Number Of Viable Cells Research Articles

Evaluation of the Genotoxic Effects of Grape Seed Extract and Marine Collagen Peptide on the Fibroblast Cell Line: An In Vitro Study.

Introduction Collagen plays a vital role in maintaining the structural integrity of dentin, and its modification with bioactive compounds can enhance its mechanical properties and bonding capabilities. Aim This study aimed to evaluate the genotoxic effects of grape seed extract (GSE) and marine collagen peptide(MCP) on dental pulp-derived primary cells. Methodology Human dental pulp stem cells were isolated,cultivated, and then treated with GSE and marine collagen peptides. DNA fragmentation was assessed using DAPI (4',6-diamidino-2-phenylindole) staining. Statistical analysis was performed using SPSS version 20 (IBM Corp., Armonk, NY, USA). Results The results showed that GSE exhibited a minimum level of cell death compared to marine collagen peptides. The viable cell count increased steadily over three days in all groups, with the control group showing the highest number of viable cells. The differences in viable cell count among the groups were statistically significant. Conclusion This study suggests that GSE and marine collagen peptides are highly biocompatible with dental pulp cells and could be considered for further clinical studies.

New Approach to the Cryopreservation of GV Oocytes and Cumulus Cells through the Lens of Preserving the Intercellular Gap Junctions Based on the Bovine Model.

Differences in structural and functional properties between oocytes and cumulus cells (CCs) may cause low vitrification efficiency for cumulus-oocyte complexes (COCs). We have suggested that the disconnection of CCs and oocytes in order to further cryopreservation in various ways will positively affect the viability after thawing, while further co-culture in vitro will contribute to the restoration of lost intercellular gap junctions. This study aimed to determine the optimal method of cryopreservation of the suspension of CCs to mature GV oocytes in vitro and to determine the level of mRNA expression of the genes (GJA1, GJA4; BCL2, BAX) and gene-specific epigenetic marks (DNMT3A) after cryopreservation and in vitro maturation (IVM) in various culture systems. We have shown that the slow freezing of CCs in microstraws preserved the largest number of viable cells with intact DNA compared with the methods of vitrification and slow freezing in microdroplets. Cryopreservation caused the upregulation of the genes Cx37 and Cx43 in the oocytes to restore gap junctions between cells. In conclusion, the presence of CCs in the co-culture system during IVM of oocytes played an important role in the regulation of the expression of the intercellular proteins Cx37 and Cx43, apoptotic changes, and oocyte methylation. Slow freezing in microstraws was considered to be an optimal method for cryopreservation of CCs.

Resazurin assay as a suitable method for testing the antimicrobial activity of photocatalytic surfaces

The antimicrobial activity of different types of photocatalytic surfaces (titania porous and nonporous phthalocyanine films) was tested using a resazurin assay. The gram-negative bacteria Escherichia coli was chosen as the model microorganism. A bacteria inoculum with a concentration of 1.75 × 106 CFU/ml was applied to hydrogel carriers and these were subsequently placed on the study surfaces. Half of the samples were kept in the dark and the other half were illuminated. After 4 h, the carriers were transferred to vials, resazurin added and the number of viable cells in the mixture counted. For nonporous surfaces, the effect of the addition of a biocide was investigated. The authors observed that as the amount of biocide in the layer increased, the proportion of viable cells in the samples kept in the dark decreased. These results are in good agreement with general assumptions. For the titania surfaces, the antimicrobial activity (characterized by ΔR) obtained through resazurin assay was compared to that obtained through the use of standard techniques. The final activity was very similar (the ΔR from the resazurin assay was 1.54 and the standard test gave a result of 1.56). The results confirm the reliability of the proposed method, and the use of carriers give us the possibility to test much larger installed surfaces without a need to cause damage to them.

