Nonviable Cell Research Articles

Seasonal changes in the viability and abundance of bacterial cells in the snowpack ecosystem of a High Arctic ice cap

ABSTRACT Microbes play an essential role in nutrient turnover within Arctic environments, and their contribution to biogeochemical cycles can depend on several factors, including but not limited to cell viability. In this study, we employed the SYBR-PI dual cell stain to epifluorescence microscopy to enumerate proportions of potentially viable and non-viable bacterial cell populations within a melting snowpack on an ice cap, Foxfonna in Svalbard. Non-viable cells dominated on Foxfonna (2.5 ± 0.36 × 107 cells m−2) during the June to early July period, when biological production was usually at its peak. Furthermore, non-viable cells also dominated the total cell abundance within superimposed ice (223 ± 242 cells mL−1) and glacial ice (695 ± 717 cells mL−1) beneath the snow. We propose that the rapid, early loss of cell viability was caused by a number of abiotic and biotic factors. Hence, necromass (dead cell residue) contributed to the export of organic matter to downstream ecosystems.

Chitosan nanoparticle-mediated drug delivery for linoleic acid isolated from Melastomastrum capitatum Fern. leaf extract against MCF-7 and OV7 cancer cells

The number of cancer cases worldwide is rising, resulting in approximately 40 million deaths each year due to the inability to effectively deliver drugs that specifically target cancer cells. The study's aim was to utilize chitosan nanoparticles containing linoleic acid extracted from Melastomastrum capitatum leaves to specifically target MCF-7 and OV7 cancer cells. The study conducted toxicity tests on Wistar rats to evaluate the potential harmful effects of the extract at different doses. It also examined the extract's impact on vital organs through histopathological analysis. Chitosan nanoparticles containing the extract were developed using the spray drying method. In addition, the study analyzed various characteristics of the chitosan-loaded extract, such as % yield, drug entrapment efficiency, morphology, particle size, thermal properties, drug release, and swelling index. In vitro studies were conducted on MCF-7 and OV7 cell lines to assess the extract's anticancer properties. In vivo studies were performed on tumor-induced Wistar rats, with five rats in each of the five groups, using hemocytometer trypan blue cell counting. The study found that the crude leaf extract of M. capitatum (MCE) was well tolerated at a dose of 5000 mg/kg in rats, with only partial congestion observed in the lungs and testis. The extract exhibited higher cytotoxicity against the ovarian cancer cell line OV7 compared to the breast cancer cell line MCF-7, with an IC50 value of 10.13±2.20. In vitro studies showed that a chitosan nanoparticle-loaded version of the extract (CSLA3) induced apoptosis and increased caspase-3 activity in OV7 cells. In vivo studies showed that treatment with the chitosan-loaded extract significantly increased mean survival time and percentage increase in life span in tumor-induced rats. Tumor cell volume and viable cell counts decreased, while non-viable cell count increased in the treated group. The study concluded that CSLA3 effectively inhibited the growth of ovarian cancer cells and demonstrated the potential of chitosan nanoparticles to deliver anticancer plant extracts. This also supports the traditional use of M. capitatum leaf extract as a treatment for ovarian cancer.

Noni enhances the anticancer activity of cyclophosphamide and suppresses myelotoxicity and hepatotoxicity in tumor-bearing mice

