Live Cell Counts Research Articles

Experimental Study on Compatibility of Human Bronchial Epithelial Cells in Collagen-Alginate Bioink for 3D Printing.

This paper reports an experimental study on the compatibility of human bronchial epithelial (HBE) cells in a collagen-alginate bioink. The compatibility was assessed using the culture well method with three bioink compositions prepared from a 10% alginate solution and neutralized TeloCol-10 mg/mL collagen stock solution. Cell viability, quantified by (live cell count-dead cell count)/live cell count within the HBE cell-laden hydrogel, was evaluated using the live/dead assay method from Day 0 to Day 6. Experimental results demonstrated that the collagen-alginate 4:1 bioink composition exhibited the highest cell viability on Day 6 (85%), outperforming the collagen-alginate 1:4 bioink composition and the alginate bioink composition, which showed cell viability of 75% and 45%, respectively. Additionally, the live cell count was highest for the collagen-alginate 4:1 bioink composition on Day 0, a trend that persisted through Days 1 to 6, underscoring its superior performance in maintaining cell viability and promoting cell proliferation. These findings show that the compatibility of HBE cells with the collagen-alginate 4:1 bioink composition was higher compared with the other two bioink compositions.

Bioprinted keratinocyte and stem cell laden constructs for skin tissue engineering

Treating large-scale skin wounds continues to be a significant therapeutic challenge. Oftentimes, insufficient autologous material is available to achieve full coverage. Since biofabrication offers the chance of reproducible and precise large-scale production, the aim was to develop a customized 3D bioprinted skin construct containing immortalized HaCaT keratinocytes and ADSC to test the principal feasibility of this approach. Keratinocytes in different hydrogels containing varying concentrations of alginate, fibrin, collagen, gelatin, gelatin methacryloyl (GelMA) and hyaluronic acid as well as on a prefabricated collagen-elastin-matrix were cultured for a period of seven days. The metabolic activity of cultured keratinocytes was then evaluated during the co-cultivation of HaCaT and ADSC in a transwell model. The metabolic activity in all groups increased over the test period. The hydrogels containing alginate/hyaluronic acid/gelatin (Alg/HA/Gel) and GelMA demonstrated a good printability and the highest diffusion rates. There was no significant difference in pore size when comparing all hydrogels with each other. On the basis of the performed printability and diffusion assays as well as on the SEM analysis and rheological measurements Alg/HA/Gel and GelMA were chosen as hydrogels for the bioprinted 3D model. Fibrin hydrogel was integrated into the biofabricated constructs due to its excellent metabolic activity in the transwell model. As a second step, hydrogel stability, cell survival and metabolic activity in bioprinted 3D models containing keratinocytes and ADSC were evaluated over a period of 14 days. On day 14 the metabolic activity as well as the live cell count within the bioprinted constructs of the cocultured groups was significantly higher compared with day 1. The biofabricated constructs made of GelMA showed a higher cell viability compared with those made of Alg/HA/Gel. Additionally, to evaluate the cell migration out of the constructs, the metabolic activity and viability of the cells on the well bottom, was examined. After 14 days, an average of 50% of the well bottom was covered by HaCaT, which were initially printed in coculture into the constructs. These findings indicate that constructs made of GelMA containing keratinocytes and ADSC may offer a promising therapeutic option in the treatment of large chronic wounds.

Production of set-type probiotic and prebiotic yogurt-like products using Enterococcus faecium and Enterococcus faecalis strains in combination with pumpkin waste

This study investigated the effect of pumpkin powder (2 %, 4 %, and 6 %) and Enterococcus faecium and Enterococcus faecalis probiotics on the physicochemical, microbiological, and sensory properties of yogurt samples during 28 days of storage at 4 °C. The prebiotic effect of pumpkin powder (Cucurbita pepo) and the probiotic effect of Enterococcus faecium and E. faecalis were determined. Adding pumpkin powder to yogurt did not significantly alter the pH, acidity, fat, protein, and ash content (p > 0.05). Water holding was not changed during the storage time in the samples of probiotic yogurts, but as the pumpkin powder content increased, the water holding capacity also increased (p < 0.05). This situation did lead to a reduction in syneresis (p < 0.05). The lowest gumminess value at the end of storage was found in the D2 sample (p < 0.05), and the highest adhesiveness value was found in the D4 sample (p < 0.05). Furthermore, throughout the 28-day storage period, E. faecium and E. faecalis maintained a live cell count of ≥6 log CFU g-1 in the probiotic product. As a result of the statistical evaluation, there was a decrease in E. faecium in the D4, S2, and S4 samples, and then it increased again (p > 0.05) during the storage time. As a result of the statistical evaluation, it was determined that the smell, consistency in the spoon, consistency in the mouth, flavor, and acidity changes during the storage were not substantial (p > 0.05). In conclusion, it was found that pumpkin, a byproduct of the pumpkin seed industry, has the potential to act as a prebiotic and improve the properties of dairy products. Additionally, the study suggests that E. faecium and E. faecalis strains could be suitable for probiotic yogurts.