An automated approach for hemocytometer cell counting based on image-processing method

Evaluating cell viability plays an important role in routine cell culture, toxicology, and drug development. Trypan blue stain is one of the simplest assays that are used to determine the number of viable cells in a cell suspension. The stained cell suspension can then be counted under a microscope using a hemocytometer. Hemocytometers have a fixed volume, making it possible to calculate the concentration of cells in the sample and measure viability. Most of the related studies are conducted using manual counting methods. However, manual counting is time-consuming and laborious. While various automated cell counting devices have been proposed, they tend to be too expensive for widespread deployment. Although low-cost automated cell counting methods presented in the literatures provide high accuracy and cost-effective alternatives, only cells in the field of view of microscope with unknown volume are counted. This will make the concentration inestimable. Accordingly, the present study proposes a low-cost but accurate method for hemocytometer cell counting using an image-processing approach. The accuracy of the proposed method is demonstrated by comparing the cell counting results obtained for human cervical carcinoma (HeLa) cells with the manual counts. It is shown that the proposed approach has a mean absolute percentage error of less than 6.26 % for samples containing a mixture of live and dead cells. Moreover, the proposed system is equipped with a simple and intuitive human machine interface such that the counting task can be performed by individuals with no prior experience in the image-processing field.

Screening of Amino Acids as a Safe Energy Source for Isolated Rat Pancreatic Acini.

Amino acids play an essential role in protein synthesis, metabolism, and survival of pancreatic acini. Adequate nutritional support is important for acute pancreatitis treatment. However, high concentrations of arginine and lysine may induce acute pancreatitis. The study aimed to identify the most suitable l -amino acids as safe energy sources for pancreatic acinar cells. Pancreatic acini were isolated from male Wistar rats. Effects of amino acids (0.1-20 mM) on uncoupled respiration of isolated acini were studied with a Clark electrode. Cell death was evaluated with fluorescent microscopy and DNA gel electrophoresis. Among the tested amino acids, glutamate, glutamine, alanine, lysine, and aspartate were able to stimulate the uncoupled respiration rate of isolated pancreatic acini, whereas arginine, histidine, and asparagine were not. Lysine, arginine, and glutamine (20 mM) caused complete loss of plasma membrane integrity of acinar cells after 24 hours of incubation. Glutamine also caused early (2-4 hours) cell swelling and blebbing. Aspartate, asparagine, and glutamate only moderately decreased the number of viable cells, whereas alanine and histidine were not toxic. DNA fragmentation assay and microscopic analysis of nuclei showed no evidence of apoptosis in cells treated with amino acids. Alanine and glutamate are safe and effective energy sources for mitochondria of pancreatic acinar cells.

Antimicrobial action, cytotoxicity and erosive potential of hypochlorous acid obtained from an electrolytic device compared with sodium hypochlorite.

This study aimed to compare the antimicrobial action, cytotoxicity, cleaning ability, and erosion of dentine of hypochlorous acid (HClO) obtained from an electrolytic device at two different concentrations (Dentaqua) and three concentrations of sodium hypochlorite (NaOCl). Microbiological test-The root canals of sixty single-rooted extracted human teeth were inoculated with Enterococcus faecalis and divided into 6 groups (n = 10), according to decontamination protocol: DW (control); 1% NaOCl; 2.5% NaOCl; 5.25% NaOCl; 250 ppm HClO and 500 ppm HClO. The colony-forming units were counted to evaluate the decontamination potential of each group, calculating the reduction in bacterial percentage. Cytotoxicity test-Cytotoxicity was evaluated after inoculation of the same tested protocols in fibroblastic cells for 3 min, calculating the cell viability percentages. Specifical statistical analysis was performed (α = 5%). Cleaning ability and erosion-Fifty-six single-rooted bovine lower incisors were divided into seven groups of 8 roots each, being the test groups 1% NaOCl; 2.5% NaOCl; 5,25% NaOCl; 250 ppm HClO and 500 ppm HClO, and a negative and positive control. Negative control was not contaminated, and the other groups were inoculated with Enterococcus faecalis. SEM images were ranked as from the cleanest to the least clean. Erosion was also assessed, being ranked from the least to the most eroded dentine. The highest bacterial reduction was observed in experimental groups, with no statistical differences between them (p > 0.05). The highest number of viable cells was observed in control group, followed by 250 ppm HClO and 500 ppm HClO groups, with statistical differences between them (p < 0.05). 1% NaOCl; 2.5% NaOCl; 5.25% NaOCl and 500 ppm HClO displayed the cleanest areas. All sodium hypochlorite groups displayed erosion with higher ranks with greater concentration, while hypochlorous acid did not display any erosion regardless the concentration. It is possible to conclude that HClO obtained from an electrolytic device presented high antimicrobial activity and low cytotoxicity in both tested concentrations. 500 ppm HClO did not display erosion and showed great cleaning ability. The use of 500 ppm hypochlorous acid may reduce unfavorable behavior of sodium hypochlorite whilst maintaining its antimicrobial action.