Background and aimMorinda citrifolia fruit juice (noni) is an herbal remedy documented to have antioxidant properties. It has been suggested that prevention of carcinogen-DNA adduct formation and the antioxidant activity of NJ may contribute to the cancer preventive effect. In the present study, the antitumor activity of noni was investigated in the presence of cyclophosphamide (CYL) in vitro and in vivo.MethodsIn vitro breast cancer cells (MDA-MB-468) were used to measure the percentage of inhibition and the IC50. The in vivo antitumor activity of noni was studied by monitoring the mean survival time (MST), percentage increase in life span (%ILS), viable and non-viable cell count, tumor volume, body weight, and hematological and serum biochemical parameters in mice. Treatment with noni and CYL exhibited dose- and time-dependent cytotoxicity toward breast cancer cells.ResultsIndividual treatment of noni and CYL exhibited dose- and time-dependent cytotoxicity on breast cancer cell lines, while in combination therapy of noni and CYL, noni enhances cytotoxic effect of CYL at 48 h than that at 24 h. Similar result was found in in vivo studies, the results of which revealed that alone treatment of CYL and noni suppressed tumor growth. However, combination treatment with CYL and noni presented better tumor inhibition than that of alone treatment of CYL and noni. On the contrary, CYL alone drastically attenuated hematological parameters, i.e., RBC, WBC, and Hb compared to normal and control groups, and this change was reversed and normalized by noni when given as combination therapy with CYL. Moreover, the levels of serum biochemical markers, i.e., AST, ALP, and ALT, were significantly increased in the control and CYL-treated groups than those in the normal group. In the combination treatment of noni and CYL, the above biochemical marker levels significantly decreased compared to CYL alone-treated group.ConclusionsThe present study suggested that CYL treatment can cause serious myelotoxicity and hepatic injury in cancer patients. In conclusion, the combined use of noni with CYL potentially enhances the antitumor activity of CYL and suppresses myelotoxicity and hepatotoxicity induced by CYL in tumor-bearing mice.

A Method for Quantifying Phytophthora Oospore Viability Using Fluorescent Dyes and Automated Image Analysis

Phytophthora are eukaryotic microbes that cause disease in a wide range of agriculturally and ecologically important plants. During the Phytophthora disease cycle, thick-walled oospores can be produced via homothallic or heterothallic reproduction. These resting spores can survive in the soil for several years in the absence of a host plant, thus providing a long-term inoculum for disease. The ability to quantitatively evaluate oospore viability is an important part of many phytopathology studies. Here, we tested six fluorescent viability dyes for their ability to differentially stain Phytophthora agathidicida oospores: SYTO 9, FUN-1, fluorescein diacetate (FDA), 5-carboxyfluorescein diacetate (CFDA), propidium iodide, and TOTO-3 iodide. Each dye was first tested individually with untreated or heat-treated oospores as proxies for viable and nonviable oospores, respectively. SYTO9, FUN-1, CFDA and propidium iodide stained untreated and heat-treated oospores indiscriminately. In contrast, FDA (a green-fluorescent viable cell stain) and TOTO-3 (a red-fluorescent nonviable cell stain) differentially stained untreated or heat-treated oospores with no cross-fluorescence. We then tested the efficacy of dual-viability staining in conjunction with a pipeline for automated image analysis. To validate the method, untreated and heat-treated oospores were mixed at specific ratios, dual-stained, and analyzed using the pipeline. Linear regression of the resulting data showed a clear correlation between the expected and measured oospore ratios ( dy/dx = 0.95, R2 = 0.88). Overall, the combination of dual-fluorescence staining and automated image analysis provides a high-throughput method for quantitatively assessing oospore viability and therefore can facilitate further studies on this key part of the Phytophthora disease cycle. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Immunomodulatory function of licensed human bone marrow mesenchymal stromal cell-derived apoptotic bodies

