Kidney Cell Cultures Research Articles

Nitrogen doped carbon dots for in vitro intracellular redox modulation via optical stimulation.

Carbon dots (CDs) are promising candidates as oxygen photosensitizers, in cancer therapeutic applications due to their high quantum yield, superior chemical and photostability, low cytotoxicity and ease of chemical functionalization/tuning. Nitrogen doping can further improve oxygen photosensitization performance. Besides photodynamic therapy, however, the possibility to finely and remotely regulate the intracellular redox balance by using physical stimuli has been attracting more and more interest not only for nanotheranostic application, but also as a novel, fully biocompatible therapeutic tool. Here, we report on the synthesis of nitrogen-doped CDs by solvothermal methods starting from abundant, bioderived, low-cost precursors, and we characterize their interface with in vitro cultures of human embryonic kidney (HEK-293) cells, a widely accepted model of non-tumoral cells. While not affecting cell proliferation, synthesized CDs efficiently modulate, under visible light and physiological eustress conditions, intracellular calcium ion dynamics and reactive oxygen species concentration, resulting in a 4-fold increase. The reported results may broaden the application of CDs beyond photodynamic therapy, unveiling new opportunities in the field of redox medicine assisted by carbon-based nanomaterials and optical stimulation.

Inhibitory activity of Euterpe oleracea Mart. fruit extract in West Nile virus infection

West Nile virus (WNV) is a neurovirulent arbovirus whose epidemic capacity is enhanced by the wide occurrence of competent vectors and susceptible avian amplifying hosts. In this study, we investigated the antiviral potential of Euterpe oleracea Mart. fruit extract (EoFE) in WNV infection of monkey kidney (Vero) cell cultures. A chromatographic authentication of the extract revealed a typical two-peak fingerprint attributable to the major anthocyanins of the fruit. As assessed by plaque assays in Vero cells, the extract showed a significant concentration-dependent antiviral effect when present throughout the infection procedure, reaching a maximum inhibition of 66.8 % at 2 mg/mL without significant cytotoxicity or direct action on virus particles. A time-of-addition assay revealed that this anti-WNV effect was mostly exerted after virus entry, as incubation of Vero cells with EoFE before or during virus addition resulted in a nonsignificant decrease of infection efficiency. These results demonstrated a promising potential of EoFE in inhibiting WNV infection that can be further explored as an antiviral strategy.

Cell Type-Specific Effects of Zinc on Human Kidney Cells in Culture

Cell Type-Specific Effects of Zinc on Human Kidney Cells in Culture

Antibody Response of Mice to Bali Isolate of Canine Parvovirus Propagated in Madin-Darby Canine Kidney Cell Culture

Canine parvovirus (CPV) infection is still common among dogs, leading to severe disease with high mortality. The potential of a local isolate of CPV as an effective vaccine to prevent the disease warrants investigation. This study aimed to determine the antibody response in mice against a Bali isolate of CPV propagated in the Madin-Darby Canine Kidney (MDCK) cell culture. The virus was purified using polyethylene glycol (PEG)-6000 and mixed with an Aluminum hydroxide adjuvant. Fifteen 7-week female mice were divided into three treatment groups: treatment group 1 (PEG-purified virus and Adjuvant), treatment group 2 (crude unpurified virus and adjuvant), and treatment group 3 (adjuvant without virus), with five replicates per group. The Bali isolate of CPV was successfully replicated in MDCK cells, achieving a titer of 210-211 hemagglutination (HA) units after eight serial passages through the cell culture. The virus was confirmed as CPV by immunocytochemistry test using a monoclonal antibody and hemagglutination inhibition (HI) test using chicken anti-CPV polyclonal antibody. Following the first immunization, the antibody endpoint titer in mice immunized with PEG-purified CPV (5.6) was significantly higher than those immunized with crude unpurified CPV (4.2) and adjuvant without CPV (1.4). Similarly, after the second immunization, the antibody endpoint titer in mice immunized with PEG-purified CPV (7.6) also remained significantly higher than those immunized with crude unpurified CPV (6.4) and adjuvant without CPV (0.8). Significant increases in antibody endpoint titer were observed after the second immunization in mice immunized with PEG-purified CPV and crude unpurified CPV, but not in those given adjuvant without CPV. The Bali isolate of CPV propagated in MDCK cell culture induced a robust antibody response in mice, suggesting it’s a potential as an alternative vaccine candidate for preventing CPV infection in dogs.

