Confocal Laser Scanning Research Articles

Lithium-Modified TiO2 Surface by Anodization for Enhanced Protein Adsorption and Cell Adhesion.

Promoting osseointegration is an essential step in improving implant success rates. Lithium has gradually gained popularity for promoting alkaline phosphatase activity and osteogenic gene expression in osteoblasts. The incorporation of lithium into a titanium surface has been reported to change its surface charge, thereby enhancing its biocompatibility. In this study, we applied anodization as a novel approach to immobilizing Li on a titanium surface and evaluated the changes in its surface characteristics. The objective of this study was to determine the effect of Li treatment of titanium on typical proteins, such as albumin, laminin, and fibronectin, in terms of their adsorption level as well as on the attachment of osteoblast cells. Titanium disks were acid-etched by 66 wt % H2SO4 at 120 °C for 90 s and set as the control group. The etched samples were placed in contact with an anode, while a platinum bar served as the counter electrode. Both electrodes were mounted on a custom electrochemical cell filled with 1 M LiCl. The samples were anodized at constant voltages of 1, 3, and 9 V. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) results showed no significant differences in the topography. However, the ζ potentials of the 3 V group were higher than those of the control group at a physiological pH of 7.4. Interestingly, the adsorption level of the extracellular matrix protein was mostly enhanced on the 3 V-anodized surface. The number of attached cells on the Li-anodized surfaces increased. The localization of vinculin at the tips of the stretching cytoplasmic projections was observed more frequently in the osteoblasts on the 3 V-anodized surface. Although the optimal concentration or voltage for Li application should be investigated further, this study suggests that anodization could be an effective method to immobilize lithium ions on a titanium surface and that modifying the surface charge characteristics enables a direct protein-to-material interaction with enhanced biological adhesion.

Phase behaviour and structure formation of alginate-gelatin composite gels

Alginate-gelatin composite gels are widely used in food and biomedical applications. These composite gels are particularly interesting for their ability to establish complex functionalities, such as in bioinks for 3D bioprinting. In order to understand and optimise their techno-functionalities, it is crucial to study the structure formation, which is influenced by molecular interactions and phase behaviour.Therefore, the aim of this study was to characterise the effect of mixing ratio and temperature (21 and 40 °C) on the phase behaviour and structure formation of alginate-gelatin composite gels using internal alginate gelation with Ca-EDTA and D-glucono-δ-lactone. We compared pure alginate gels and composite gels using oscillatory rheology, pH measurements during gelation and microstructural analysis through confocal laser scanning microscopy.The temperature-dependent state of the gelatin - liquid or gelled - strongly influenced the gelation velocity of alginate. At 40 °C, liquid gelatin delayed the gelation process by buffering the pH decrease. This effect was most pronounced in the presence of excess gelatin, which had a strong antagonistic effect on gel strength. At 21 °C, gelled gelatin resulted in a faster alginate gelation and the formation of phase-separated networks at all mixing ratios. This was also accompanied by antagonistic effects on the gel strength. The phase-separated networks showed different microstructures with high-contrast regions depending on the mixing ratio, which correlated with variations in the turbidity of the gels. These insights into the structure-function relationship of alginate-gelatin composite gels may enable the customisation of specific network types for the use in 3D bioprinting.

Insights into the EPS production and distribution of planktonic and attached Sulfobacillus thermosulfidooxidans cells during bioleaching

Bacterial leaching can effectively promote the bioleaching of chalcopyrite by secreting extracellular polymeric substances (EPS). In this study, the production and distribution of EPS on the surface of planktonic and attached cells of Sulfobacillus thermosulfidooxidans (S. thermosulfidooxidans) during the process of chalcopyrite concentrate bioleaching were observed in situ using Field emission scanning electron microscope (FE-SEM) and Confocal laser scanning microscope (CLSM). The TOC content and key components of EPS were extracted during the chalcopyrite bioleaching process. Moreover, the main functional groups of extracellular polymorphs were identified through Fourier transmission-infrared spectroscopy (FT-IR). The results showed a similar trend in the evolution of EPS content in planktonic and attached cells. However, the EPS content secreted by the cells attached was more than that of planktonic cells during the entire process of bioleaching. Furthermore, the results of FE-SEM indicated that the EPS secreted by the attached cells is thicker and stickier, with better adsorption performance. The FT-IR spectrum analysis revealed that the O–H, C–H, and CO bonds are the main functional groups of EPS. This work further clarifies the role of EPS in the bioleaching process.

Fate and biofilm formation of Salmonella enterica subsp. enterica serovar Thompson on fresh strawberries stored under refrigeration and room temperatures

Fresh strawberry is a popular, frequently fruit consumed, which is largely appreciated for its organoleptic characteristics and health benefits. However, different outbreaks caused by food-borne pathogens have been attributed to strawberry consumption. The persistence of bacteria on fruits like strawberries could be related to their ability to adhere and form biofilms on the surface. The present study aimed to evaluate the fate and biofilm formation capacity of Salmonella enterica subsp. enterica serovar Thompson in post-harvest strawberries stored at 4 and 7 °C for 192 h and 20 °C for 72 h. Fresh strawberries were spot inoculated to achieve an initial concentration of around 5 log CFU/g of S. Thompson. During storage at 20 °C, a 2-log reduction per gram was observed. At 7 and 4 °C, the S. Thompson population decreased to 1.7 and 2.0 log, respectively after 192 h of storage. A more marked decrease of the pathogen was observed at 4 °C, in comparison with a slight decay at 7 or 20 °C. On the other hand, the observations with scanning electron microscopy and confocal laser scanning microscopy demonstrated the formation of biofilm on the epidermis of the strawberry stored at all temperatures. This could be considered a mechanism adopted by the bacteria against stress during storage. Prevention of biofilm formation and disruption of biofilm in strawberries become essential for food safety.