BackgroundMesenchymal stromal cells (MSCs) show great potential for immunomodulatory and anti-inflammatory treatments. Clinical trials have been performed for the treatment of Type 1 diabetes, graft-versus-host disease and organ transplantation, which offer a promise of MSCs as an immunomodulatory therapy. Nevertheless, their unstable efficacy and immunogenicity concerns present challenges to clinical translation. It has emerged that the MSC-derived secretome, which includes secreted proteins, exosomes, apoptotic bodies (ABs) and other macromolecules, may have similar therapeutic effects to parent MSCs. Among all of the components of the MSC-derived secretome, most interest thus far has been garnered by exosomes for their therapeutic potential. However, since MSCs were reported to undergo apoptosis after in vivo transplantation and release ABs, we speculated as to whether ABs have immunomodulatory effects. In this study, cytokine licensing was used to enhance the immunomodulatory potency of MSCs and ABs derived from licensed MSCs in vitro were isolated to explore their immunomodulatory effects as an effective non-viable cell therapy. ResultsIFN-γ and IFN-γ/TGF-β1 licensing enhanced the immunomodulatory effect of MSCs on T cell proliferation. Further, TGF-β1 and IFN-γ licensing strengthened the immunomodulatory effect of MSC on reducing the TNF-α and IL-1β expression by M1 macrophage-like THP-1 cells. Additionally, we discovered the immunomodulatory effect mediated by MSC-derived apoptotic bodies. Licensing impacted the uptake of ABs by recipient immune cells and importantly altered their phenotypes. ConclusionABs derived from IFN-γ/TGF-β1-licensed apoptotic MSCs significantly inhibited T cell proliferation, induced more regulatory T cells, and maintained immunomodulatory T cells but reduced pro-inflammatory T cells.

THE ABILITY OF Lactiplantibacillus plantarum TSD-10 AND DR-162 TO REDUCE AFLATOXIN AND MICROBIAL CONTAMINATION OF CORN

Feed safety becoming the main factor to produce good quality food. The purpose of this study was to evaluate the ability of lactic acid bacteria in degrading aflatoxin B1, suppressing the growth of contaminant bacteria, and its application on feed. Biological degradation of aflatoxin B1 (AFB1) in liquid cultures and Escherichia coli and Saccharomyces aureus inhibition by Lactobacillus plantarum were examined. Fermentation of corn for 24 hours by L. plantarum was used to determine the ability of LAB to decrease E. coli, coliform, mold, and AFB1 on a solid substrate. This study showed that both L. plantarum TSD-10 and DR-162 could inhibit E. coli growth at 3% and 5% supernatant addition. The addition of 5% L. plantarum DR-162 and TSD-10 could inhibit S. aureus at 97.40% and 97.28%, respectively. Furthermore, viable cells of L. plantarum DR-162 and TSD-10 removed 56.97% and 64.65% AFB1 within 72 h of incubation, respectively. The AFB1 reduction by nonviable cell was lower than the viable cell. The population of E. coli, coliform, and mold completely disappeared in both fermentation treatment of corn by L. plantarum DR-162 and TSD-10 at 3% and 5%. AFB1 significantly decreased by 17.57% and 22.60% on 5% L. plantarum DR-162 and TSD-10 treatment.

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Production of Lactobacillus plantarum ghosts by conditional expression of a prophage-encoded holin.

Bacterial ghosts (BGs) are non-viable empty bacterial cell envelopes with intact cellular morphology and native surface structure. BGs made from pathogenic bacteria are used for biomedical and pharmaceutical applications. However, incomplete pathogenic cell inactivation during BG preparation raises safety concerns that could limit the intended use. Therefore, safer bacterial cell types are needed for BG production. Here, we produced BGs from the food-grade Gram-positive bacterium Lactobacillus plantarum TBRC 2-4 by conditional expression of a prophage-encoded holin (LpHo). LpHo expression was regulated using the pheromone-inducible pSIP system and LpHo was localized to the cell membrane. Upon LpHo induction, a significant growth retardation and a drastic decrease in cell viability were observed. LpHo-induced cells also showed membrane pores by scanning electron microscopy, membrane depolarization by flow cytometry, and release of nucleic acid contents in the cell culture supernatant, consistent with the role of LpHo as a pore-forming protein and L. plantarum ghost formation. The holin-induced L. plantarum BG platform could be developed as a safer alternative vehicle for the delivery of biomolecules.