Triphenyltin(IV) compounds bearing modulated azo-carboxylato ligands: Synthesis, structural characterization, in vitro cytotoxicity, BSA/DNA binding affinity, and in silico studies

Three novel triphenyltin(IV) compounds with modulated azo-carboxylato ligands: triphenylstannyl (E)-4-((2-hydroxynaphthalen-1-yl)diazenyl)benzoate, 1, triphenylstannyl (E)-4-((4-hydroxyphenyl)diazenyl)benzoate, 2, and triphenylstannyl (E)-4-((4-(dimethylamino)phenyl)diazenyl)benzoate, 3, were synthesized and characterized by elemental analysis, FTIR and NMR (1H, 13C, 119Sn) spectroscopy. The structures and spectra of compounds were predicted by Density Functional Theory (DFT) methods at B3LYP-D3BJ/6–311++G(d,p)(H,C,N,O)/LanL2DZ(Sn) level of theory. Furthermore, the antitumor potential of ligand precursors, HL1–HL3, and appropriate organotin(IV) compounds 1–3 was evaluated across mouse melanoma B16F1, human breast adenocarcinoma MCF-7, human colorectal HT-29 and human prostate PC3 cell lines using MTT and CV assays. The organotin(IV) compounds exhibit enhanced cellular uptake and efficacy in reducing viable cell numbers when compared to free acids. Specifically, compound 3 demonstrates a notable impact at lower nanomolar concentrations on all tested cell lines. Moreover, 3 induces cell death in MCF-7 cells by inhibiting cell division and promoting the overproduction of cellular nitric oxide (NO), ultimately leading to caspase-independent apoptosis. Importantly, this process occurs without concurrent activation of autophagy or the generation of ROS/RNS species. The binding affinity of 1–3 with calf thymus DNA (CT-DNA) and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy and molecular docking simulations, suggesting their capacity to interact with these biomolecules.

Construction of a convenient microbial-growth observation system based on microscopic image analysis applicable to the evaluation of preservative effects

The pour-plate method, a conventional approach to the evaluation of food microbial contamination, is costly and time-consuming. We have developed a novel and efficient system to evaluate microbial growth on the surfaces of foods using a membrane filter and microscope followed by image analysis. While Wickerhamomyces anomalus cells directly inoculated onto loin ham were undetectable because of the complex surface structure of the food, the placement of a hydrophilic polytetrafluoroethylene membrane filter on the loin ham enabled us to clearly observe the yeast cells on the filter by microscopy without affecting their growth. Under microscopic observation, cells on the membrane filter as well as those on the agar plate were always observed with surrounding annular dark regions, which were separated from the background by clear boundaries. We defined the areas inside the boundaries, including the cells and the annular dark regions, as the “bounded areas”. In image analysis, the numbers of pixels in the bounded areas increased exponentially in parallel with the viable cell count (CFU/g), suggesting that the pixel numbers were proportional to the viable cell numbers. We applied this system to investigate the adverse effects of sorbic acid preparation (SAP), a common food preservative, on yeast growth. The yeast was incubated on filter-overlaid agar medium in the presence or absence of SAP, resulting in a dose-dependent reduction in the specific pixel increase rate (k [/h]) determined by image analysis as well as in the specific growth rate (μ [/h]) estimated by the agar-plate-based conventional method. Regression analysis revealed a strong correlation between the k and μ values (R2, 0.985) normalized to the corresponding values without SAP. Our system could be applied to estimate the growth rate of microbes on foods and to examine the effects of environmental factors on microbial growth by using the k value as an index. Furthermore, this system would contribute to enhancing food safety, extending shelf life, and solving the important global problem of food loss.

Evaluation of Cytotoxicity of Allium sativum (Garlic Extract) against Human Dental Pulp Fibroblasts.

