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Processes ◽  
2022 ◽  
Vol 10 (1) ◽  
pp. 162
Author(s):  
Tessy A. H. Hick ◽  
Corinne Geertsema ◽  
Maurice G. L. Henquet ◽  
Dirk E. Martens ◽  
Stefan W. Metz ◽  
...  

Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne virus that causes a severe febrile illness with long-lasting arthralgia in humans. As there is no vaccine to protect humans and limit CHIKV epidemics, the virus continues to be a global public health concern. The CHIKV envelope glycoproteins E1 and E2 are important immunogens; therefore, the aim of this study is to produce trimeric CHIKV spikes in insect cells using the baculovirus expression system. The CHIKV E1 and E2 ectodomains were covalently coupled by a flexible linker that replaces the 6K transmembrane protein. The C-terminal E1 transmembrane was replaced by a Strep-tag II for the purification of secreted spikes from the culture fluid. After production in Sf9 suspension cells (product yields of 5.8–7.6 mg/L), the CHIKV spikes were purified by Strep-Tactin affinity chromatography, which successfully cleared the co-produced baculoviruses. Bis(sulfosuccinimidyl)suberate cross-linking demonstrated that the spikes are secreted as trimers. PNGase F treatment showed that the spikes are glycosylated. LC–MS/MS-based glycoproteomic analysis confirmed the glycosylation and revealed that the majority are of the mannose- or hybrid-type N-glycans and <2% have complex-type N-glycans. The LC –MS/MS analysis also revealed three O-glycosylation sites in E1. In conclusion, the trimeric, glycosylated CHIKV spikes have been successfully produced in insect cells and are now available for vaccination studies.


Author(s):  
Atul Goyal ◽  
Binh Vu ◽  
Vijay Maranholkar ◽  
Ujwal Patil ◽  
Katerina Kourentzi ◽  
...  

In the manufacture of therapeutic monoclonal antibodies (mAbs), the clarified cell culture fluid is typically loaded onto an initial protein A affinity capture column. Imperfect mass transfer and loading to maximum capacity can risk antibody breakthrough and loss of valuable product, but conservative underloading wastes expensive protein A resin. In addition, the effects of column fouling and ligand degradation require the frequent optimization of IgG loading to avoid wastage. Therefore, continuous real-time monitoring of IgG flowthrough is of great interest. We previously developed a fluorescence-based monitoring technology that allows mix-and-read mAb detection in cell culture fluid. Here we report the use of reporters immobilized on CNBr-activated Sepharose 4B resin for continuous detection of IgG in column breakthrough. The column effluent is continuously contacted with immobilized fluorescein-labeled Fc-binding ligands to produce an immediately detectable change in fluorescence intensity. The technology allows rapid and reliable monitoring of IgG in a flowing stream of clarified cell culture fluid emerging from a Protein A column, without prior sample preparation. We observed a significant change in fluorescence intensity at 0.5 g/L human IgG, sufficient to detect a 5% breakthrough of a 10 g/L load, within 2 minutes at a flow rate of 0.5 mL/min.


2021 ◽  
Vol 5 (4) ◽  
pp. 220-227
Author(s):  
Oksana Mykchaylova ◽  
Nataliia Poyedіnok

