scholarly journals Electro-Optical Analysis of Cell Viability of Tularemia Microbe Vaccine Strain

2020 ◽  
pp. 50-55
Author(s):  
O. A. Volokh ◽  
S. V. Borisova ◽  
D. N. Bibikov ◽  
E. M. Kuznetsova ◽  
Yu. I. Samokhvalova ◽  
...  
Keyword(s):  
Cell Viability ◽  
Vaccine Strain ◽  
Specific Activity ◽  
Total Concentration ◽  
Advanced Technology ◽  
Polarizability Anisotropy ◽  
Vaccine Production ◽  
Optical Analysis ◽  
Bacteriological Method ◽  
Tularemia Vaccine

Objective: to study the possibility of applying electro-optical analysis for the assessment of cell viability of tularemia microbe vaccine strain at different stages of experimental live tularemia vaccine production.Materials and methods. The research object was a cell culture of Francisella tularensis 15 NIIEG.  Investigations were carried out at all stages of experimental live tularemia vaccine (ELTV) manufacturing according to an advanced technology: cultivation, concentrating, diafiltration, mixing with drying media, stabilization, and storage (two-year period of observation). Electro-optical analysis by the parameter “polarizability anisotropy” of bacterial cell was conducted with the help of EloTrace (EloSystems, Germany). Total concentration of cells was evaluated using density metering at 590 nm and spectrometry – at 650 nm. Viability was assessed through inoculation of plates with FT-agar.Results and discussion. The experiment has demonstrated that the change in polarizability anisotropy of the cell at the frequencies of 900 kHz and 2100 kHz, reflecting the state of cytoplasm and cytoplasmic membrane, respectively, is the earliest response to changes in vital indicators of bacterial culture in the process of cultivation. Thereby, the decrease in viability of F. tularensis cells occurrs well before the decrease in cell concentration. We have shown the preservation of viability of F. tularensis 15 NIIEG cells at all stages of experimental live tularemia vaccine production. Electro-optical analysis allows for registering the changes in vital parameters of microorganism cells in real-time mode, while the assessment of viability applying bacteriological method takes up to 5 days. Different stages of tularemia vaccine manufacturing have impact on the vital indicators of F. tularensis cells, and electro-optical analysis is a prospect method of control of such parameter as “Specific activity (the number of live microbial cells)”.

Determination of Specific Activity of Cholera Chemical Vaccine Components using Cell Culture

2020 ◽  
Vol 36 (3) ◽  
pp. 82-89
Author(s):  
O.V. Gromova ◽  
O.S. Durakova ◽  
S.V. Generalov ◽  
L.F. Livanova ◽  
O.A. Volokh
Keyword(s):  
Cell Culture ◽  
Vibrio Cholerae ◽  
Production Control ◽  
Specific Activity ◽  
Methodological Approach ◽  
Toxin Production ◽  
Submerged Cultivation ◽  
Vaccine Production ◽  
Antigenic Activity