Vital pulp therapy procedures in primary dentition focuses on preservation and maintenance of pulp tissue that has been compromised due to caries, trauma, etc. Several pulp dressing materials have been used in primary teeth and some natural materials from the field of traditional medicine have also been introduced as medicaments in vital pulp therapy. The understanding of biologic and cytotoxic properties of newer materials is important for safe clinical usage. The biologic compatibility of these newer materials is imperative to limit or avoid tissue irritation or degeneration. To evaluate the cytotoxic effects of Allium sativum on cultured human primary dental pulp fibroblasts. Primary pulp fibroblasts were cultured from the pulp tissue obtained from extracted deciduous primary canines and central incisor teeth. The freshly prepared concentrations of 1000, 500, 250, 125, and 62.5 µg/mL A. sativum extract were added to the 96-well plate in triplicates to which culture medium containing fourth passage cell suspension was added previously. Cells without treatment served as control, while cells treated with 5% dimethyl sulfoxide (DMSO) served as toxic control. After the addition of experimental and control agents, cells were incubated for 24 and 48 hours at 37°C in 5% CO2 atmosphere. After the incubation period, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine the number of viable cells. Absorbance was read with a microplate reader at 570 nm wavelength and the relative viability of dental pulp fibroblasts at various concentrations was expressed as color intensity of the experimental wells relative to that of control. The percentage of cell viability was also calculated accordingly. The MTT assay results revealed that A. sativum extract, in all the concentrations tested at both the time intervals maintained a cell viability of greater than 90%. At 24 hours, the mean absorbance value of untreated control wells was recorded as 0.84400 ± 0.00916 with 100% cell viability. Among all the concentrations of garlic extract tested, highest mean absorbance value of 0.83933 ± 0.00550 with 99.44% cell viability was recorded for 62.5 µg/mL concentration. At 48 hours, the mean absorbance value of untreated control wells was recorded as 1.22767 ± 0.01106 with 100% cell viability, and the highest mean absorbance value of 1.22567 ± 0.01006 with 99.83% cell viability was recorded for 62.5 µg/mL concentration. The cell viability did not seem to be affected by the concentration of A. sativum extract at 24 hours. However, at 48 hours, the sensitivity of the cells was observed to be dependent on the concentration of A. sativum with a decrease in the viability of cells noted with the increase in concentration. A. sativum extract is noncytotoxic in nature and preserves the vitality of cultured human primary dental pulp fibroblasts making it a suitable material for use in vital pulp therapy procedures of primary teeth. Devaraju R, Reddy D, Paul ST, et al. Evaluation of Cytotoxicity of Allium sativum (Garlic Extract) against Human Dental Pulp Fibroblasts. Int J Clin Pediatr Dent 2024;17(2):143-148.

Solid-State Fermentation Using Bacillus licheniformis-Driven Changes in Composition, Viability and In Vitro Protein Digestibility of Oilseed Cakes

The solid-state fermentation (SSF) efficiency of Bacillus licheniformis ATCC 21424 (BL) on various agro-industrial by-products such as oilseed cakes [hemp (HSC), pumpkin (PSC), and flaxseed (FSC)] was evaluated by examining the nutritional composition, reducing sugars, and in vitro protein digestibility (IVPD) for use in animal nutrition. SSF significantly decreased crude protein, along with changes in the total carbohydrates (p &lt; 0.05) for all substrates fermented. An increase in crude fat for HSC (1.04%) and FSC (1.73%) was noted, vs. PSC, where the crude fat level was reduced (−3.53%). Crude fiber does not differ significantly between fermented and nonfermented oilseed cakes (p &gt; 0.05). After fermentation, neutral detergent fiber (NDF) and acid detergent fiber (ADF) significantly increased for HSC and FSC (p &lt; 0.05), as well as for PSC despite the small increase in ADF (4.46%), with a notable decrease in NDF (−10.25%). During fermentation, pH shifted toward alkalinity, and after drying, returned to its initial levels for all oilseed cakes with the exception of PSC, which maintained a slight elevation. Further, SSF with BL under optimized conditions (72 h) increases the reducing sugar content for FSC (to 1.46%) and PSC (to 0.89%), compared with HSC, where a reduction in sugar consumption was noted (from 1.09% to 0.55%). The viable cell number reached maximum in the first 24 h, followed by a slowly declining phase until the end of fermentation (72 h), accompanied by an increase in sporulation and spore production. After 72 h, a significant improvement in water protein solubility for HSC and FSC was observed (p &lt; 0.05). The peptide content (mg/g) for oilseed cakes fermented was improved (p &lt; 0.05). Through gastro-intestinal simulation, the bacterial survivability rate accounted for 90.2%, 101.5%, and 85.72% for HSC, PSC, and FSC. Additionally, IVPD showed significant improvements compared to untreated samples, reaching levels of up to 65.67%, 58.94%, and 80.16% for HSC, PSC, and FSC, respectively. This research demonstrates the advantages of oilseed cake bioprocessing by SSF as an effective approach in yielding valuable products with probiotic and nutritional properties suitable for incorporation into animal feed.

