating from colonies previously contaminated with prochloraz. As exclusively upregulation was registered, prochloraz alone most likely acts as a challenge that induces activation of immune pathways in newly emerged bees. The combination of both stressors (prochloraz and
infection) exerted the greatest effect on six-day-old honey bees. Among ten genes with significantly altered expression, half were upregulated and half downregulated.
as a sole stressor had the weakest effects on immune gene expression modulation with only three genes significantly dysregulated. In conclusion, food contaminated with prochloraz consumed in larval stage could present a threat to the development of immunity and detoxification mechanisms in honey bees.
The mushroom Pleurotus ostreatus has been reported to produce the hemolytic proteins ostreolysin (OlyA), pleurotolysin A (PlyA) and pleurotolysin B (PlyB). The present study of the native and ...recombinant proteins dissects out their lipid-binding characteristics and their roles in lipid binding and membrane permeabilization. Using lipid-binding studies, permeabilization of erythrocytes, large unilamellar vesicles of various lipid compositions, and electron microscopy, we show that OlyA, a PlyA homolog, preferentially binds to membranes rich in sterol and sphingomyelin, but it does not permeabilize them. The N-terminally truncated Δ48PlyB corresponds to the mature and active form of native PlyB, and it has a membrane attack complex-perforin (MACPF) domain. Δ48PlyB spontaneously oligomerizes in solution, and binds weakly to various lipid membranes but is not able to perforate them. However, binding of Δ48PlyB to the cholesterol and sphingomyelin membranes, and consequently, their permeabilization is dramatically promoted in the presence of OlyA. On these membranes, Δ48PlyB and OlyA form predominantly 13-meric oligomers. These are rosette-like structures with a thickness of ∼9 nm from the membrane surface, with 19.7 nm and 4.9 nm outer and inner diameters, respectively. When present on opposing vesicle membranes, these oligomers can dimerize and thus promote aggregation of vesicles. Based on the structural and functional characteristics of Δ48PlyB, we suggest that it shares some features with MACPF/cholesterol-dependent cytolysin (CDC) proteins. OlyA is obligatory for the Δ48PlyB permeabilization of membranes rich in cholesterol and sphingomyelin.
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•Ostreolysin A (OlyA) binds selectively to cholesterol/sphingomyelin rich membranes.•OlyA recruits a MACPF/PF protein, Δ48-pleurotolysin B, to the membranes.•Δ48-pleurotolysin B forms 13-meric rosette-like pores on membranes.
The role of chondrocytes in the development of infectious arthritis is not well understood. Several examples of mycoplasma-induced arthritis in animals indicate that chondrocytes come into direct ...contact with bacteria. The objective of this study was to analyze the interaction of an arthrogenic Mycoplasma synoviae strain WVU 1853 with chicken chondrocytes. We found that M. synoviae significantly reduces chondrocyte respiration. This was accompanied by alterations in chondrocyte morphology, namely cell shrinkage and cytoplasm condensation, as well as nuclear condensation and formation of plasma membrane invaginations containing nuclear material, which appeared to cleave off the cell surface. In concordance with these apoptosis-like events in chondrocytes, transcription was increased in several pro-apoptotic genes. Twenty-four hours after infection, strong upregulation was assayed in NOS2, Mapk11, CASP8 and Casp3 genes. Twenty-four and 72 h incubation of chondrocytes with M. synoviae induced upregulation of AIFM1, NFκB1, htrA3 and BCL2. Casp3 and NOS2 remained upregulated, but upregulation ceased for Mapk11 and CASP8 genes. Increased production of nitric oxide was also confirmed in cell supernates. The data suggests that chicken chondrocytes infected with M. synoviae die by apoptosis involving production of nitric oxide, caspase 3 activation and mitochondrial inactivation. The results of this study show for the first time that mycoplasmas could cause chondrocyte apoptosis. This could contribute to tissue destruction and influence the development of arthritic conditions. Hence, the study gives new insights into the role of mycoplasma infection on chondrocyte biology and development of infectious arthritis in chickens and potentially in humans.
Chickens, as a source of desired antibodies, represent an alternate animal system that offers some advantages with respect to animal care, high productivity and special suitability of avian ...antibodies for certain diagnostic purposes. Despite being an excellent counterpart
to mammal IgG chicken IgY antibodies still represent an underused resource. This may be due to the lack of information concerning the possibility of production and application of IgY or their use is being hampered by problems with keeping the chickens and with IgY isolation. As a suggestion how to overcome IgY isolation problems a new immunoaffinity isolation method is presented here. The main purpose of the present work is to provide information on developments and possibilities in the production of chicken IgY.
Polyclonal, monoclonal and recombinant forms of IgY, successfully produced so far, as well as their applications are summarised. This article should be a contribution to the efforts of the European Centre for the Validation of Alternative Methods (ECVAM), whose main goal is to promote the scientific and regulatory acceptance of alternative methods, which are of importance to the bioscience and which reduce, refine or replace the use of laboratory animals.
The main goal in biosimilar development is to increase Chinese Hamster Ovary (CHO) viability and productivity while maintaining product quality. Despite media and feed optimization during process ...development, depletion of amino acids still occurs. The aim of the work was to optimize an existing industrial fed batch process by preventing shortage of amino acids and to gather knowledge about CHO metabolism. Several process outputs were evaluated such as cell metabolism, cell viability, monoclonal antibodies (mAbs) production, and product quality. First step was to develop and supplement an enriched feed containing depleted amino acids. Abundance of serine and glucose increased lactate production resulting in low viability and low productivity. In the next step, we developed an amino acid feed without serine to avoid the metabolic boost. Supplemented amino acids improved cell viability by 9%; however, mAb production did not increase significantly. In the final step, we limited glucose concentration (<5.55 mmol/L) in the cell culture to avoid the metabolic boost while supplementing an amino acid feed including serine. Data analysis showed that we were able to (a) replace depleted amino acids and avoid metabolic boost, (b) increase viability by 12%, (c) enhance mAb production by 0.5 g/L (total by approximately 10 g), and (d) extend the overall process time of an already developed bioprocess.
When nanoparticles enter the body, their interactions with cells are almost unavoidable. Unintended nanoparticle interaction with immune cells may elicit a molecular response that can have toxic ...effects and lead to greater susceptibility to infectious diseases, autoimmune disorders, and cancer development. As evidenced by several studies, nanoparticle interactions with biological systems can stimulate inflammatory or allergic reactions and activate the complement system. Nanoparticles can also stimulate immune response by acting as adjuvants or as haptens. Immunosuppressive effects have also been reported. This article gives a brief review of in vitro and in vivo research evidencing stimulatory or suppressive effects of nanoparticles on the immune system of mammals. In order to ensure safe use of nanosized particles, future research should focus on how their physical and chemical properties influence their behaviour in the biological environment, as they not only greatly affect nanoparticle-immune system interactions but can also interfere with experimental assays.
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