Screening the Protective Agents Able to Improve the Survival of Lactic Acid Bacteria Strains Subjected to Spray Drying Using Several Key Enzymes Responsible for Carbohydrate Utilization.

The aim of this study was to identify the most effective protectants for enhancing the viability of specific lactic acid bacteria (LAB) strains (Lactobacillus delbrueckii subsp. bulgaricus CICC 6097, Lactiplantibacillus plantarum CICC 21839, Lactobacillus acidophilus NCFM) by assessing their enzymatic activity when exposed to spray drying (inlet/outlet temperature: 135 °C/90 °C). Firstly, it was found that the live cell counts of the selected LAB cells from the 10% (w/v) recovered skim milk (RSM) group remained above 107 CFU/g after spray drying. Among all the three groups (1% w/v RSM group, 10% w/v RSM group, and control group), the two enzymes pyruvate kinase (PK) and lactate dehydrogenase (LDH) were more sensitive to spray drying than hexokinase (HK) and β-galactosidase (β-GAL). Next, transcriptome data of Lb. acidophilus NCFM showed that 10% (w/v) RSM improved the down-regulated expressions of genes encoding PK (pyk) and LDH (ldh) after spray drying compared to 1% (w/v) RSM. Finally, four composite protectants were created, each consisting of 10% (w/v) RSM plus a different additive-sodium glutamate (CP-A group), sucrose (CP-B group), trehalose (CP-C group), or a combination of sodium glutamate, sucrose, and trehalose (CP-D group)-to encapsulate Lb. acidophilus NCFM. It was observed that the viable counts of strain NCFM (8.56 log CFU/g) and enzymatic activity of PK and LDH in the CP-D group were best preserved compared to the other three groups. Therefore, our study suggested that measuring the LDH and PK activity could be used as a promising tool to screen the effective spray-dried protective agent for LAB cells.

Understanding the transition to viable but non-culturable state in Escherichia coli W3110: a comprehensive analysis of potential spectrochemical biomarkers

The viable but non-culturable (VBNC) state is considered a survival strategy employed by bacteria to endure stressful conditions, allowing them to stay alive. Bacteria in this state remain unnoticed in live cell counts as they cannot proliferate in standard culture media. VBNC cells pose a significant health risk because they retain their virulence and can revive when conditions normalize. Hence, it is crucial to develop fast, reliable, and cost-effective methods to detect bacteria in the VBNC state, particularly in the context of public health, food safety, and microbial control assessments. This research examined the biomolecular changes in Escherichia coli W3110 induced into the VBNC state in artificial seawater under three different stress conditions (temperature, metal, and antibiotic). Initially, confirmation of VBNC cells under various stresses was done using fluorescence microscopy and plate counts. Subsequently, lipid peroxidation was assessed through the TBARS assay, revealing a notable increase in peroxidation end-products in VBNC cells compared to controls. ATR-FTIR spectroscopy and chemomometrics were employed to analyze biomolecular changes, uncovering significant spectral differences in RNA, protein, and nucleic acid concentrations in VBNC cells compared to controls. Notably, RNA levels increased, while protein and nucleic acid amounts decreased. ROC analyses identified the 995 cm− 1 RNA band as a consistent marker across all studied stress conditions, suggesting its potential as a robust biomarker for detecting cells induced into the VBNC state under various stressors.