In Vitro Evaluation of Anti-Hemolytic and Cytotoxic Effects of Traditional Mexican Medicinal Plant Extracts on Human Erythrocytes and Cell Cultures.

Plant extracts of fifteen plants of ethnomedicinal use in Mexico were analyzed to provide scientific knowledge of their medicinal properties through the evaluation of different biological activities such as anti-hemolytic, antioxidant, and cytotoxic effects in normal cells. Therefore, methanolic extracts were obtained from each of the plants by the Soxhlet extraction. The hemolytic activity in human erythrocytes was evaluated, as was their potential to protect the erythrocyte membrane against the 2,2'-azobis (2-methylpropionamidine) dihydrochloride (AAPH) and 1,1-diphenyl-2-picryl hydrazyl (DPPH) radicals. Finally, the toxicity of the extracts in normal cell cultures of African green monkey kidney cells (Vero) and peripheral blood mononuclear cells (PBMC) was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction method. Most of the extracts showed low hemolytic activity and high anti-hemolytic activity as well as high selectivity indices (SI) and antioxidant effects. Extracts of H. inuloides, J. dioica, and J. spicigera induced cell proliferation of the Vero cells. K. daigremontiana, A. adstringens, S. mexicanum, J. spicigera, L. tridentata, and M. tenuiflora extracts showed PBMC cell proliferation. In the present study, it was observed that the evaluated extracts did not present hemolytic activity, and some presented low toxicity when Vero and PBMC cell cultures were exposed. In conclusion, traditionally used plants possess beneficial health properties, and it is hoped that this study will serve as a basis for understanding the biological effects of traditionally used plants and may complement future studies.

Serum-Free Media Formulation Using Marine Microalgae Extracts and Growth Factor Cocktails for Madin-Darby Canine Kidney and Vero Cell Cultures.

The development of serum-free media (SFM) is critical to advance cell culture techniques used in viral vaccine production and address the ethical concerns and contamination risks associated with fetal bovine serum (FBS). This study evaluated the effects of marine microalgal extracts and growth factor cocktails on the activity of Madin-Darby canine kidney (MDCK) and Vero cells. Five marine microalgal species were used: Spirulina platensis (SP), Dunaliella salina (DS), Haematococcus pluvialis (HP), Nannochloropsis salina (NS), and Tetraselmis sp. (TS). DS and SP extracts significantly increased the proliferation rate of both MDCK and Vero cells. DS had a proliferation rate of 149.56% and 195.50% in MDCK and Vero cells, respectively, compared with that in serum-free medium (SFM). Notably, DS and SP extracts significantly increased superoxide dismutase (SOD) activity, which was 118.61% in MDCK cells and 130.08% in Vero cells for DS, and 108.72% in MDCK cells and 125.63% in Vero cells for SP, indicating a reduction in intracellular oxidative stress. Marine microalgal extracts, especially DS and SP, are feasible alternatives to FBS in cell culture as they promote cell proliferation, ensure safety, and supply essential nutrients while reducing oxidative stress.

In vitro analysis of quercetin-like compounds from mistletoe Dendrophthoe pentandra (L.) Miq as a potential antiviral agent for Newcastle disease

Background Recent evidence suggests that some flavonoid compounds obtained from crude methanol extract of mistletoe leaves (Dendrophthoe pentandra L. Miq), also known as Benalu Duku (BD), have antimicrobial effects. Thus, the plant has the potential to eliminate viruses that may cause outbreaks in chicken farms. This study aimed to prove the in vitro ability of flavonoid compounds, namely quercetin-like compounds (QLCs), to eliminate field viruses, specifically the Newcastle disease virus (NDV). Methods This research was performed in two stages. An in vitro test was used with a post-test of the control groups designed at a significance of 0.05. BD leaves (5 kg) were extracted using a maceration method with methanol and then separated into hexane, chloroform, ethyl acetate, and methanol fractions. The final extracted products were separated using semi-preparative high-performance liquid chromatography (HPLC) to obtain QLCs. The QLCs were identified and compared with quercetin using HPLC, proton and carbon nuclear magnetic resonance spectrometry, Fourier transform infrared spectrophotometry and ultra-performance liquid chromatography-mass spectrometry. The activity of QLCs was tested in vitro against the NDV at a virulence titter of 10−5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. Results Solutions of 0.05% (w/v) QLCs were discovered to have antiviral activity against NDVs, with an average cytopathogenic effect antigenicity at a 10−5 dilution (p<0.05). Conclusions QLCs from flavonoids from the leaves of BD have in vitro antiviral bioactivity against NDV at a virulence titter of 10-5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. QLCs may have the potential to be developed as medicinal compounds for the treatment of other human or animal viral infections.