Inclusion of Antifungal and Probiotic Lactiplantibacillus plantarum Strains in Edible Alginate Coating as a Promising Strategy to Produce Probiotic Table Grapes and Exploit Biocontrol Activity

The use of lactic acid bacteria (LAB) for the probiotic enrichment of minimally processed fruit is a well-established practice in the literature. In addition, several LAB demonstrated a strain-specific ability to control harmful microorganisms and decay agents, improving shelf life, maintaining quality, and promoting the safety of fruits and vegetables. Edible coatings can help modulate the phenomena of gas exchange and water loss by fruits, representing protection from physical damage and spoilage phenomena linked to oxidation and the development of undesired microorganisms. At the same time, the coating can represent an innovative delivery matrix for the LAB strains of potential interest to improve safety and quality in the postharvest management of fruits. In this work, five Lactiplantibacillus plantarum strains, previously characterised for their probiotic and antifungal activity, were incorporated into a sodium alginate coating to develop edible probiotic coatings with antifungal properties for table grapes cv. Italia. The bacterial transfer and their survival were evaluated by comparing coated and uncoated table grapes during 14 days of cold storage at 4 °C. The alginate edible coating increased the number of viable cells transferred to the surface of the berries from about 5 to more than 7 Log CFU/g, with a crucial impact on the potential functional attributes of the final product. The ability of the functionalised coatings to counteract the decay development was evaluated on table grape berries artificially contaminated with Aspergillus niger CECT 2805. A significant reduction in lesion diameter was observed in the alginate coating with L. plantarum 11-A, with a reduction from 15.40 ± 1.14 mm of uncoated berries to 8.40 ± 1.14 mm of berries coated with L. plantarum 11-A. The lesion diameter reduction was also accompanied by a reduction in the symptoms of infection, such as browning around the wound. These results suggest the application of selected strains of L. plantarum as promising bio-resources to enhance the overall value of ready-to-eat fruits and vegetables, particularly in combination with edible coating as a carrier matrix. While a strain-dependent effect was not detected with respect to the improvement in the number of cells in the edible coating, a variability depending on the biotype used was detected for the properties linked to biocontrol, suggesting that the inclusion in edible packaging may represent an innovative criterion in the selection of lactobacilli to be applied postharvest.

In vitro anticancer activity of melanin-like nanoparticles for multimodal therapy of glioblastoma

Abstract Glioblastoma (GBM) is one of the most aggressive and hard to treat cancers. Traditional anti-cancer treatment methods have low efficiency and the lifespan after diagnosis is only 12–18 months. Brain tumor cells overexpress many proteins that play an important role in tumor progression and can be used as therapeutic targets. One of the promising approaches in cancer treatment is down-regulation of an extracellular matrix glycoprotein – Tenascin-C (TN-C) through RNA interference therapy. However, the effective delivery of double stranded RNA with one strand complementary to TN-C mRNA sequence is difficult due to rapid degradation by nucleases and low intracellular uptake. Polydopamine (PDA), a biomimetic polymer characterized by high biocompatibility and simple modification ability, is commonly used in nanobiomedicine to create a drug/gene delivery vehicle. Furthermore, photothermal characteristics of this polymer enable its application in photothermal therapy (PTT), which is a great option for cancer treatment. Here we synthesize PDA nanoparticles (NPs) coated with polyamidoamine dendrimers generation 3.0 (DD3.0) for therapeutic anti-TN-C RNA and doxorubicin delivery. As prepared PDA@DD3.0 NPs are then used in combined drug delivery, gene silencing, and PTT of GBM. The obtained materials are analyzed in terms of physicochemical and photothermal properties as well as their cytotoxicity, using human GBM cells. The results demonstrate that the obtained nanocarriers are effective non-viral vehicle for combined therapeutic approach for killing glioma cells via anti-TN-C RNA delivery and combined chemo-PTT therapy (CT-PTT). The application of PDA@DD3.0 NPs contributed to the 3-fold reduction in the proliferation rate of GBM cells, a decrease in the level of TN-C expression (by 30%) and a reduction in the number of viable cells by up to 20%.