Cancer Stem Cell and Epithelial Mesenchymal Transition (EMT) in Canine Transmissible Venereal Tumour- A Preliminary Study

Background: Cancer stem cell (CSC) and Epithelial to Mesenchymal Transition (EMT) transforms conversion of normal cells in the tumor microenvironment into CSC which is the main cause of embryogenesis, carcinogenesis and metastasis. Methods: Nineteen canine patients with recurrent transmissible venereal tumor (TVT) and metastasis of inguinal lymph node were the subjects of present study. Preoperative work up and clinical staging involving radiography, ultrasonography, hematobiochemical analysis and histopathology or biopsy were done to confirm the type of cancerous tissue. Tumor tissues were collected in ice cold RPMI-1640 and phosphate buffer solution. Flow cytometry analysis was performed following established protocol from surgically excised tissue sample. Trypan blue dye exclusion test was done to quantify the live/dead cell count. CD24PE, CD44FITC, CD133APC and EpCAM antibody with conjugates were chosen markers for flow cytometry analysis. Result: Nineteen canine patients were diagnosed with Transmissible Venereal tumor (TVT) (common sexually transmitted disease in male dog) through biopsy of excised surgical tissue following Haematoxylin and Eosin staining protocol. Recurrence was seen even after adjuvant chemotherapy with Vincristine injection @ 0.25 mg/m2 at weekly interval for 5 weeks. Trypan blue dye exclusion test confirms 98% live cell count in transmissible venereal granuloma after digestion and incubation for flow cytometry. Flow cytometry analysis of the cells from the TVT showed surface biomarkers for EpCAM+ cells, EpCAM CD24+ cells, EpCAM CD44+ cells, EpCAM CD133+ indicating cells with CSC and EMT properties. Presence of CSC and EMT cells in this patient was responsible for chemoresistance, distant metastasis and recurrence. Identification of specific CSC in a malignant cancer will yields deeper insight into biological aspects of metastasis and formulation of targeted therapy in fighting with cancer.

Effects of 4-hydroxy-2(3H)-benzoxazolone on proliferation and apoptosis of pancreatic cancer L3.6 cells

This study explored the effects of 4-hydroxy-2(3H)-benzoxazolone(HBOA) on the proliferation and apoptosis of pancreatic cancer cells and its molecular mechanism. The L3.6 cells cultured in vitro were treated with HBOA of 0-1.0 mmol·L~(-1). The cell viability was detected by the cell counting kit-8(CCK-8) method, and the half inhibitory concentration(IC_(50)) was analyzed to determine the drug concentration and time. The cell morphology was observed under an inverted microscope and by acridine orange(AO) staining. The ability of proliferation and self-renewal were evaluated through live cell counting and colony formation experiments. The cell cycle progression and cell apoptosis rate were detected by flow cytometry. The morphology of cell apoptosis was observed by scanning electron microscopy. The mRNA expression of proliferating cell nuclear antigen(PCNA), cyclinA1, cyclinA2, cyclin dependent kinase 2(CDK2), and cyclin dependent kinase inhibitor 1A(P21) were determined by qPCR. The level of reactive oxygen species(ROS), lipid peroxide, and mitochondrial membrane potential were measured by flow cytometry. The activity of protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathway was detected by Western blot. Compared with the control group, the cells treated with HBOA exhibited a significant decrease in viability. Then the optimal concentration and intervention time of HBOA were determined to be 0.4 mmol·L~(-1), 0.6 mmol·L~(-1), and 48 h. Compared with the control group, groups with HBOA of 0.4 mmol·L~(-1 )and 0.6 mmol·L~(-1) showed a significant suppression in cell proliferation and colony formation ability, down-regulated mRNA of PCNA, cyclinA1, cyclinA2, and CDK2, up-regulated P21 mRNA, S-phase cell cycle arrest, and increased cell apoptosis rate. There was an appearance of apoptotic bodies, increased ROS and lipid peroxide, decreased mitochondrial membrane potential(with a significant decrease in 0.6 mmol·L~(-1) group), and down-regulated p-Akt and p-mTOR proteins. The results show that HBOA inhibits the proliferation of pancreatic cancer L3.6 cells and induces cell apoptosis, which may be related to the increase in reactive oxygen species and the inhibition of the Akt/mTOR pathway.

Mass Production of Rg1-Loaded Small Extracellular Vesicles Using a 3D Bioreactor System for Enhanced Cardioprotective Efficacy of Doxorubicin-Induced Cardiotoxicity.