In vitro analysis of quercetin-like compounds from mistletoe Dendrophthoe pentandra (L.) Miq as a potential antiviral agent for Newcastle disease

Background Recent evidence suggests that some flavonoid compounds obtained from crude methanol extract of mistletoe leaves (Dendrophthoe pentandra L. Miq), also known as Benalu Duku (BD), have antimicrobial effects. Thus, the plant has the potential to eliminate viruses that may cause outbreaks in chicken farms. This study aimed to prove the in vitro ability of flavonoid compounds, namely quercetin-like compounds (QLCs), to eliminate field viruses, specifically the Newcastle disease virus (NDV). Methods This research was performed in two stages. An in vitro test was used with a post-test of the control groups designed at a significance of 0.05. BD leaves (5 kg) were extracted using a maceration method with methanol and then separated into hexane, chloroform, ethyl acetate, and methanol fractions. The final extracted products were separated using semi-preparative high-performance liquid chromatography (HPLC) to obtain QLCs. The QLCs were identified and compared with quercetin using HPLC, proton and carbon nuclear magnetic resonance spectrometry, Fourier transform infrared spectrophotometry and ultra-performance liquid chromatography-mass spectrometry. The activity of QLCs was tested in vitro against the NDV at a virulence titter of 10−5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. Results Solutions of 0.05% (w/v) QLCs were discovered to have antiviral activity against NDVs, with an average cytopathogenic effect antigenicity at a 10−5 dilution (p<0.05). Conclusions QLCs from flavonoids from the leaves of BD have in vitro antiviral bioactivity against NDV at a virulence titter of 10-5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. QLCs may have the potential to be developed as medicinal compounds for the treatment of other human or animal viral infections.

Adding yeast extract to culture medium enhances replication of the avian coronavirus infectious bronchitis virus in chicken embryo kidney cells

Infectious bronchitis virus (IBV), an avian coronavirus, can be isolated and cultured in tracheal organ cultures (TOCs), embryonated eggs and cell cultures, the first two of which are commonly used for viral isolation. Previous studies have suggested that foetal bovine serum (FBS) can inhibit coronavirus replication in cell cultures. In this study, the replication of IBV in chicken embryo kidney (CEK) cell cultures and the Leghorn hepatocellular carcinoma (LMH) cell line was assessed using two different cell culture media containing FBS or yeast extract (YE) and two different IBV strains. The highest concentrations of viral genomes were observed when the cell culture medium (CEK) contained YE. Similar results were observed in LMH cells. Examination of the infectivity by titration demonstrated that the cell lysate from CEK cell cultures in a medium including YE contained a higher median embryo infectious dose than that from CEK cell cultures in a medium containing FBS. These results indicate that improved replication of IBV in cell cultures can be achieved by replacing FBS with YE in the cell culture medium.

Combustion-Derived Pollutants Linked with Kidney Disease in Low-Lying Flood-Affected Areas in the Balkans.

Tens of thousands of people in southern Europe suffer from Balkan endemic nephropathy (BEN), and four times as many are at risk. Incidental ingestion of aristolochic acids (AAs), stemming from the ubiquitousAristolochia clematitis(birthwort) weed in the region, leads to DNA adduct-induced toxicity in kidney cells, the primary cause of BEN. Numerous cofactors, including toxic organics and metals, have been investigated, but all have shown small contributions to the overall BEN relative to non-BEN village distribution gradients. Here, we reveal that combustion-derived pollutants from wood and coal burning in Serbia also contaminate arable soil and test as plausible causative factors of BEN. Using a GC-MS screening method, biomass-burning-derived furfural and coal-burning-derived medium-chain alkanes were detected in soil samples from BEN endemic areas levels at up to 63-times and 14-times higher, respectively, than in nonendemic areas. Significantly higher amounts were also detected in colocated wheat grains. Coexposure studies with cultured kidney cells showed that these pollutants enhance DNA adduct formation by AA, - the cause of AA nephrotoxicity and carcinogenicity. With the coincidence of birthwort-derived AAs and the widespread practice of biomass and coal burning for household cooking and heating purposes and agricultural burning in rural low-lying flood-affected areas in the Balkans, these results implicate combustion-derived pollutants in promoting the development of BEN.