Evaluation of the anti-cancer study of chloroform seed extract of Datura stramonium on EAC cell lines

Including cancer, native medicinal herbs have long been employed for illness prevention and treatment. Herbal supplements are promoted for their wide range of biological benefits, from preventing cancer cell proliferation to boosting the immune system. Datura stramonium L., a member of the Solanaceae family, is a common and easily accessible shrub that may grow to a height of 6 feet on fertile soil and a width of 3 feet in poor soil. The purpose of the current study is to determine whether or not a Chloroform seeds extract of Datura stramonium may inhibit cancer caused by the EAC cell line in albino mice. The collection of plant material and subsequent chloroform extraction using a Soxhlet equipment made this investigation possible. The percentage of cell growth inhibition, tumour weight, packed cell volume, average increase in body weight, change in food intake, mean survival time (MST), percentage increase in life span (% ILS), and viable and non-viable tumour cell count were all significantly different between the 100 mg/kg and 200 mg/kg doses of Datura stramonium in E.A.C. bearing mice. Here there is therapeutic promise for treating illnesses generated by oxidative stress, and the current investigation confirms that Datura stramonium L. is a rich source of considerable natural oxidants with anticancer agents.

Immunotoxicity and proliferation of human peripheral blood mononuclear cells of HIV-positive patient after treatment with aqueous extract of polyherbal formulation

Medicinal plants have been widely used for medicinal purposes in the treatment of various ailments including human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). This study evaluated the effect of aqueous extracts of polyherbal formulation (PHF) on peripheral blood mononuclear cells (PBMCs) of an HIV+ patient. In this study, 4 ml of blood was collected from HIV+ patient who has not commenced antiretroviral treatment. PBMCs were isolated using the Ficoll-Paque method and counted using the trypan blue assay. The isolated PBMCs were treated with different concentrations of the PHF (25 50, 100, and 200 μg/ml). Cytotoxicity and proliferation were determined using the WST-8 assay. The percentage viability in this study was 96.74%, whereas the non-viable cell percentage was 3.26%. The lowest percentage of cytotoxicity for the PHF was 0.05% at 25 µg/ml of the extract, while the highest was 5.35% at 200 µg/ml. The mean absorbance scores of the formulation's various concentrations and the control group did not significantly differ from one another (F=0.622, df = 5, p=0.69). There was significant difference (t= -4.887, df =6, 95% CI= -0.1566 to -0.0521, p = 0.003) in mean absorbance scores between control and treatment. The polyherbal formulation exerts the lowest cytotoxicity of 0.05% and significant proliferation on the PBMCs at 25 µg/ml. The PHF has potential immunoproliferative activity with a good safety margin. An in-depth study of the bioactivity of the PHF on individual immune cells is recommended.

Oroxylum indicum stem bark extract exerts antitumor potential against Ehrlich’s ascites carcinoma in Swiss albino mice

Introduction and Aim: Constant efforts are exerted to explore unique bioactive principles from natural sources that possess more effective and specific antineoplastic activities. In the present study, we aimed to evaluate the antitumor activity of stem bark extract of Oroxylum indicum in mice bearing Ehrlich ascites carcinoma (EAC). Materials and Methods: Ninety female Swiss albino mice were categorized into fifteen groups (n=6). The animals were inoculated with 1x106 EAC cells. Tumor control animals received sterile water once daily for 10 consecutive days. Positive control group was injected with Cisplatin (CP) (one dose – 3.5 mg/kg body weight). The treatment groups were administered with O. indicum (OI) stem bark ethanol extract once daily with 50mg/kg, 200mg/kg and 400mg/kg body weight for eleven consecutive days. The blood parameters and serum hepatic enzymes activity was determined. The percentage increase in weight, the median survival time, the increase in median life span was calculated. The cytotoxic effect of CP and OI extract was determined. Results: There was significant reduction in the white blood cells count in OI and CP treatment group compared to increased level in EAC control group. The RBC count and Hemoglobin level which was significantly decreased (p<0.05) in the tumour mice, was enhanced in the drug treatment groups. The EAC control group showed significant increase in tumour cell count (p<0.05) whereas, treatment of EAC tumor bearing mice with OI and CP significantly increased the non-viable tumor cell count (p<0.05). Conclusion: OI stem bark ethanol extract reduced the toxic implications of Ehrlich ascites carcinoma, reverted the haematological and biochemical changes induced by tumour. These results call for additional research on isolating and identifying the responsible bioactive elements in order to clarify the underlying processes of the anticancer impact.