Том 36(2020) №3 стр. 82-89; DOI 10.21519/0234-2758-2020-36-3-82-89А.В. Гаева1*, О.В. Громова1, О.С. Дуракова1, С.В. Генералов1, Л.Ф. Ливанова1, О.А. Волох1 Определение специфической активности компонентов холерной химической вакцины с использованием культуры клеток 1ФКУЗ «Российский научно-исследовательский противочумный институт «Микроб»» Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека, Саратов 410005 *[email protected] Поступила - 2019-11-26; После доработки - 2020-03-16; Принята к публикации - 2020-05-15 Список литературы Описаны методы определения динамики продукции токсинов штаммом Vibrio cholerae 569B при глубинном культивировании в биореакторе и антигенной активности специфической фракции холерогена-анатоксина по анатоксинсвязыванию с использованием клеточных культур. Показана высокая степень соответствия результатов, полученных методами, применяемыми для контроля этапов производства холерной химической вакцины и рассмотренными в данной работе. Отмечено, что применение клеточной линии СНО-К1 наиболее перспективно для замены биомоделей на промежуточных этапах контроля активных компонентов холерной химической вакцины. Разработанный методический подход впервые предлагается использовать на этапах производства холерной бивалентной химической вакцины. культура клеток, Vibrio cholerae, холерная химическая вакцина, контроль производства, холера. Vol 36(2020) N 3 p. 82-89; DOI 10.21519/0234-2758-2020-36-3-82-89A.V. Gaeva1*, O.V. Gromova1, O.S. Durakova1, S.V. Generalov1, L.F. Livanova1, O.A. Volokh1 Determination of Specific Activity of Cholera Chemical Vaccine Components using Cell Culture 1Russian Research Anti-Plague Institute «Microbe» of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Saratov, 410005 *[email protected] Received - 26.11.2019; Accepted - 15.05.2020 References The methods has been described to determine the dynamics of toxin production by the Vibrio cholerae 569B strain during submerged cultivation in bioreactor and of the antigenic activity of specific choleragen anatoxin fraction by anatoxin binding levels using cell cultures. High degree of consistency was observed between the results obtained via the method under consideration and those obtained via control methods at different stages of cholera chemical vaccine production. It was shown that the CHO-K1 cell line is the most promising substitute for biomodels at the intermediate stages of control of active cholera chemical vaccine components. The developed methodological approach was first proposed for use at the stages of cholera chemical bivalent vaccine manufacturing. cell culture, Vibrio cholerae, cholera chemical vaccine, production control, cholera.

scholarly journals In Vitro Cytotoxicity of Oleanolic/Ursolic Acids-Loaded in PLGA Nanoparticles in Different Cell Lines

Pharmaceutics ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 362 ◽  
Author(s):  
Amélia M. Silva ◽  
Helen L. Alvarado ◽  
Guadalupe Abrego ◽  
Carlos Martins-Gomes ◽  
Maria L. Garduño-Ramirez ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Specific Activity ◽  
Polymeric Nanoparticles ◽  
Hepatoma Cell ◽  
Plga Nanoparticles ◽  
Hepatoma Cell Line ◽  
Human Hepatoma Cell

Oleanolic (OA) and ursolic (UA) acids are recognized triterpenoids with anti-cancer properties, showing cell-specific activity that can be enhanced when loaded into polymeric nanoparticles. The cytotoxic activity of OA and UA was assessed by Alamar Blue assay in three different cell lines, i.e., HepG2 (Human hepatoma cell line), Caco-2 (Human epithelial colorectal adenocarcinoma cell line) and Y-79 (Human retinoblastoma cell line). The natural and synthetic mixtures of these compounds were tested as free and loaded in polymeric nanoparticles in a concentration range from 2 to 32 µmol/L. The highest tested concentrations of the free triterpene mixtures produced statistically significant cell viability reduction in HepG2 and Caco-2 cells, compared to the control (untreated cells). When loaded in the developed PLGA nanoparticles, no differences were recorded for the tested concentrations in the same cell lines. However, in the Y-79 cell line, a decrease on cell viability was observed when testing the lowest concentration of both free triterpene mixtures, and after their loading into PLGA nanoparticles.

scholarly journals SUPPRESSION OF MANGANESE SUPEROXIDE DISMUTASE ACTIVITY IN ROTENONE-TREATED HUMAN GLIOBLASTOMA T98G CELLS REDUCES CELL VIABILITY

2018 ◽  
Vol 11 (1) ◽  
pp. 48
Author(s):  
Septelia Inawati Wanandi ◽  
Novi Silvia Hardiany ◽  
Nurjati Chairani Siregar ◽  
Mohamad Sadikin
Keyword(s):  
Superoxide Dismutase ◽  
Cell Viability ◽  
Manganese Superoxide Dismutase ◽  
Specific Activity ◽  
High Dose ◽  
Human Glioblastoma ◽  
Manganese Superoxide ◽  
Rotenone Treatment ◽  
Intracellular Ros