Small extracellular vesicles (sEVs) obtained from human umbilical cord mesenchymal stromal cells (MSCs) have shown cardioprotective efficacy in doxorubicin-induced cardiotoxicity (DIC). However, their clinical application is limited due to the low yield and high consumption. This study aims to achieve large-scale production of sEVs using a three-dimensional (3D) bioreactor system. In addition, sEVs were developed to deliver Ginsenoside Rg1 (Rg1), a compound derived from traditional Chinese medicine, Ginseng, that has cardioprotective properties but limited bioavailability, to enhance the treatment of DIC. The 3D bioreactor system with spinner flasks was used to expand human umbilical cord MSCs and collect MSC-conditioned medium. Subsequently, sEVs were isolated from the conditioned medium using differential ultra-centrifugation (dUC). The sEVs were loaded with Ginsenoside Rg1 by electroporation and evaluated for cardioprotective efficacy using Cell Counting Kit-8 (CCK-8) analysis, Annexin V/PI staining and live cell count of H9c2 cells under DIC. Using the 3D bioreactor system with spinner flasks, the expansion of MSCs reached ~600 million, and the production of sEVs was up to 2.2 × 1012 particles in five days with significantly reduced bench work compared to traditional 2D flasks. With the optimized protocol, the Ginsenoside Rg1 loading efficiency of sEVs by electroporation was ~21%, higher than sonication or co-incubation. Moreover, Rg1-loaded sEVs had attenuated DOX-induced cardiotoxicity with reduced apoptosis compared to free Ginsenoside Rg1 or sEVs. The 3D culture system scaled up the production of sEVs, which facilitated the Rg1 delivery and attenuated cardiomyocyte apoptosis, suggesting a potential treatment of DOX-induced cardiotoxicity.

Lactobacillus bulgaricus-loaded and chia mucilage-rich gum arabic/pullulan nanofiber film: An effective antibacterial film for the preservation of fresh beef

In recent years, the development of probiotic film by incorporating probiotics into edible polymers has attracted significant research attention in the field of active packaging. However, the influence of the external environment substantially reduces the vitality of probiotics, limiting their application. Therefore, to improve the probiotic activity, this study devised a novel nanofiber film incorporating chia mucilage protection solution (CPS), gum arabic (GA), pullulan (PUL), and Lactobacillus bulgaricus (LB). SEM images indicated the successful preparation of the nanofiber film incorporating LB. CPS incorporation significantly improved the survival ability of LB, with a live cell count reaching 7.62 log CFU/g after 28 days of storage at 4 °C — an increase of 1 log CFU/g compared to the fiber film without CPS. The results showed that the fiber film containing LB inhibited Escherichia coli and Staphylococcus aureus. Finally, the novel probiotic nanofiber film was applied to beef. The results showed that the shelf life of the beef during the experiments was extended for 2 days at 4 °C. Therefore, the novel probiotic film containing LB was suitable for meat preservation.

Evaluation of kefir grain microbiota, grain viability, and bioactivity from fermenting dairy processing by-products

Four dairy foods processing by-products (acid whey permeate (AWP), buttermilk (BM), sweet whey permeate (SWP), and sweet whey permeate with added milk fat globule ingredient (SWP+MFGM)) were fermented for 4 weeks and compared with traditional kefir milks for production of novel kefir-like dairy products. AU: Sweet whey permeates and SWP supplemented with 1.5% milk fat globule membrane (MFGM) showed to be the most viable by-products for kefir grain fermentation, exhibiting diverse abundance of traditional kefir microorganisms and positive indicators of bioactive properties. Grain viability was assessed with shotgun metagenomics, texture profile analysis, live cell counts, and scanning electron microscopy. Assessed bioactivities of the kefir-like products included antibacterial, antioxidant, potential anticancerogenic properties, and membrane barrier effect on human colorectal adenocarcinoma Caco-2 cells. All kefir grains were most abundant in Lactobacillus kefiranofaciens when analyzed with shotgun metagenomics. When analyzed with live cell counts on selective media, AWP kefir-like product had no countable Lactococcus spp. indicating suboptimal conditions for kefir grain microbiota survival and application for fermented dairy starter culture bacterium. Live cell counts were affirmed with kefir grain surface scanning electron microscopy images. SWP had the most adhesive kefir grain surface while SWP+MFGM had the largest exopolysaccharide (EPS) yield from grain extraction. All kefir and kefir-like products were able to achieve a 6-log reduction against Listeria innocua and Escherichia coli. Traditional milk kefirs had the highest antioxidant capacity for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. AWP had a significantly higher DPPH antioxidant activity and SWP had the lowest Trolox equivalence concentration in the ABTS assay. Sweet whey and supplemented milk fat sweet whey had upregulation of Cldn-1 and Ocln-1 gene expression, which correspond with a significant increase in transepithelial electrical resistance.