Inactivation of Salmonella in ready-to-eat cabbage slices packaged in a plastic container using an integrated in-package treatment of hydrogen peroxide and cold plasma

An in-package cold plasma (CP) treatment integrated with hydrogen peroxide (H 2 O 2 ) treatment was developed for the inactivation of foodborne pathogens in cabbage slices packaged in polyethylene terephthalate (PET) containers. The effects of H 2 O 2 -CP treatment on microbial inactivation, microbial cell membrane integrity, cell oxidation, and the antioxidant activity and color of cabbage slices were investigated. As the treatment time increased from 1 to 2 and 3 min, the respective reduction level of Salmonella increased from 0.6 to 0.9 log CFU/g ( p < 0.05). Moreover, the levels of Salmonella reduction were 0.8, 0.9, 1.8, and 1.5 log CFU/g when the H 2 O 2 concentrations in the H 2 O 2 pad within the package were 3, 10, 20, and 30%, respectively. The values for cell lipid peroxidation, reactive oxygen species generation, and number of non-viable cells with damaged membranes, increased following CP and H 2 O 2 -CP treatments. Meanwhile, H 2 O 2 -CP treatment did not influence the antioxidant activities nor the color of cabbage slices. However, the concentration of malondialdehyde in cabbage slices was increased by H 2 O 2 -CP treatment. The results suggest that H 2 O 2 -CP treatment is an effective strategy for disinfecting cabbage slices without altering their antioxidant activities and color. • In-package cold plasma (CP) and H 2 O 2 treatment inactivates Salmonella effectively. • Increased treatment time enhances reduction of Salmonella in cabbage slices. • H 2 O 2 decreases the number of Salmonella in a concentration dependent manner. • CP/H 2 O 2 -CP treatments increase lipid peroxidation, ROS, and non-viable cell number. • H 2 O 2 -CP treatment does not impact antioxidant activities or color of cabbage slices.

Anti-salmonella properties of kefir yeast isolates: An in vitro screening for potential infection control

The rise of antibiotic resistance has increased the need for alternative ways of preventing and treating enteropathogenic bacterial infection. Various probiotic bacteria have been used in animal and human. However, Saccharomyces boulardii is the only yeast currently used in humans as probiotic. There is scarce research conducted on yeast species commonly found in kefir despite its claimed potential preventative and curative effects. This work focused on adhesion properties, and antibacterial metabolites produced by Kluyveromyces lactis and Saccharomyces unisporus isolated from traditional kefir grains compared to Saccharomyces boulardii strains. Adhesion and sedimentation assay, slide agglutination, microscopy and turbidimetry assay were used to analyze adhesion of Salmonella Arizonae and Salmonella Typhimurium onto yeast cells. Salmonella growth inhibition due to the antimicrobial metabolites produced by yeasts in killer toxin medium was analyzed by slab on the lawn, turbidimetry, tube dilution and solid agar plating assays. Alcohol and antimicrobial proteins production by yeasts in killer toxin medium were analyzed using gas chromatography and shotgun proteomics, respectively. Salmonella adhered onto viable and non-viable yeast isolates cell wall. Adhesion was visualized using scanning electron microscope. Yeasts-fermented killer toxin medium showed Salmonella growth inhibition. The highest alcohol concentration detected was 1.55%, and proteins with known antimicrobial properties including cathelicidin, xanthine dehydrogenase, mucin-1, lactadherin, lactoperoxidase, serum amyloid A protein and lactotransferrin were detected in yeasts fermented killer medium. These proteins are suggested to be responsible for the observed growth inhibition effect of yeasts-fermented killer toxin medium. Kluyveromyces lactis and Saccharomyces unisporus have anti-salmonella effect comparable to Saccharomyces boulardii strains, and therefore have potential to control Salmonella infection.