  Objective: Glioma is the most common human primary brain tumor which is highly resistant to oxidative stress-based anticancer. The aim of this study was to analyze the effect of rotenone-induced reactive oxygen species (ROS) on the modulation of manganese superoxide dismutase (MnSOD) expression and cell viability in human glioblastoma (GBM) T98G cells.Methods: In this in vitro experimental study, T98G cells were treated with high-dose rotenone (0.5, 5, and 50 μM, respectively). Following rotenone treatment and intracellular ROS, both peroxide and superoxide radicals were determined. Moreover, we analyzed MnSOD mRNA expression, protein, and specific activity, as well as cell survival including viability, proliferation, apoptosis, and mitochondrial structure.Results: High-dose rotenone treatment of T98G cells significantly increased intracellular ROS and MnSOD mRNA, but its protein and specific activity definitely decreased. The treatment also led to a reduction of cell viability, enhancement of apoptosis, and disruption of mitochondrial integrity.Conclusion: Overproduction of ROS in rotenone-treated human GBM T98G cells could suppress the MnSOD protein level and activity even though mRNA synthesis has been increased. This modulation led to reduced survival of T98G cells through induction of cell death rather than inhibition of cell proliferation.

scholarly journals Complete Genome Sequence of Mycobacterium bovis BCG SL222 Sofia, the First WHO Reference Reagent for the M. bovis BCG Vaccine of the Russian BCG-I Substrain

2021 ◽  
Vol 10 (3) ◽  
Author(s):  
Stefan Panaiotov ◽  
Yordan Hodzhev ◽  
Vladimir Tolchkov ◽  
Alexander Mihailov ◽  
Roumen Kofinov ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Vaccine Strain ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Bcg Vaccine ◽  
Vaccine Production ◽  
Content Type ◽  
Mycobacterium Bovis Bcg

ABSTRACT Mycobacterium bovis BCG SL222 Sofia is a substrain descending from the Russian BCG-I vaccine strain. Here, we report the complete genome sequence of BCG SL222 Sofia, which will facilitate identity assurance and will contribute to more consistent manufacturing, standardization, and differentiation of substrains used in vaccine production.

scholarly journals Effect of Various Stabilizers on Viability of Lyophilized Pasteurella multocida B:3,4 for Use as Hemorrhagic Septicemia Vaccine

2021 ◽  
Vol 25 (03) ◽  
pp. 567-574
Author(s):  
Sajid Mahmood Sajid
Keyword(s):  
Cell Viability ◽  
Vaccine Strain ◽  
Pasteurella Multocida ◽  
Live Vaccine ◽  
Live Vaccine Strain ◽  
Suitable Temperature ◽  
Effective Stabilizer ◽  
Skimmed Milk ◽  
Subsequent Storage ◽  
Storage Temperatures

Hemorrhagic septicemia (HS) is a devastating disease of cattle and buffaloes. The live aerosol vaccine is the best option to control HS. However, stability and viability of live vaccine is an issue. The present study was conducted to investigate the effect of three extraneous stabilizers trehalose, skimmed milk and lactalbumin on the viability of the live vaccine strain Pasteurella multocida B:3,4. The viability of the strain was evaluated using various concentrations (5, 10, 15 and 20%) of these three stabilizers. Moreover, viability of P. multocida B:3,4 was also determined at four different storage temperatures (-20, 4, 25 and 37°C). The duration of lyophilization cycle was also standardized for highest survival of cells. The data showed that trehalose and lactalbumin ensued percentage of viability as 91.89±0.08 and 80.38±2.57 respectively. Skimmed milk as stabilizer did not prove to defend cells during lyophiliztion and subsequent storage and exhibited cell viability approximately 0.47±0.009%. The study indicated that most effective stabilizer for lyophiliztion of P. multocida B:3,4 was trehalose at 15% concentration and was most suitable temperature for storage of lyophilized P. multocida B:3,4. © 2021 Friends Science Publishers

scholarly journals Immunogenicity and Reactogenicity of Francisella tularensis Strain 15/23-1 recA - the Candidate for a New Live Tularemia Vaccine