모싯잎 에탄올 추출물을 첨가한 산업용 배지에서 배양한 유산균 발효물의 항비만 효과

This study investigated the anti-obesity effects of lactic acid bacteria ferments cultured in industrial medium with ethanol extract of ramie leaf (Boehmeria nivea L.). On the 4th day of fermentation, the maximum live cell counts were 8.75-8.85 log CFU/mL, pH was 3.74-3.79, and total acidity was 2.07-2.19%. The fermentation of lactic acid bacteria on the fourth day resulted in the amount of lactic acid reaching 1,676.03-1,910.12 mg%. The lipase inhibitory activities of Lactiplantibacillus plantarum (L. plantarum) JBLAB0101 (FRLPLA) and Lactobacillus rhamnosus GG (LGG, (FRLLGG)) ferments were 30.10%, and 25.63%, respectively, at a concentration of 0.5 mg/mL. The lipid accumulation, leptin production, PPAR-γ and SREBP-1c mRNA levels were decreased to 37.54%, 54.64%, 24.18%, and 31.32%, respectively, at 200 μg/mL concentration of FRLPLA. These results suggest that anti-obesity effect could be increased by lactic acid bacteria in industrial medium with extract of ramie leaf.

Poly-Gamma-Glutamic Acid Nanopolymer Effect against Bacterial Biofilms: In Vitro and In Vivo Study.

In this study, a biodegradable poly-gamma-glutamic-acid nanopolymer (Ɣ-PGA NP) was investigated for its activity against clinical strains of Gram-positive (Staphylococcus aureus and Streptococcus pyogenes) and Gram-negative (Klebsiella pneumoniae and Escherichia coli), and reference strains of S. aureus ATCC 6538, S. pyogenes ATCC 19615 (Gram-positive), and Gram-negative E. coli ATCC 25922, and K. pneumoniae ATCC 13884 bacterial biofilms. The minimum inhibitory concentration (MIC) effect of Ɣ-PGA NP showed inhibitory effects of 0.2, 0.4, 1.6, and 3.2 μg/mL for S. pyogenes, S. aureus, E. coli, and K. pneumoniae, respectively. Also, MIC values were 1.6, 0.8, 0.2, and 0.2 μg/mL for K. pneumoniae ATCC 13884, E. coli ATCC 25922, S. aureus ATCC 6538, and S. pyogenes ATCC 19615, respectively. Afterwards, MBEC (minimum biofilm eradication concentration) and MBIC (minimum biofilm inhibitory concentration) were investigated to detect Ɣ-PGA NPs efficiency against the biofilms. MBEC and MBIC increased with increasing Ɣ-PGA NPs concentration or time of exposure. Interestingly, MBIC values were at lower concentrations of Ɣ-PGA NPs than those of MBEC. Moreover, MBEC values showed that K. pneumoniae was more resistant to Ɣ-PGA NPs than E. coli, S. aureus, and S. pyogenes, and the same pattern was observed in the reference strains. The most effective results for MBEC were after 48 h, which were 1.6, 0.8, 0.4, and 0.2 µg/mL for K. pneumoniae, E. coli, S. aureus, and S. pyogenes, respectively. Moreover, MBIC results were the most impactful after 24 h but some were the same after 48 h. MBIC values after 48 h were 0.2, 0.2, 0.2, and 0.1 μg/mL for K. pneumoniae, E. coli, S. aureus, and S. pyogenes, respectively. The most effective results for MBEC were after 24 h, which were 1.6, 0.8, 0.4, and 0.4 µg/mL for K. pneumoniae ATCC 13884, E. coli ATCC 25922, S. aureus ATCC 6538, and S. pyogenes ATCC 19615, respectively. Also, MBIC results were the most impactful after an exposure time of 12 h. MBIC values after exposure time of 12 h were 0.4, 0.4, 0.2, and 0.2 μg/mL for K. pneumoniae ATCC 13884, E. coli ATCC 25922, S. aureus ATCC 6538, and S. pyogenes ATCC 19615, respectively. Besides that, results were confirmed using confocal laser scanning microscopy (CLSM), which showed a decrease in the number of living cells to 80% and 60% for MBEC and MBIC, respectively, for all the clinical bacterial strains. Moreover, living bacterial cells decreased to 70% at MBEC while decreasing up to 50% at MBIC with all bacterial refence strains. These data justify the CFU quantification. After that, ImageJ software was used to count the attached cells after incubating with the NPs, which proved the variation in live cell count between the manual counting and image analysis methods. Also, a scanning electron microscope (SEM) was used to detect the biofilm architecture after incubation with the Ɣ-PGA NP. In in vivo wound healing experiments, treated wounds of mice showed faster healing (p < 0.00001) than both the untreated mice and those that were only wounded, as the bacterial count was eradicated. Briefly, the infected mice were treated faster (p < 0.0001) when infected with S. pyogenes > S. aureus > E. coli > K. pneumoniae. The same pattern was observed for mice infected with the reference strains. Wound lengths after 2 h showed slightly healing (p < 0.001) for the clinical strains, while treatment became more obvious after 72 h > 48 h > 24 h (p < 0.0001) as wounds began to heal after 24 h up to 72 h. For reference strains, wound lengths after 2 h started to heal up to 72 h.