In vitro analysis of quercetin-like compounds from mistletoe Dendrophthoe pentandra (L.) Miq as a potential antiviral agent for Newcastle disease

Background Recent evidence suggests that some flavonoid compounds obtained from crude methanol extract of mistletoe leaves (Dendrophthoe pentandra L. Miq), also known as Benalu Duku (BD), have antimicrobial effects. Thus, the plant has the potential to eliminate viruses that may cause outbreaks in chicken farms. This study aimed to prove the in vitro ability of flavonoid compounds, namely quercetin-like compounds (QLCs), to eliminate field viruses, specifically the Newcastle disease virus (NDV). Methods This research was performed in two stages. An in vitro test was used with a post-test of the control groups designed at a significance of 0.05. BD leaves (5 kg) were extracted using a maceration method with methanol and then separated into hexane, chloroform, ethyl acetate, and methanol fractions. The final extracted products were separated using semi-preparative high-performance liquid chromatography (HPLC) to obtain QLCs. The QLCs were identified and compared with quercetin using HPLC, proton and carbon nuclear magnetic resonance spectrometry, Fourier transform infrared spectrophotometry and ultra-performance liquid chromatography-mass spectrometry. The activity of QLCs was tested in vitro against the NDV at a virulence titter of 10−5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. Results Solutions of 0.05% (w/v) QLCs were discovered to have antiviral activity against NDVs, with an average cytopathogenic effect antigenicity at a 10−5 dilution (p<0.05). Conclusions QLCs from flavonoids from the leaves of BD have in vitro antiviral bioactivity against NDV at a virulence titter of 10-5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. QLCs may have the potential to be developed as medicinal compounds for the treatment of other human or animal viral infections.

Impact of juice processing of Costa Rican guava (Psidum friedrichsthalianum) on the physicochemical properties, total phenols and antioxidant capacity

Psidium plants are commonly consumed in processed forms, leading to a need to evaluate the impact of processing on bioactive compounds. The purpose of this study is to assess the effect of Costa Rican guava juice processing on physicochemical and antioxidant characteristics. Five samples corresponding to distinct phases in the juice processing were evaluated. Total polyphenols were measured using the Folin-Ciocalteu method, while antioxidant activity was assessed using various methodologies, including chemical methods (DPPH and ORAC) and cell assays with kidney cell cultures, liver homogenates, and erythrocytes. Results revealed variations in the impact of the juice processing stages on total polyphenols and for each antioxidant assay, evidencing that different polyphenols counteract in each technique. Significant decreases (ranging from 27 % to 58 %, p < 0.05) were primarily observed during the pressing stage, while thermal treatments resulted in non-significant reductions. This study demonstrated a decline in antioxidant activity during the processing of Costa Rican guava juice underscoring the need for continued efforts to optimize processing conditions and enhance the retention of health-promoting properties in the juice.

#2485 CellMatchR—RNA-seq based matching of kidney cell lines to kidney reference cell types