Antitumor activity of Albizia lebbeck L. against Ehrlich ascites carcinoma in vivo and HeLa and A549 cell lines in vitro.

The aim of the present study was to explore the antitumor activity of the ethanolic extract of Albizia lebbeck L. pods against Ehrlich ascites carcinoma (EAC) in Swiss albino mice and its cytotoxic effect against HeLa and A549 cell lines in vitro. Antitumor activity of ethanolic extract of A. lebbeck L. (ALEE) pods was evaluated in Swiss albino mice against EAC cell lines at the doses of 200 and 400 mg/kg body weight which were given by intraperitoneal route of administration and was compared with 5-fluorouracil (5-FU), the reference standard. The extract and 5-FU were administered for 14 consecutive days. After 24 h of the last dose and 18 h of fasting, the mice were sacrificed and the antitumor effect of ALEE was assessed by evaluating tumor volume, viable and nonviable tumor cell count, increase in life span, and hematological parameters of EAC-bearing hosts.In vitro cytotoxicity has been assessed using (2,3-bis[2-Methoxy-4-nitro-5sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt assay method and was compared with cisplatin, the reference standard. ALEE showed direct cytotoxicity on EAC cells in a dose-dependent manner. ALEE exhibited a significant (P < 0.001) decrease in the body weight, tumor volume, viable cell count, tumor weight, and elevated the life span of EAC tumor-bearing mice. Hematological profile such as red blood cell, hemoglobin, white blood cell, and platelet count was reverted to the normal level in ALEE-treated mice. The results showed that the ethanolic extract of A. lebbeck L. has a powerful antitumor activity because it was effective in significantly inhibiting the tumor growth in both in vivo and in vitro cancer cell lines.

Using direct epifluorescent microscopic count for rapid enumeration of viable yeast and bacteria in injured conditions

Traditional method for assessing cell count requires an incubation period of 2-3 days. The direct microscopic count (DMC) method gives rapid enumeration of yeast and bacteria. However, the application is limited, since it is not possible to distinguish accurately between viable and nonviable cell. In this report, several nucleic acid dyes from Molecular Probe Inc. were used to stain the cells for enumeration of live and dead yeast in injured conditions, in which the dye can stain the DNA of nucleus and mitochondria, which then fluoresce yellow under fluorescence microscope, as well as stain the cytoplasm of actively growing cells to fluoresce green, while the cytoplasm of inactive cells fluoresced orange. The direct microscopic count multiplied by the live cell count ratio obtained by this direct epifluoroscent microscopic count (DEMC) method gives the estimated viable yeast count. According to our results, the correlation coefficient (R2) between the cell count obtained by the DEMC method and that by the standard plate count was 0.91 for yeast and 0.96 for bacteria in frozen and heating conditions, respectively. Requiring only 30 min, this method can also be used for the rapid enumeration of viable bacteria in injured conditions.