Cytotoxicity and Degradation Resistance of Cryo- and Hydrogels Based on Carboxyethylchitosan at Different pH Values.

Background: The use of chitosan-based gels is still limited due to their restricted solubility in acid solutions, where the molecules have a positive charge. The functionalization of chitosan makes it possible to significantly expand the possibilities of using both the polymer itself and hydrogels based on its derivatives. Objective: To evaluate the effect of the conditions for the production of cryo- and hydrogels based on carboxyethylchitosan (CEC) crosslinked with glutaraldehyde on gel swelling and its resistance to degradation depending on pH and cytotoxic effects and to test the hypothesis that the amount of crosslinking agent during synthesis may affect the cytotoxicity of the gel. Methods: Gels' swelling values and degradation resistance were determined using the gravimetric method. The cytotoxic effect was evaluated during the co-cultivation of gels in the presence of human fibroblasts using light optical microscopy and flow cytometry. Results: All CEC-based cryogels had a higher equilibrium swelling value and degradation time than the CEC hydrogel in the pH range from 4.6 to 8.0. This demonstrates the superiority of cryogels relative to CEC-based hydrogels in terms of swelling potential and degradation resistance, while an increase in the number of crosslinks with glutaraldehyde contributes to longer swelling of the cryogel. The positive control (intact fibroblasts) and all gel samples were statistically identical in the number of viable cells. On the third day, the viability of the fibroblast cells was consistently high (above 95%) and did not differ between all tested CEC-based gels. And in general, the cell morphology analysis results corresponded with the results obtained in the flow cytometry-based cytotoxicity test. We also did not find proof in our experiment to support our hypothesis that the amount of crosslinking agent during synthesis may affect the cytotoxicity of the material.

Effects of film-forming components on the viability of probiotics and the application of synbiotic pectin film in preserving Da Xanh pomelo and Thai jackfruit fresh-cut.

Minimally processed products are highly convenient, and fresh-cut fruits coated with the synbiotic film have many advantages. This study investigated the film-forming components and preservation ability of Da Xanh pomelo and Thai jackfruit fresh-cut by synbiotic pectin film. The results showed that PA70 film combined with 1.5% FOS (fructooligosaccharides) had the highest number of viable cells of L. plantarum after 30days of storage at 5°C. The number of probiotic cells existing on fresh-cut products of Da Xanh pomelo and Thai jackfruit was always high (> 8 log CFU/g) and stable during 10days of storage. In addition, jackfruit and pomelo fresh-cut preserved with probiotic film also showed probiotic activity in simulated stomach and small intestine medium with the number of probiotic cells (> 6 log CFU/g) and survival cell ratio after 4h in small intestine medium reached 81.20 ± 0.92% (pomelo) and 82.16 ± 0.94% (Thai jackfruit).

Antimicrobial action and cytotoxicity of hypochlorous acid obtained from an innovative electrolytic device – An in vitro study

ObjectiveThis study evaluated the antimicrobial effect and cytotoxicity of hypochlorous acid(HClO) obtained from an innovative electrolytic device. DesignThe root canals of fifty extracted human teeth were inoculated with Enterococcus faecalis and divided into 5 groups (n = 10): DW (control); 2% chlorhexidine gel(CHX); 2.5% sodium hypochlorite(NaOCl); 250 ppm HClO and 500 ppm HClO. The counting of colony forming units evaluated the decontamination potential of each group. Cytotoxicity was evaluated after inoculation of tested protocols in fibroblastic cells for 3 min, calculating the cell viability. Specific statistical analysis was performed (α = 5%). ResultsThe highest bacterial reduction was observed in experimental groups, with no statistical differences from each other (p > 0.05). The highest number of viable cells was observed in control group, followed by 250 ppm HClO and 500 ppm HClO groups, with statistical differences from each other (p < 0.05). ConclusionsIt could be concluded that HClO presented high antimicrobial activity and low cytotoxicity at both tested concentrations.