Modulating efficacy and cytotoxicity of lipoamino fatty acid nucleic acid carriers using disulfide or hydrophobic spacers.

Double pH-responsive xenopeptides comprising polar ionizable succinoyl tetraethylene pentamine (Stp) motifs and lipophilic ionizable lipoamino fatty acids (LAFs) were recently found to efficiently transfect mRNA and pDNA at low doses. However, potency was often accompanied with cytotoxicity at higher doses. Insertion of bioreducible disulfide building blocks (ssbb) or non-reducible hydrophobic spacers between polar and apolar ionizable domains of LAF-Stp carriers should mitigate toxicity of xenopeptides. Carriers showed stable nucleic acid complexation and endosomal pH-dependent lytic activities, both of which were abolished after reductive cleavage of ssbb-containing carriers. For pDNA, U-shaped carriers with one Stp and two LAF units or bundle carriers with two Stps and four LAFs displayed highest potency. For mRNA, best transfection was achieved with bundle carriers with one Stp and four LAFs. Both the ssbb and hydrophobic spacer containing analogs displayed improved metabolic activity, reduced membrane damage, and improved cell growth. The ssbb carriers were most beneficial regarding living cell count and low apoptosis rates. Mechanistically, inserted spacers decelerated the transfection kinetics and altered the requirement of endosomal protonation. Overall, mRNA and pDNA carriers with improved biocompatibility have been designed, with their high potency illustrated in transfection of various cell lines including low passage number colon carcinoma cells.

Prospects for using flow cytometry in the quality control of live plague vaccines

Scientific relevance. The number of live bacteria is a quality parameter controlled at all stages of live plague vaccine production. Currently, live microbial cell counting uses a bacteriological method. However, flow cytometry has the potential to increase analytical accuracy and reduce testing time.Aim. This study aimed at testing the applicability of flow cytometry to assessing the quality of live plague vaccines.Materials and methods. The study quantified live microbial cells in 5 experimental batches of live plague vaccine as part of their quality control using the bacteriological method according to the State Pharmacopoeia of the Russian Federation (FS.3.3.1.0022.15). Cytofluorometry of the samples used the SynaptoGreen fluorescent dye.Results. The study quantified live microbial cells in live plague vaccine samples using the bacteriological method and flow cytometry. The results obtained by the bacteriological method ranged from 27.8±2.2 to 56.5±3.1% with an average of 39.8±5.4%. The results obtained by flow cytometry ranged from 29.2±1.2 to 59.1±2.1% with an average of 41.7±5.5%. The statistical analysis showed no significant difference between the results of vaccine quality control by both methods, as well as a high coefficient of determination.Conclusions. The results show that flow cytometry is an appropriate method for the quantification of live microbial cells as part of the quality control of plague vaccines. Being quick, easy, and highly informative, flow cytometry is preferable to traditional methods.

Ammidin ameliorates myocardial hypoxia/reoxygenation injury by inhibiting the ACSL4/AMPK/mTOR-mediated ferroptosis pathway