Abstract Background and Aims Cultured kidney cell lines are broadly used to model the physiology and pathophysiology of the kidney. Due to immortalization, passaging and culture conditions, a cell line's gene expression profile might differ greatly from the primary cells it was originally derived from. This might be further enhanced by experimental treatments with compounds or genetic modifications. It is therefore a relevant question if a given cell culture system is still the appropriate model for a certain disease or scientific investigations. Reasoning that RNA sequencing (RNA-seq) based transcriptomes are generated as part of many experimental setups and hence may be used to address this question, we developed and tested an RNA-seq based approach, CellMatchR. This approach matches kidney cell lines, primary cells and even tissue specimen to known kidney reference cell types to determine which primary kidney cell type is the most similar and to what degree global gene expression or the expression of selected marker genes differs. Method CellMatchR uses published murine and human kidney single cell- and tubule-level transcriptomic datasets from healthy mice and human donors as references. Single cell datasets were further processed to pseudobulk references for each of the contained cell types. Then RNA-seq data from cell lines or tissues of interest (test data) was compared to the reference (pseudo)bulk data with Spearman correlations of gene counts-per-million values (CPM) or Euclidean distance using log-transformed CPM values. Both approaches were systematically tested for various combinations of test and reference datasets across different species and using global gene expression (i.e. all genes measured in reference and test datasets) and a set of 315 manually curated, kidney cell type marker genes. Results We sequentially used different kidney tissue types, primary cells and cell lines as positive controls and matched them to our reference datasets. Spearman correlations of gene expression rankings showed the highest correlation coefficients with biologically correct reference cell types (examples in Fig. 1A-C). We found Spearman correlations to be superior to Euclidean distances as method of comparison. Analyses based on global gene expression compared to our curated set of 315 kidney cell type marker genes yielded similar results. Notably, correlation coefficients were higher and showed less variation between reference cell types when using the global gene set. Matchings across species, i.e. using murine test and human reference data or vice versa still yielded mostly correct results. However, correlation coefficients were generally lower (i.e. rho = 0.9 vs. 0.6) and varied more across reference datasets if comparisons were performed across species. Using published RNA-seq data for different cell lines of the proximal tubule (e.g. human HK-2 and opossum OKH cells) we observed low similarities with proximal tubule cells (Fig. 1D), whereas two tested cell lines of the collecting duct (mIMCD-3 and mpkCCD cells) plausibly showed the highest similarity to medullary cell types like collecting duct and Loop of Henle cells. The most similar reference cell types did not change in mIMCD-3 cells when kept in 2-dimensional vs. 3-dimensional culture conditions, and in mpkCCD cells when the osmolality of the culture medium was changed from 300 to 600 mosmol/kg. Also, a Pkd1 knockout in mIMCD-3 cells did not change the most similar reference cell type in our analyses (Fig. 1E and F). Conclusion Our CellMatchR approach uses publicly available kidney single cell and bulk RNA-seq datasets and combines these with simple, computationally fast yet effective statistical methods to determine similarities of kidney cell lines and tissue samples to reference cell types of interest. It can easily be implemented by trained users or integrated into online resources for usage with a visual interface. It relies on RNA-seq data from the cell lines of interest and hence presents a feasible complementary method to check the general similarity of cell culture models to kidney cell types and assess their stability across experimental conditions.

A Single-Step Method for Harvesting Influenza Viral Particles from MDCK Cell Culture Supernatant with High Yield and Effective Impurity Removal

Influenza vaccines, which are recommended by the World Health Organization (WHO), are the most effective preventive measure against influenza virus infection. Madin–Darby canine kidney (MDCK) cell culture is an emerging technology used to produce influenza vaccines. One challenge when purifying influenza vaccines using this cell culture system is to efficiently remove impurities, especially host cell double-stranded DNA (dsDNA) and host cell proteins (HCPs), for safety assurance. In this study, we optimized ion-exchange chromatography methods to harvest influenza viruses from an MDCK cell culture broth, the first step in influenza vaccine purification. Bind/elute was chosen as the mode of operation for simplicity. The anion-exchange Q chromatography method was able to efficiently remove dsDNA and HCPs, but the recovery rate for influenza viruses was low. However, the cation-exchange SP process was able to simultaneously achieve high dsDNA and HCP removal and high influenza virus recovery. For the SP process to work, the clarified cell culture broth needed to be diluted to reduce its ionic strength, and the optimal dilution rate was determined to be 1:2 with purified water. The SP process yielded a virus recovery rate exceeding 90%, as measured using a hemagglutination units (HAUs) assay, with removal efficiencies over 97% for HCPs and over 99% for dsDNA. Furthermore, the general applicability of the SP chromatography method was demonstrated with seven strains of influenza viruses recommended for seasonal influenza vaccine production, including H1N1, H3N2, B (Victoria), and B (Yamagata) strains, indicating that the SP process could be utilized as a platform process. The SP process developed in this study showed four advantages: (1) simple operation, (2) a high recovery rate for influenza viruses, (3) a high removal rate for major impurities, and (4) general applicability.