Optimization of preparation conditions for Salsola laricifolia protoplasts using response surface methodology and artificial neural network modeling

BackgroundSalsola laricifolia is a typical C3–C4 typical desert plant, belonging to the family Amaranthaceae. An efficient single-cell system is crucial to study the gene function of this plant. In this study, we optimized the experimental conditions by using Box-Behnken experimental design and Response Surface Methodology (RSM)-Artificial Neural Network (ANN) model based on the previous studies.ResultsAmong the 17 experiment groups designed by Box-Behnken experimental design, the maximum yield (1.566 × 106/100 mg) and the maximum number of viable cells (1.367 × 106/100 mg) were obtained in group 12, and the maximum viability (90.81%) was obtained in group 5. Based on these results, both the RSM and ANN models were employed for evaluating the impact of experimental factors. By RSM model, cellulase R-10 content was the most influential factor on protoplast yield, followed by macerozyme R-10 content and mannitol concentration. For protoplast viability, the macerozyme R-10 content had the highest influence, followed by cellulase R-10 content and mannitol concentration. The RSM model performed better than the ANN model in predicting yield and viability. However, the ANN model showed significant improvement in predicting the number of viable cells. After comprehensive evaluation of the protoplast yield, the viability and number of viable cells, the optimal results was predicted by ANN yield model and tested. The amount of protoplast yield was 1.550 × 106/100 mg, with viability of 90.65% and the number of viable cells of 1.405 × 106/100 mg. The corresponding conditions were 1.98% cellulase R-10, 1.00% macerozyme R-10, and 0.50 mol L−1 mannitol. Using the obtained protoplasts, the reference genes (18SrRNA, β-actin and EF1-α) were screened for expression, and transformed with PEG-mediated pBI121-SaNADP-ME2-GFP plasmid vector. There was no significant difference in the expression of β-actin and EF1-α before and after treatment, suggesting that they can be used as internal reference genes in protoplast experiments. And SaNADP-ME2 localized in chloroplasts.ConclusionThe current study validated and evaluated the effectiveness and results of RSM and ANN in optimizing the conditions for protoplast preparation using S. laricifolia as materials. These two methods can be used independently of experimental materials, making them suitable for isolating protoplasts from other plant materials. The selection of the number of viable cells as an evaluation index for protoplast experiments is based on its ability to consider both protoplast yield and viability. The findings of this study provide an efficient single-cell system for future genetic experiments in S. laricifolia and can serve as a reference method for preparing protoplasts from other materials.

Conservation techniques for promising bacteria in the oil industry to preserve biological properties

Background: The conservation of valuable bacterial strains is crucial for various scientific, industrial, and environmental applications. Microorganisms with oil-emulsifying and oil-displacing properties are potentially significant for biotechnologies applied in the oil industry, particularly in such areas as bioremediation and tertiary enhanced oil recovery. To supply enterprises with pure cultures of microorganisms, they should be constantly maintained in the collection conditions in an active state while monitoring the preservation of their biotechnological properties. Therefore, keeping microorganism strains in working conditions and preserving their valuable properties are important for almost any work with microorganisms, ranging from primary research to their use in the production of various biopreparations.&#x0D; Aim: The article focuses on studying a method for preserving bacteria that are useful in the oil industry. This method involves modifying the technique of microencapsulating microorganism cells in alginate gel by adding glycerin, which is used as an agent with biostatic action.&#x0D; Materials and methods: The subject of research are eight hydrocarbon-oxidizing cultures of microorganisms that were sourced from the Department of Biotechnology of the al-Farabi Kazakh National University. Of these, four cultures were spore-bearing, while the other four were non-spore-bearing. The research employed microbiological methods of cultivation and storage of microorganisms in both solid and liquid media under aerobic conditions. In addition, Cooper's method was used to determine oil emulsification index and statistical methods for data analysis.&#x0D; Results: It has been found that adding glycerin (15% vol.) as a biostatic to the gel-forming matrix of sodium alginate can ensure long-term (up to 6 months) cell viability for the studied bacteria in the range of 88-96% while maintaining functionality of immobilized cells. The values of the bacteria’s oil emulsification remained at the pre-conservation levels, whereas traditional storage methods result in a lower number of viable cells after six months. It should be noted that after six months of being stored in encapsulated form with glycerin, the viability of non-sporе-forming Pseudomonas cultures is lower (88-91%) than spore-forming Bacillus (95-98%). This correlation is also is observed for traditional methods.&#x0D; Conclusion: The modern method of preserving bacteria allows for their long-term storage while maintaining functionality and viability.

Water-soluble microencapsulation using gum Arabic and skim milk enhances viability and efficacy of Pediococcus acidilactici probiotic strains for application in broiler chickens.