ObjectiveThe aim of the present study was to investigate the therapeutic effect of ammidin on hypoxia/reoxygenation (H/R) injury in primary neonatal rat cardiomyocytes by observing the role of ferroptosis in the process of H/R injury, and to verify its target and regulatory signaling pathways.MethodsThe network pharmacology analysis was used to predict the biological processes, core targets and related signaling pathways of Angelica dahurica in the treatment of ferroptosis. Cell viability was assessed using live cell imaging and cell counting kit-8. Lactate dehydrogenase (LDH), reactive oxygen species (ROS) production, and malondialdehyde (MDA), superoxide dismutase (SOD) and mitochondrial membrane potential (MMP) content were determined to assess the level of ferroptosis. Western blotting was performed to measure protein expression.ResultsNetwork pharmacology predicted that Acyl-CoA synthetase long chain family member 4 (ACSL4) was highly associated with myocardial H/R injury in the intersection of Angelica dahurica and ferroptosis. The top three active components of Angelica dahurica were found to be mandenol, alloisoimperatorin and ammidin, among which ammidin was found to have the strongest binding to the target proteins of the ACSL4/AMPK/mTOR pathway. H/R reduced the viability of cardiomyocytes, while the inhibition of ferroptosis by ferrostatin-1 alleviated the H/R-induced inhibition of cardiomyocyte viability. This was evidenced by the increased cell viability, SOD release, MMP level and glutathione peroxidase 4 (GPX4) protein expression, as well as the decreased LDH and MDA release and ROS production and ACSL4 protein expression (P < 0.05). To verify the existence of ferroptosis in myocardial hypoxia/reoxygenation injury. In addition, ammidin increased cell viability and GPX4 protein expression (P < 0.05), decreased ROS generation, and MDA and MTT expression (P < 0.05), then inhibited ferroptosis, and finally alleviated myocardial H/R injury by regulating the ACSL4/AMPK signaling pathway.ConclusionsNetwork pharmacology was used to predict the correlation between ammidin and ferroptosis following myocardial H/R injury. It was demonstrated that ammidin may regulate ferroptosis by inhibiting the ACSL4/AMPK/mTOR signaling pathway and reduce H/R injury in cardiomyocytes.

Effect of freeze-dried protectants on the survival rate and fermentation performance of fermented milk's directed vat set starters

A directed vat set (DVS) starter was proposed to improve the drawbacks of liquid starters in fermented production and enhance the survival rates of B. animalis subsp. lactis BZ11, S. thermophilus Q-1, and Lactiplantibacillus plantarum LB12. The protective agent formula was optimized using the response surface method (RSM), with the survival rate as the benchmark. The best combination of cryoprotectants was determined to be BZ11: 10 % skimmed milk powder, 3 % sodium glutamate, and 15 % trehalose; LB12: 10 % skim milk powder, 5 % glutamate sodium, and 10 % trehalose; Q-1: 10 % skimmed milk powder, 3 % sodium glutamate, and 10 % trehalose. The survival rate of BZ11 significantly increased to 92.87 ± 1.25 %. The DVS fermented milk did not differ significantly from the control group regarding cholesterol removal, live cell counts and pH (p > 0.05). All DVS can be stored for at least 2500 d at −20 °C—this DVS starter for fermented milk benefits from its large-scale and automated commercial production.

Construction of walnut protein/tea polyphenol/alginate complex for enhancing heat and gastrointestinal tolerance of lactic acid bacteria

The presence of live cells in the intestinal tract is crucial for the beneficial effects of probiotic products containing lactic acid bacteria. However, many bacterial cells lose viability during production, storage, and gastric passage. Protein-based hydrogels and polysaccharide-based capsules effectively enhance the viability of live probiotic cells in products, but the underlying mechanisms and in vivo effectiveness are unclear. In this study, a complex was developed by self-aggregating walnut protein, tea polyphenol, and Lactobacillus rhamnosus cells under acidic conditions, followed by alginate coating. Complex formation was facilitated by hydrogen bonding and hydrophobic interactions. The complex had a low thermal diffusion coefficient of 4.9 × 10−7 m2/s, making the bacterial cells resistant to temperature fluctuation. Cell survival rate was 75.5% after 12 h at 55 °C and 92.8% after freeze-drying. Shelf-life predictions were 14 years at 4 °C. and 2.3 months at 25 °C, with a live cell count exceeding 106 CFU/g. The product demonstrated a 60% increase in live bacterial cells compared to free cells in simulated gastric fluid, and achieved complete release in simulated intestinal fluid within 120 min. This method was successfully applied to L. rhamnosus, L. casei and L. plantarum. Oral administration of the L. rhamnosus product significantly increased the abundance of Lactobacillus and L. rhamnosus in the colon and cecum of mice. These findings present a new method for producing highly active probiotic products using plant materials and provide insights into the underlying mechanisms from a thermodynamic perspective, contributing to the industrialization and advancement of probiotics.

In-vitro polymicrobial oral biofilm model represents clinical microbial profile and disease progression during implant-related infections.