2015 ◽  
Vol 14 (6) ◽  
pp. 74-86 ◽  
Author(s):  
A. N. Mokrievich ◽  
G. M. Titareva ◽  
T. I. Kombarova ◽  
E. A. Ganina ◽  
T. B. Kravchenko ◽  
...  
Keyword(s):  
Vaccine Strain ◽  
Guinea Pigs ◽  
Virulent Strain ◽  
Gamma Interferon ◽  
Reca Gene ◽  
Experimental Animals ◽  
Significant Difference ◽  
The Difference ◽  
Spleen And Lymph Nodes ◽  
Tularemia Vaccine

F. tularensis strain 15/23-1 recA is different from the vaccine strain F. tularensis line 15 NIIEG by lacking one from the two copies of iglC gene and recA gene has reduced its reactogenicity for experimental animals (BALB/c mice and guinea pigs): transient weight reduction is less expressed, as well as leukopenia and thrombocytopenia during the first week after the immunization, compared to vaccine strain. The concentration of gamma-interferon in mice blood serum after 7 days immunized with strain 15/23-1 recA, was 5 times less than in mice immunized with strain 15 NIIEG. The virulence of the modified strain for mice is less than for the strain 15 NIIEG by more than two orders of magnitude. According to histological studies F. tularensis strain 15/23-1 recA stimulates morphologically a more expressed immune rearrangement in the spleen and lymph nodes as compared to F. tularensis strain 15 NIIEG. The difference of strain 15/23-1 recA from vaccine is that it is not detected in the spleens of experimental animals at 21 days after immunization. The level of specific antibodies in sera of immunized mice and guinea pigs were almost identical in both variants and there was no significant difference in the amount of gamma interferon synthesized by splenocytes of mice under specific induction. Immune mice were protected against subcutaneous challenge with virulent strain of tularemia 503 (subsp. holarctica) and protective immunity formed by the strains was lasting more than 180 days.

scholarly journals SUPPRESSION OF MANGANESE SUPEROXIDE DISMUTASE ACTIVITY IN ROTENONE-TREATED HUMAN GLIOBLASTOMA T98G CELLS REDUCES CELL VIABILITY

2018 ◽  
Vol 11 (1) ◽  
pp. 48
Author(s):  
Septelia Inawati Wanandi ◽  
Novi Silvia Hardiany ◽  
Nurjati Chairani Siregar ◽  
Mohamad Sadikin
Keyword(s):  
Superoxide Dismutase ◽  
Cell Viability ◽  
Manganese Superoxide Dismutase ◽  
Specific Activity ◽  
High Dose ◽  
Human Glioblastoma ◽  
Manganese Superoxide ◽  
Rotenone Treatment ◽  
Intracellular Ros

  Objective: Glioma is the most common human primary brain tumor which is highly resistant to oxidative stress-based anticancer. The aim of this study was to analyze the effect of rotenone-induced reactive oxygen species (ROS) on the modulation of manganese superoxide dismutase (MnSOD) expression and cell viability in human glioblastoma (GBM) T98G cells.Methods: In this in vitro experimental study, T98G cells were treated with high-dose rotenone (0.5, 5, and 50 μM, respectively). Following rotenone treatment and intracellular ROS, both peroxide and superoxide radicals were determined. Moreover, we analyzed MnSOD mRNA expression, protein, and specific activity, as well as cell survival including viability, proliferation, apoptosis, and mitochondrial structure.Results: High-dose rotenone treatment of T98G cells significantly increased intracellular ROS and MnSOD mRNA, but its protein and specific activity definitely decreased. The treatment also led to a reduction of cell viability, enhancement of apoptosis, and disruption of mitochondrial integrity.Conclusion: Overproduction of ROS in rotenone-treated human GBM T98G cells could suppress the MnSOD protein level and activity even though mRNA synthesis has been increased. This modulation led to reduced survival of T98G cells through induction of cell death rather than inhibition of cell proliferation.