This study aimed to develop and evaluate the effectiveness of a water-soluble microencapsulation method for probiotic strains using gum Arabic (GA) and skim milk (SKM) over a three-month storage period following processing. Four strains of Pediococcus acidilactici (BYF26, BYF20, BF9, and BF14) that were typical lactic acid bacteria (LAB) isolated from the chicken gut were mixed with different ratios of GA and SKM as coating agents before spray drying at an inlet temperature 140°C. After processing, the survivability and probiotic qualities of the strains were assessed from two weeks to three months of storage at varied temperatures, and de-encapsulation was performed to confirm the soluble properties. Finally, the antibacterial activity of the probiotics was assessed under simulated gastrointestinal conditions. As shown by scanning electron microscopy, spray-drying produced a spherical, white-yellow powder. The encapsulation efficacy (percent) was greatest for a coating containing a combination of 30% gum Arabic: 30% skim milk (w/v) (GA:SKM30) compared to lower concentrations of the two ingredients (p<0.05). Coating with GA:SKM30 (w/v) significantly enhanced (p<0.05) BYF26 survival under simulated gastrointestinal conditions (pH 2.5 to 3) and maintained higher survival rates compared to non-encapsulated cells under an artificial intestinal juices condition of pH 6. De-encapsulation tests indicated that the encapsulated powder dissolved in water while keeping viable cell counts within the effective range of 106 for 6 hours. In addition, following three months storage at 4°C, microencapsulation of BYF26 in GA:SKM30 maintained both the number of viable cells (p<0.05) and the preparation's antibacterial efficacy against pathogenic bacteria, specifically strains of Salmonella. Our prototype water-soluble probiotic microencapsulation GA:SKM30 effectively maintains LAB characteristics and survival rates, demonstrating its potential for use in preserving probiotic strains that can be used in chickens and potentially in other livestock.

Standard Operating Procedure to Optimize Resazurin-Based Viability Assays

The resazurin assay, also known as the Alamar Blue assay, stands as a cornerstone technique in cell biology, microbiology, and drug development. It assesses the viability of cells through the conversion of resazurin into highly fluorescent resorufin. The resulting fluorescence intensity provides a reliable estimate of viable cell numbers. Cytotoxicity assays, such as the resazurin-based method, play a crucial role in the screening of potential drug candidates and in the assessment of pharmaceutical and chemical toxicity. In recent years, inconsistencies have arisen in pharmacogenomic studies, often due to poorly optimized laboratory protocols. These inconsistencies hinder progress in understanding how substances affect cell health, leading to unreliable findings. Thus, the need for standardized and rigorously optimized protocols is evident to ensure consistent and accurate results in cytotoxicity studies. This manuscript describes a standardized procedure for optimizing resazurin-based viability assays to improve the reliability of cytotoxicity data. This optimization approach focuses on critical experimental parameters and data quality, aiming to achieve a level of measurement imprecision of less than 20%. In conclusion, to address the critical issues of reproducibility and reliability, protocol standardization, such as the one described in this manuscript, can greatly enhance the credibility of cytotoxicity studies, ultimately advancing drug safety assessments.

Suppression of planktonic and biofilm of Escherichia coli by the synergistic lantibiotics-polymyxins combinations.

Escherichia coli are generally resistant to the lantibiotic's action (nisin and warnerin), but we have shown increased sensitivity of E. coli to lantibiotics in the presence of subinhibitory concentrations of polymyxins. Synergistic lantibiotic-polymyxin combinations were found for polymyxins B and M. The killing of cells at the planktonic and biofilm levels was observed for two collection and four clinical multidrug-resistant E. coli strains after treatment with lantibiotic-polymyxin B combinations. Thus, 24-h treatment of E. coli mature biofilms with warnerin-polymyxin B or nisin-polymyxin B leads to five to tenfold decrease in the number of viable cells, depending on the strain. AFM revealed that the warnerin and polymyxin B combination caused the loss of the structural integrity of biofilm and the destruction of cells within the biofilm. It has been shown that pretreatment of cells with polymyxin B leads to an increase of Ca2+ and Mg2+ ions in the culture medium, as detected by atomic absorption spectroscopy. The subsequent exposure to warnerin caused cell death with the loss of K+ ions and cell destruction with DNA and protein release. Thus, polymyxins display synergy with lantibiotics against planktonic and biofilm cells of E. coli, and can be used to overcome the resistance of Gram-negative bacteria to lantibiotics.