Background. According to the World Health Organization antibiotic resistance is among the top ten threats to human health, food safety and development. Today antibiotic resistance has reached alarmingly high levels all over the world. Meanwhile, the increase in the synthetic drugs' production has led to the pathogenic mycobiota's rapid adaptation to the created chemicals, which have a narrow focus of application. That is why in modern biotechnology and pharmacology much attention is paid to natural producers of biologically active compounds, in particular – to xylotrophic fungi. It has been experimentally proven that the xylotrophic macromycete Fomitopsis officinalis or tinder fungus can be considered to be a promising producer of pharmacological substances with a broad spectrum of action. Studies of active metabolites, contained in the mycelial mass, culture fluid of the medicinal xylotrophic macromycete F. officinalis, and determination of their biological action remain relevant. Objective. The objective was to determine the antimicrobial activity of culture fluid and mycelial mass of F. officinalis different strains from the mushrooms collection (IBK Mushroom Culture Collection of the M.G. Kholodny Institute of Botany, NAS of Ukraine) against gram-negative and gram-positive bacteria species. Methods. An in vitro study of the antimicrobial activity of ethyl acetate extracts of culture fluid and aqueous-ethyl extracts of mycelial mass for F. officinalis strains IBK-5004, IBK-2497, IBK-2498 against gram-positive Staphylococcus aureus (B-918), Bacillus subtilis (В-901) and gram-negative Escherichia coli (B-906), Bacillus subtilis (B-900), Klebsiella pneumoniae (M-123) bacteria by disc-diffusion method was conducted. Results. High antimicrobial activity of tinder fungus culture fluid and mycelial mass extracts against Staphylococcus aureus was established after the 21st day of cultivation, while on the 28th day the zone of growth retardation was maximal (15–25 mm). The highest indices were recorded in F. officinalis IBK-5004 (20–25 mm) and IBK-2498 (20–24 mm) strains. Antimicrobial activity against Klebsiella pneumoniae in culture fluid extracts was manifested on the 21st and 28th days of cultivation. The highest antimicrobial activity against Klebsiella pneumoniae was observed in the culture fluid of the strain F. officinalis IBK-5004, the diameter of the growth retardation zone was 18 mm on the 28th day of cultivation. Mycelial mass's extracts showed moderate activity on the 14th day of cultivation (7-8 mm); maximal activity was recorded on the 28th day (12–22 mm). The most active strain was Fomitopsis officinalis IBK-2498. No antimicrobial activity against test organisms was detected in the following studied strains: Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis. Conclusions. It has been established that the mycelial mass and culture fluid extracts of F. officinalis IBK-5004, IBK-2497, IBK-2498 strains have high antimicrobial activity against Staphylococcus aureus and moderate antimicrobial activity against Klebsiella pneumoniae on the 21st and 28th day of cultivation.


2021 ◽  
Vol 19 (4(76)) ◽  
pp. 47-53
Author(s):  
Liubov V. Regeda ◽  
Nina A. Bisko ◽  
Nina V. Gurinovych

Aim. To determine the value of the antioxidant activity of the biomass and culture fluid extracts of strains of seven species of Pholiota genus: P. adiposa, P. alnicola, P. aurivella, P. limonella, P. nameko, P. squarrosa, P. subochracea, which stored in the Mushroom Culture Collection (IBK) of the M. G. Kholodny Institute of Botany of the National Academy of Sciences of Ukraine.Materials and methods. The antioxidant properties of the biomass and culture fluid extracts of strains of Pholiota genus were determined by the method of Elfahri et al. using DPPH (1,1-diphenyl-2-picrylhydrazyl). Mycelia of the strains studied were grown by the surface method on a liquid glucose-pepton-yeast medium. The culture fluid was separated from the mycelial biomass by filtration through a capron filter. The absorption of methanol extracts of the culture fluid and the biomass of the strains studied was measured at 517 nm on a SF 46 LOMO spectrophotometer.Results and discussion. Comparing the data obtained we can conclude that the antioxidant effect is significantly higher in the case of methanol biomass extracts – the indicators ranged from 65.98 ± 0.98 % (P. nameko) to 83.6 ± 1.4 % (P. alnicola). As for the culture fluid extracts, the maximum values were recorded in the case of P. limonella (38.3 ± 1.14 %), and the minimum values were observed for P. subochracea (7.37 ± 0.46 %).Conclusions. For the first time, the value and limits of variation in the antioxidant activity of the biomass (65-83 %) and culture fluid extracts (7.4-38 %) have been determined for strains of medicinal fungal species P. adiposa, P. alnicola, P. aurivella, P. limonella, P. nameko, P. squarrosa, P. subochracea.


2021 ◽  
Vol 12 ◽  
Author(s):  
Yu-Chieh Lin ◽  
Chun-Yi Wu ◽  
Hung-Tse Huang ◽  
Mei-Kuang Lu ◽  
Wei-Shou Hu ◽  
...  