Sensitivity of Oncogenic KRAS-Expressing Cells to CDK9 Inhibition

2021 ◽  
pp. 247255522110088
Author(s):  
Lick Pui Lai ◽  
Viviane Brel ◽  
Kanika Sharma ◽  
Julia Frappier ◽  
Nadia Le-Henanf ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Specific Activity ◽  
Mammalian Target Of Rapamycin ◽  
Mouse Embryonic Fibroblast ◽  
Wild Type ◽  
Compound Library ◽  
Cell Viability Assay ◽  
Viability Assay

Oncogenic forms of KRAS proteins are known to be drivers of pancreatic, colorectal, and lung cancers. The goal of this study is to identify chemical leads that inhibit oncogenic KRAS signaling. We first developed an isogenic panel of mouse embryonic fibroblast (MEF) cell lines that carry wild-type RAS, oncogenic KRAS, and oncogenic BRAF. We validated these cell lines by screening against a tool compound library of 1402 annotated inhibitors in an adenosine triphosphate (ATP)-based cell viability assay. Subsequently, this MEF panel was used to conduct a high-throughput phenotypic screen in a cell viability assay with a proprietary compound library. All 126 compounds that exhibited a selective activity against mutant KRAS were selected and prioritized based on their activities in secondary assays. Finally, five chemical clusters were chosen. They had specific activity against SW620 and LS513 over Colo320 colorectal cancer cell lines. In addition, they had no effects on BRAFV600E, MEK1, extracellular signal-regulated kinase 2 (ERK2), phosphoinositide 3-kinase alpha (PI3Kα), AKT1, or mammalian target of rapamycin (mTOR) as tested in in vitro enzymatic activity assays. Biophysical assays demonstrated that these compounds did not bind directly to KRAS. We further identified the mechanism of action and showed that three of them have CDK9 inhibitory activity. In conclusion, we have developed and validated an isogenic MEF panel that was used successfully to identify RAS oncogenic or wild-type allele-specific vulnerabilities. Furthermore, we identified sensitivity of oncogenic KRAS-expressing cells to CDK9 inhibitors, which warrants future studies of treating KRAS-driven cancers with CDK9 inhibitors.

scholarly journals Drug repurposing: phosphate prodrugs of anticancer and antiviral FDA-approved nucleosides as novel antimicrobials

2020 ◽  
Vol 75 (10) ◽  
pp. 2864-2878
Author(s):  
Fabrizio Pertusati ◽  
Elisa Pileggi ◽  
Jennifer Richards ◽  
Mandy Wootton ◽  
Thijs Van Leemputte ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Cell Viability ◽  
Specific Activity ◽  
Drug Repurposing ◽  
Resistance Mechanisms ◽  
Antibiofilm Activity ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Nucleoside Drugs

Abstract Objectives Following a drug repurposing approach, we aimed to investigate and compare the antibacterial and antibiofilm activities of different classes of phosphate prodrugs (HepDirect, cycloSal, SATE and mix SATE) of antiviral and anticancer FDA-approved nucleoside drugs [zidovudine (AZT), floxouridine (FUDR) and gemcitabine (GEM)] against a variety of pathogenic Gram-positive and -negative bacteria. Methods Ten prodrugs were synthesized and screened for antibacterial activity against seven Gram-negative and two Gram-positive isolates fully susceptible to traditional antibiotics, alongside six Gram-negative and five Gram-positive isolates with resistance mechanisms. Their ability to prevent and eradicate biofilms of different bacterial pathogens in relation to planktonic growth inhibition was also evaluated, together with their effect on proliferation, viability and apoptosis of different eukaryotic cells. Results The prodrugs showed decreased antibacterial activity compared with the parent nucleosides. cycloSal-GEM-monophosphate (MP) prodrugs 20a and 20b were the most active agents against Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus) and retained their activity against antibiotic-resistant isolates. cycloSal-FUDR-MP 21a partially retained good activity against the Gram-positive bacteria E. faecalis, Enterococcus faecium and S. aureus. Most of the prodrugs tested displayed very potent preventive antibiofilm specific activity, but not curative. In terms of cytotoxicity, AZT prodrugs did not affect apoptosis or cell viability at the highest concentration tested, and only weak effects on apoptosis and/or cell viability were observed for GEM and FUDR prodrugs. Conclusions Among the different prodrug approaches, the cycloSal prodrugs appeared the most effective. In particular, cycloSal (17a) and mix SATE (26) AZT prodrugs combine the lowest cytotoxicity with high and broad antibacterial and antibiofilm activity against Gram-negative bacteria.

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