Analysis of Toxicity of Recombinant Pneumolysin from Streptococcus pneumoniae.

We studied toxicity of recombinant Streptococcus pneumoniae pneumolysin protein in experiments on mice and its cytopathogenic effect on cultures of Vero green monkey kidney cells and human lung carcinoma A549 cells in vitro. In vivo and in vitro experiments proved the absence of compromised toxicity and direct cytopathogenic action of the recombinant protein.

Life in the Slow Lane: The Rate of Protein Synthesis Is Slower in the Protoendothermic Tenrec than the Boreoeutherian Golden-Mantled Ground Squirrel

In order to address mechanisms of how hibernating mammals depress protein synthesis during torpor, we developed primary cell cultures from golden-mantled ground squirrels (Spermophilus [Callospermophilus] lateralis) and tenrecs (Tenrec ecaudatus). During torpor in squirrels, there is an acute uncoupling of translational initiation and elongation at body temperature (Tb) = 18°C as animals enter torpor. As a result, there are robust changes to the polysome profile of squirrels depending on state. Protein synthesis is depressed to 0.23 to 0.5% of active rates during torpor. In the bizarre, Afrotherian Tenrec ecaudatus, metabolism and Tb are remarkably plastic. Tenrecs may be fully active with a Tb of 12 °C or hibernating at a Tb of 28 °C. Moreover, oxygen consumption rates may vary 25 fold in active tenrecs. With this high variability in Tb, one might expect that these tenrecs might not be able to use Tb to control protein synthesis. Liver polysome profiles were confusing- there were no real changes between states or temperature. Rather, all tenrecs had a rather modest polysome profile with little heavy material typical of very active protein synthesis. To address this, we developed liver (do not grow well) and kidney cell (grow very well) cultures from ground squirrels and tenrecs. Our results indicate that the translation rate in tenrec primary cells is ~30 % of the translation rate in golden-mantled ground squirrel (p &lt; 0.05). We interpret these results as tenrecs living ‘Life in the Slow Lane.’ By experiencing these low rates of a process critical to homeostasis, we contend tenrecs are avoiding rapid changes as a consequence of their rapid changes in Tb or metabolism. In effect, ‘driving slowly’ allows a tenrec to avoid major accidents which would imperil the animal. Perhaps not surprisingly, tenrec litters are enormous with as many as 32 confirmed young in a single litter. This research was supported by the Translational Research Institute for Space Health (TRISH). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of Heavy Metal Co-Exposure on the formation of DNA Adducts from Aristolochic Acid I: Implications for Balkan Endemic Nephropathy Development.

Accumulated evidence has shown that Balkan endemic nephropathy (BEN) is a multifactorial environmental disease, with exposure to aristolochic acids (AA), and the associated DNA adduct formation, as a key causative factor of BEN development. Here, we show that coexposure to arsenic, cadmium, and iron increases the DNA adduct formation of AA in cultured kidney cells, while exhibiting both an exposure concentration and duration dependence. In contrast, coexposure to calcium and copper showed a decreasing DNA adduct formation. Because DNA damage is responsible for both the nephrotoxicity and carcinogenicity of AA, these results shed greater light on the endemic nature of BEN.

In vitro analysis of quercetin-like compounds from mistletoe Dendrophthoe pentandra (L.) Miq as a potential antiviral agent for Newcastle disease

Background Recent evidence suggests that some flavonoid compounds obtained from crude methanol extract of mistletoe leaves (Dendrophthoe pentandra L. Miq), also known as Benalu Duku (BD), have antimicrobial effects. Thus, the plant has the potential to eliminate viruses that may cause outbreaks in chicken farms. This study aimed to prove the in vitro ability of flavonoid compounds, namely quercetin-like compounds (QLCs), to eliminate field viruses, specifically the Newcastle disease virus (NDV). Methods This research was performed in two stages. An in vitro test was used with a post-test of the control groups designed at a significance of 0.05. BD leaves (5 kg) were extracted using a maceration method with methanol and then separated into hexane, chloroform, ethyl acetate, and methanol fractions. The final extracted products were separated using semi-preparative high-performance liquid chromatography (HPLC) to obtain QLCs. The QLCs were identified and compared with quercetin using HPLC, proton and carbon nuclear magnetic resonance spectrometry, Fourier transform infrared spectrophotometry, and ultra-performance liquid chromatography–mass spectrometry. The activity of QLCs was tested in vitro against the NDV at a virulence titer of 10−5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. Results Solutions of 0.05% (w/v) QLCs were discovered to have antiviral activity against NDVs, with an average cytopathogenic effect antigenicity at a 10−5 dilution (p&lt;0.05). Conclusions QLCs from flavonoids from the leaves of BD have in vitro antiviral bioactivity against NDV at a virulence titer of 10-5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. QLCs may have the potential to be developed as medicinal compounds for the treatment of other human or animal viral infections.