Effect of seawater variability on endemic bacterial biofouling of a reverse osmosis membrane coated with iron nanoparticles (FeNPs)

Biofouling presents difficult problems for seawater reverse osmosis (SWRO). Although iron nanoparticles (FeNPs) exhibit anti-biofouling properties, this may vary with geographic location. The efficacy of an FeNP coating for SWRO membranes is studied at laboratory scale in two sites – Sea of Cortez, Mexico (27.917°N, 110.776°W), and El Sol Beach, Chile (33.01°S, 71.553°W) – with significant differences in physicochemical parameters. Constant concentrations (109CFU mL−1) of native strains Bacillus halotolerans MCC1 and Bacillus sp. TA011_2 (EU308309), were respectively used at each of the sites to promote biofouling. The biofilm thickness, total, viable and non-viable cell counts, and organic matter were analyzed. More biofouling (>74%) was observed at the Mexican site due to differences in temperature and pH, but the coating presented stronger biocide effect (by 29%) there. Temperature, dissolved oxygen and pH had a more significant effect than salinity on biofouling FeNP coated membranes.

Effects of plant-derived immunomodulators and lizak preparation in vitro taken separately or in combination on the immune factors of palatine tonsils of children with chronic tonsillitis

Introduction: Chronic inflammatory diseases of the upper respiratory tract of both microbial and viral origin occur due to the immune deficiency of systemic and local nature. Among a large number of immunomodulation agents, a special role belongs to plant preparations, which are characterized by relative harmlessness and high efficiency, both in case of systemic and local use and they are often combined with antimicrobial agents, therefore the aim of the study was to investigate their effect on the reactions and condition of lymphoid cells of tonsilswith separate and combined use in vitro. Methods: Cell suspensions were prepared mechanically and adjusted to a concentration of 2 million/ml in medium 199 with additives. Then, Lizak preparation was added to the cells and starch solution was used in control. Imupret and Esberitox preparations were added to the culture with or without Lizak preparation. After cultivation, the levels of proinflammatory cytokine – Interleukin-1β, pro-allergic factor – Interleukin-4, Th-1 – derivative of cytokine – interferon-γ were studied in the supernatant using the «Tsitokin LLC» reagent kits (RF), as well as tissue regeneration factor – transforming growth factor –TGF-1β (Austria). The preparation was prepared from the cell pellet, in which the relative nonviable cell count was determined in a sample with trypan blue using a light microscope (Olympus CX21FS1).The statistical analysis was performed using Student's t-distribution. Results: Plant-derived preparations did not have any effect on cell viability in culture, did not reduce the level of the pro-inflammatory cytokine interleukin-1β and did not stimulate the production of the regeneration factor TGF-1β. At the same time, the combined use of plant-derived preparations and the antibacterial preparation Lizak in cell culture of tonsils was accompanied by an increase in cell viability compared to the use of Lizak preparation separately, the stimulation of the regulatory antiviral factor interferon-γ and an increase in the production of tissue regeneration factor. Conclusion: The immunomodulating properties of the antibacterial preparation Lizak and plant-derived immunomodulator Esberitox significantly differ in the points of application. Both plant-derived preparations stimulated the production of γ-interferon by tonsil cells in the presence of Lizak preparation, the transforming growth factor TGF-1β, the viability of tonsil cells was the highest with the combination of Lizak and Esberitox.

Identification and Quantification of Nonviable Lactobacillus pentosus Cells in a Health Food Product.

The lack of formal protocol to verify the nonviable cell probiotic product authenticity blocks its registration and supervision process. To develop a protocol for identification, enumeration, and purity determination of a Lactobacillus pentosus nonviable cell product. The 16S ribosomal RNA (rRNA) sequencing, 16S rRNA metagenomic analysis, whole-genome sequencing, matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF-MS), and FACSMicroCount™ system were applied to establish a protocol of identification, enumeration and purity determination of a Lactobacillus pentosus nonviable cell product. The 1530 bp of 16S rRNA sequence could only identify the bacteria at genus level, but the MALDI-TOF-MS could identify both the nonviable cell and fresh culture to species level with high confidence. Metagenomic analysis of the 16S rRNA amplicon could recognize Lactobacillus as the dominant genus in the nonviable cell product. The total number of matching k-mers between the nonviable cell product and the L. pentosus BGM48 in the GenBank was the highest. The 95% confidence interval of the nonviable cell concentration in the product was determined as 4.31-4.68 × 1010 cells/g through the BD FACSMicroCount system. This validation protocol offers an executable approach that can verify microbial contents in nonviable cell products and ensure the compliance with label claims. The established validation protocol could determine the nonviable cell species through MALDI-TOF-MS, the concentration through FACSMicroCount system, and the purity and strain level identification through metagenomic analysis of 16S rRNA and the genomic deoxyribonucleic acid.