Clinically relevant in-vitro biofilm models are essential and valuable tools for mechanistically dissecting the etiopathogenesis of infectious diseases and test new antimicrobial therapies. Thus, the aim of this study was to develop and test a clinically relevant in-vitro oral polymicrobial biofilm model that mimics implant-related infections in terms of microbial profile. For this purpose, 24-well plate system was used to model oral biofilms, using three different microbial inoculums to grow in-vitro biofilms: (1) human saliva from periodontally healthy patients; (2) saliva as in inoculum 1+Porphyromonas gingivalis strain; and (3) supra and subgingival biofilm collected from peri-implant sites of patients diagnosed with peri-implantitis. Biofilms were grown to represent the dynamic transition from an aerobic to anaerobic community profile. Subsequently, biofilms were collected after each phase and evaluated for microbiological composition, microbial counts, biofilm biomass, structure, and susceptibility to chlorhexidine (CHX). Results showed higher live cell count (P<.05) for biofilms developed from patients' biofilm inoculum, but biomass volume, dry weight, and microbiological composition were similar among groups (P>.05). Interestingly, according to the checkerboard DNA-DNA hybridization results, the biofilm developed from stimulated human saliva exhibited a microbial composition more similar to the clinical subgingival biofilm of patients with peri-implantitis, with proportions of the main pathogens closer to those found in the disease. In addition, biofilm developed using saliva as inoculum was shown to be susceptible to CHX with significant reduction in bacteria compared with biofilms without exposure to CHX (P<.05). The findings suggested that the in-vitro polymicrobial biofilm developed from human saliva as inoculum is a suitable model and clinically relevant tool for mimicking the microbial composition of implant-related infections.

Oral microbial colonization on titanium and polyetheretherketone dental implant healing abutments: An in vitro and in vivo study

Although polyetheretherketone (PEEK) implant healing abutments have become popular because of their esthetic, mechanical, and chemical properties, studies analyzing oral polymicrobial adhesion to PEEK abutments are lacking. The purpose of this in vitro and in vivo study was to evaluate oral microbial adhesion and colonization on titanium (Ti) and PEEK healing abutments. Ti (N=35) and PEEK substrates (N=35) were evaluated in vitro in terms of the initial adhesion (1 hour) or biofilm accumulation (48 hours) of Candida albicans and a polymicrobial inoculum using stimulated human saliva to mimic a diverse oral microbiome. Surface decontamination ability was evaluated after 24 hours of in vitro biofilm formation after exposure to an erbium-doped yttrium aluminum garnet (Er:YAG) laser. Conventional and flowable composite resin veneering on PEEK was also tested for microbial adhesion. In addition, an in vivo model with 3 healthy volunteers was conducted by using a palatal appliance containing the tested materials (3 or 4 specimens of each material per appliance) for 2 days to evaluate the effect of substrate on the microbial profile. Biofilms were evaluated by live cell counts and scanning electron microscopy images, and the microbial profile by Checkerboard deoxyribonucleic acid (DNA)-DNA hybridization. The t test and Mann-Whitney test were used to compare the groups (α=.05). PEEK and Ti materials showed similar fungal adhesion (P>.05). Although the PEEK surface limited the initial in vitro polymicrobial adhesion (approximately 2 times less) compared with Ti (P=.040), after 48 hours of biofilm accumulation, the microbial load was statistically similar (P=.209). Er:YAG laser decontamination was more effective on PEEK than on Ti surfaces, reducing approximately 11 times more microbial accumulation (P=.019). Both composite resins tested showed similar microbial adhesion (1 hour). In vivo, the PEEK material showed reduced levels of 6 bacterial species (P<.05), including the putative pathogen Treponema denticola. Although PEEK and Ti had similar bacterial and fungus biofilm attachment and accumulation, PEEK promoted a host-compatible microbial profile with a significantly reduced T. denticola load.

Monitoring the process of yogurt spoilage by dielectric measurements and spread plate method

In my research work, I primarily focused on the investigation of yogurt (made from home-made raw milk) spoilage by microbiological and dielectric measurements. During the experiment, I continuously monitored the changes in aerobic and anaerobic Lactobacillus cell counts of the product, as a possible spoilage process would cause the deteriorative microbes to displace the lactic acid bacteria, and I also monitored the changes in the dielectric properties of the sample material at 400 MHz frequency. The research results verified that there is a strong correlation between the variation in live cell counts and dielectric parameters in both aerobically and anaerobically cultured lactic acid bacteria. The main conclusions of the results are that the change in the bacterial count, and thus the deterioration process leading to it, can be indirectly monitored in the dairy product under study by low-frequency determination of both the dielectric constant and the loss factor.