Antigenic Activity of Canine Enteric Coronavirus Strain "Rich" in Experiments with Rabbits, Ferrets and Guinea Pigs

Introduction. Canine enteric coronavirus is a widespread infection, especially dangerous for the puppies of up to 12 weeks old kept in shelters and breeding kennels. The causative agent Alphacoronavirus 1 has a high mutational variability. Foreign sources have repeatedly reported on the isolation and study of the new strains of canine coronavirus infection, including the pantropic ones, causing systemic pathology and death of animals. In this regard, the currently produced vaccines are not effective enough. The present research is intended to substantiate one of the solutions to the problem. The aim of the work is to evaluate the antigenic potential of the canine enteric coronavirus strain “Rich” in terms of its reliability for being included in the combined vaccines against canine viral diseases.Materials and Methods. A purified suspension culture of the canine enteric coronavirus strain “Rich” with the infectivity titers 4.0±0.25 lg TCID50/cm3 (TCID — Tissue Culture Infectious Dose) was used in the work. Antigenic properties were studied in laboratory animals: rabbits, ferrets and guinea pigs. The virus neutralising antibody titers for the canine enteric coronavirus in the blood serum of the experimental animals was determined by a neutralisation test using the “standard” viral dose of 100 TCID50/cm3 in the feline kidney cell culture. The material for the test was obtained from the Cell Culture Bank of the Federal Centre for Animal Health.Results. In animals initially seronegative to the virus, an increase in the CCoV virus-neutralising antibodies (VNA) titers has been recorded 7 days after a single injection of the suspension of the canine enteric coronavirus strain “Rich”. Over the time, the level has been increasing and has reached its maximum value on the 21st day. The mean value in the group of rabbits has been 4.08±0.36 log2 SN50, ferrets — 3.72±0.35 log2 SN50 and 3.77±0.63 log2 SN50, guinea pigs — 4.12±0.34 log2 SN50. Then the values of the virus neutralising antibody titers have decreased, but remained at a fairly high level for 35 days (observation time). At the same time, the physiological state of the animals has not changed.Discussion and Conclusion. In general, the results of the experiments are consistent with the conclusions of the other authors. New data has been obtained by studying the antigenic properties of the strain in ferrets. The strain injected into them has demonstrated the clearly expressed antigenic activity and did not cause a general or local pathological response of the body. The “Rich” is promising for further study and can be included in the combined vaccines against canine and other animal viral diseases.

Lanthanide-modified graphene oxide and nanodiamond materials and their cytotoxicity

Integrating lanthanide elements with carbon-based nanomaterials results in the creation of hybrid materials and nanocomposites with unique properties. We report a functionalization approach free of organic solvents for modifying graphene oxide (GO) and nanodiamond (ND) with various lanthanides (La(III), Ce(III), Eu(III), Gd(III), Tb(III) and Ho(III)). The aqueous-phase impregnation with their acetates was followed first by drying at 80 °C and then by heating at 300 °C for 2 h in air conditions. Scanning electron microscopy revealed that after the initial drying, the samples exhibited the presence of larger lanthanide-containing particles, whereas after the heating at 300 °C they became indistinguishable, and thus the lanthanide species became more homogeneously distributed throughout GO and ND. The toxicity, which is commonly associated with the presence of lanthanides, was evaluated in terms of the ability to inhibit the proliferation of COS-7 monkey kidney cell cultures. Most composites demonstrated a dose-dependent toxicity, with significantly lower cytotoxic effects compared to both the thermally treated GO and ND, as well as the pristine GO. The composites functionalized with gadolinium were among the least toxic at the maximum tested concentration (100 µg/ml). The ND-based composites were not cytotoxic at 50 µg/ml, the lowest tested concentration.