Enterococcus faecalis is considered a leading cause of hospital-acquired infections. Treatment of these infections has become a major challenge for clinicians because some E. faecalis strains are resistant to multiple clinically used antibiotics. Moreover, the presence of E. faecalis biofilms can make infections with E. faecalis more difficult to eradicate with current antibiotic therapies. Thus, our aim in this study was to investigate the effects of probiotic derivatives against E. faecalis biofilm formation. Bacillus subtilis natto is a probiotic strain isolated from Japanese fermented soybean foods, and its culture fluid potently inhibited adherence to Caco-2 cell monolayers, aggregation, and biofilm production without inhibiting the growth of E. faecalis. An apparent decrease in the thickness of E. faecalis biofilms was observed through confocal laser scanning microscopy. In addition, exopolysaccharide synthesis in E. faecalis biofilms was reduced by B. subtilis natto culture fluid treatment. Carbohydrate composition analysis also showed that carbohydrates in the E. faecalis cell envelope were restructured. Furthermore, transcriptome sequencing revealed that the culture fluid of B. subtilis natto downregulated the transcription of genes involved in the WalK/WalR two-component system, peptidoglycan biosynthesis and membrane glycolipid biosynthesis, which are all crucial for E. faecalis cell envelope synthesis and biofilm formation. Collectively, our work shows that some derivatives present in the culture fluid of B. subtilis natto may be useful for controlling E. faecalis biofilms.


Algologia ◽  
2021 ◽  
Vol 31 (4) ◽  
pp. 320-336
Author(s):  
P.M. Tsarenko ◽  
◽  
N.V. Zaimenko ◽  
N.P. Didyk ◽  
N.E. Ellanska ◽  
...  

The influence of the cultural medium of the charophyte Interfillum terricola on the allelopathic, microbiological, agrophysical and agrochemical properties of the soil have been studied in model pot experiments. Allelopathic soil regime was assessed by biological testing methods for water-soluble compounds and direct biotesting, as well as by vital indicators of plants-phytometers of winter wheat (Triticum aestivum L., variety "Smuglyanka") and fodder corn (Zea mays L., variety "Kadr 267 MB"). The seeds were sown immediately after the introduction of the culture fluid. The number of germinated seeds was recorded from the 2nd to the 8th day after sowing. The vital condition of phytometer plants was evaluated at the end of the experiments by morphometric indicators of growth (leaf surface area, dry matter biomass of aboveground parts and roots) and the content of photosynthetic pigments in the leaves. When the experiment was completed, soil samples were taken to determine the cytostatic effect of water-soluble compounds and to carry out microbiological and biochemical analyzes. Phenolic compounds were isolated from the soil by ion exchange (desorption) using an ion exchanger KU-2-8 (Н+). In parallel, the electrical conductivity, redox potential, pH and content of nutrients in the soil were determined. The stimulating effect of cultural medium on seed germination, growth and development of assimilation organs of wheat and corn plants has been revealed. The strength of the effect did not depend on the concentration of growing medium, which is characteristic of signal allelopathically active substances. Allelopathic and cytostatic activity of the soil decreased with the use of Interfillum terricola growing medium. The introduction of the cultural fluid significantly affected the number of microorganisms of different ecological and trophic groups. The lowest number of microorganisms was observed at the minimum rate of introduction of microalga medium, and its increase contributed to the growth of the number of almost all studied groups of microorganisms, indicators of transformation and mineralization of organic matter. Under the influence of the cultural medium, the content of phenolic compounds in the soil decreased by 1.1–1.6 times, especially at the norm of 10 mL. The soil treated with cultural fluid had higher rates of transformation and mineralization of organic matter than untreated. The concentration of phenolic compounds in the soil decreased, apparently, due to the activation of the microbiota resulting in the intensification of the destruction processes. An increase in the electrical conductivity of the soil with the introduction of microalgae inoculum was recorded, which may indicate the release of metal ions into the substrate. This confirms the increase in Ca and Mg.