Evaluation of the interaction between microencapsulated Bifidobacterium BB-12 added in goat's milk Frozen Yogurt and Escherichia coli in the large intestine.

Goat milk and goat milk and inulin were used as encapsulating agents of Bifidobacterium BB-12 and applied in Frozen Yogurt (GF2 and GF3, respectively) in order to evaluate the antagonistic effect against Escherichia coli. GF1 is a control containing only Escherichia coli. Simulation of gastrointestinal digestion occurred sequentially. Quantification of Bifidobacterium BB-12 was performed using plate counting while E. coli count was compared with quantification by qPCR with viable and nonviable cell differentiation. The Bifidobacterium BB-12 count was <1.0, 9.23 and 9.11 log CFU g-1 for GF1, GF2 and GF3, respectively. In the ascending colon, all samples showed E. coli counts of approximately 5 log CFU g-1 by plate counting and by qPCR. Throughout the transverse (24 h) and descending colon (48 h) samples GF2 and GF3 showed decrease in E. coli number. GF3 showed higher decrease of E. coli in the descending colon because of inulin bifidogenic characteristic. The production of organic acids by bifidobacteria was directly related to the decrease in the E. coli count. In plate counts, E. coli was not detected for the GF3 sample in the descending colon. However, when quantified by qPCR the sample presented amplification that corresponded to 3 log CFU g-1. In this way, it was possible to observe the phenomenon of the viable but not-culturable cells of E. coli. Finally, it is recommended the microcapsule produced with goat milk and the inulin for application in goat milk products, due to the better antagonist effect against E. coli.

IN VITRO CYTOTOXICITY ACTIVITY OF METHANOLIC LEAF AND FRUIT EXTRACT OF ZANTHOXYLUM OVALIFOLIUM WIGHT AGAINST DALTON’S LYMPHOMA ASCITES AND EHRLICH ASCITES CARCINOMA CANCER CELL LINES

Objectives: The objective of the present study, the methanolic leaf and fruit extraction of Zanthoxylum ovalifolium, was examined for in vitro cytotoxicity using trypan blue dye exclusion technique.&#x0D; Methodology: In vitro cytotoxicity activity of methanolic leaf and fruit extracts of Z. ovalifolium. Against Dalton’s lymphoma ascites (DLA) and Ehrlich ascites carcinoma (EAC) cell lines evaluated using trypan blue exclusion method. Non-viable cell absorbs blue color of trypan blue while viable cell does not absorb the dye. The percentage of total number of viable and non-viable cells is calculated using % viability=(live cell count/total cell count)×100.&#x0D; Results: Cytotoxicity activity of methanolic leaf and fruit extract against chosen tumor cell line at different concentrations of 10, 20, 50,100, and 200 μg/ml, respectively. The result confirmed that the fruit extract at 200 showed less toxic effect against DLA and EAC cell line showed 72% and 75% of inhibition growth of cell line and the most potent 80% and 82% of cell death were found in leaf extract against the chosen tumor cell line. The present study indicates that both leaf and fruit extract of Z. ovalifolium were appreciable antitumor activity against DLA and EAC cell lines may be due to the presence of phytochemicals such as flavonoids and alkaloids present in the plant.&#x0D; Conclusion: Zanthoxylum ovalifolium wight fruit and leaf methanolic extracts showed satisfying result of cytotoxicity activity against DLA and EAC cell line. Hence, Z.ovalifolium could be suggested as an important source of anticancer agent for pharmacological industries.