Author(s):  
Michał Piegza ◽  
Kamil Szura ◽  
Wojciech Łaba

The mechanism of direct impact of Trichoderma fungi on other organisms is a multilayer process. The level of limiting the growth of other microorganisms is determined by the strain and often by the environment. Confirmation of the presence of extracellular biosurfactants in certain strains of Trichoderma considered as biocontrol agents was regarded as a crucial topic complementing the characterization of their interactive mechanisms. Selected strains of T. citrinoviride were cultured in media stimulating biosurfactant biosynthesis, optionally supplemented with lytic enzyme inducers. Results confirmed the anti-fungal properties of surface-active compounds in the tested culture fluids. Preparations that displayed high fungal growth inhibition presented marginal enzymatic activities of both chitinases and laminarinases, implying the inhibitory role of biosurfactants. Fractions from the foam of the culture fluid of the C1 strain, cultured on Saunders medium, and HL strain on MGP medium, without an additional carbon source, exhibited the most prominent ability to inhibit the growth of phytopathogens. Filamentous fungi capable of producing fungicidal compounds, including surfactants, may find applications in protecting the plants against agri-food pathogenic molds.


2021 ◽  
Vol 66 (7-8) ◽  
pp. 4-12
Author(s):  
M. V. Demiankova ◽  
V. S. Sadykova ◽  
A. A. Glukhova ◽  
T. A. Efimenko ◽  
Yu. V. Boykova ◽  
...  

Currently, the problem of antibiotic resistance of opportunistic and pathogenic microorganisms is extremely urgent. In order to find new effective natural antibiotics, it is necessary to intensify the search process. In the gradual selection of the most promising producers, we introduced the stage of determining the antibiotic activity of the culture fluid of the studied natural strains against the clinical isolates of hospital microorganisms with multiple resistance to medical antibiotics. Determining the species affiliation of potential producers allows to select those producers of a particular species that differ in the antimicrobial spectrum of activity from those described in the literature. Four strains of actinomycetes that showed activity against resistant clinical isolates of yeast Candida albicans, C.famata, C.parapsilosis and Cryptococcus neoformans were selected, namely: Nocardia soli INA 01217, Streptomyces bottropensis INA 01214, S.chromofuscus INA 01211 and S.netropsis INA 01190. The N.soli INA 01217 strain also shows antibiotic activity against the Gram-negative bacterium Escherichia coli ATCC 25922. These strains of actinobacterial producers were selected for subsequent chemical studies of the antimicrobial compounds formed by them.


Molecules ◽  
2021 ◽  
Vol 26 (19) ◽  
pp. 5978
Author(s):  
Jan Dohnálek ◽  
Jarmila Dušková ◽  
Galina Tishchenko ◽  
Petr Kolenko ◽  
Tereza Skálová ◽  
...  

Commensal bacterium Clostridium paraputrificum J4 produces several extracellular chitinolytic enzymes including a 62 kDa chitinase Chit62J4 active toward 4-nitrophenyl N,N′-diacetyl-β-d-chitobioside (pNGG). We characterized the crude enzyme from bacterial culture fluid, recombinant enzyme rChit62J4, and its catalytic domain rChit62J4cat. This major chitinase, securing nutrition of the bacterium in the human intestinal tract when supplied with chitin, has a pH optimum of 5.5 and processes pNGG with Km = 0.24 mM and kcat = 30.0 s−1. Sequence comparison of the amino acid sequence of Chit62J4, determined during bacterial genome sequencing, characterizes the enzyme as a family 18 glycosyl hydrolase with a four-domain structure. The catalytic domain has the typical TIM barrel structure and the accessory domains—2x Fn3/Big3 and a carbohydrate binding module—that likely supports enzyme activity on chitin fibers. The catalytic domain is highly homologous to a single-domain chitinase of Bacillus cereus NCTU2. However, the catalytic profiles significantly differ between the two enzymes despite almost identical catalytic sites. The shift of pI and pH optimum of the commensal enzyme toward acidic values compared to the soil bacterium is the likely environmental adaptation that provides C. paraputrificum J4 a competitive advantage over other commensal bacteria.


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