Development of a Novel Peptide with Antimicrobial and Mineralising Properties for Caries Management.

The purpose of the study is to develop a novel peptide for caries management. Gallic-Acid-Polyphemusin-I (GAPI) was synthesised by grafting Polyphemusin I (PI) and gallic acid (GA). Biocompatibility was evaluated using a Cell Counting Kit-8 Assay. Antimicrobial properties were assessed using minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC). The bacterial and fungal morphology after GAPI treatment was investigated using transmission electron microscopy (TEM). The architecture of a consortium biofilm consisting of Streptococcus mutans, Lacticaseibacillus casei and Candida albicans was evaluated using scanning electron microscopy (SEM) and confocal laser scanning microscopy. The growth kinetics of the biofilm was examined using a propidium monoazide-quantitative polymerase chain reaction. The surface and calcium-to-phosphorus molar ratio of GAPI-treated enamel after pH cycling were examined with SEM and energy-dispersive X-ray spectroscopy. Enamel crystal characteristics were analysed using X-ray diffraction. Lesion depths representing the enamel's mineral loss were assessed using micro-computed tomography. The MIC of GAPI against S. mutans, L. casei and C. albicans were 40 μM, 40 μM and 20 μM, respectively. GAPI destroyed the biofilm's three-dimensional structure and inhibited the growth of the biofilm. SEM showed that enamel treated with GAPI had a relatively smooth surface compared to that treated with water. The calcium-to-phosphorus molar ratio of enamel treated with GAPI was higher than that of the control. The lesion depths and mineral loss of the GAPI-treated enamel were less than the control. The crystallinity of the GAPI-treated enamel was higher than the control. This study developed a biocompatible, mineralising and antimicrobial peptide GAPI, which may have potential as an anti-caries agent.

Unveiling Optimal Synthesis and Structural Insights of Starch Ferulate via the Mechanoenzymatic Method.

In this study, starch ferulate was synthesized employing a mechanoenzymatic method, specifically based on the twin screw extrusion technique and lipase catalysis. The research then primarily centered on optimizing process parameters and conducting structural analysis. Optimal conditions were determined to be 8.2% ferulic acid addition, 66 °C extrusion temperature, and 3.2% lipase (N435) addition. The enzyme-catalyzed time was 30 s. The degree of substitution for starch ferulate was quantified at 0.005581 under these specific conditions. The presence of C=O bonds in the synthesized starch ferulate proved that the synthesis process was efficient. Additionally, the crystal structure underwent reconstruction. Observations through Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM) demonstrated that the mechanoenzymatic method led to an augmentation in the specific surface area of starch molecules, thereby facilitating the exposure of active sites. This breakthrough underscores the vast potential of mechanoenzymatic techniques to revolutionize the rapid and sustainable synthesis of starch ferulate, marking a pioneering stride in ester synthesis. The insights garnered from this study transcend theory, offering a visionary roadmap for the development and real-world deployment of advanced modified starch esters.

Effect of palm oil content and melting point on the freeze-thaw stability of fat substitutes

Animal or plant-based meat products are typically stored and transported using freezing. Freezing-thawing cycles can lead to detrimental changes in the texture, appearance, and mouthfeel of fat substitutes, thus affecting their quality. In this work, a protein/polysaccharide composite emulsion gel was created by emulsifying soybean isolate protein (SPI) and palm oil (PO), and incorporating curdlan (CL). It can serve as a potential replacement for animal fat in meat products, such as sausage, and plant-based meat. The study aimed to investigate the stability of the complex fat substitutes during freeze-thaw at various melting points and palm oil content. The results showed that the 52°C-palm oil samples were the most freeze-thaw stable compared to samples with other melting points. 52°C-palm oil samples were tested for texture and water holding capacity (WHC), increasing the oil content from 5g/100g–20g/100g improved the gel strength and WHC after freeze-thaw. The samples containing high melting point palm oil were less affected by temperature changes and showed higher stability as detected by low-field NMR and differential calorimetric scanning. The confocal laser scanning and rheological detection further confirmed the enhanced structural strength and freeze-thaw stability of the emulsion gels containing solid fat particles with high melting points.

Boiling vs. baking: Cooking-induced structural transformations drive differences in the in vitro starch digestion profiles that are consistent with the in vivo glycemic indexes of white and sweet potatoes

White and sweet potatoes can elicit different blood glucose responses depending on whether they are boiled or baked. This work investigated how microstructure and starch digestion in vitro relate to these differences. The main methods were INFOGEST’s semi-dynamic digestion protocol, Scanning Electron Microscopy and Confocal Laser Scanning Microscopy. The cooking method impacted microstructure, thereby significantly influencing starch digestion. Boiling and baking led to similar types of microstructural changes, including cell expansion and separation and disruption to cell walls, with the differences lying on the magnitude of such changes. Hydrolysis of white potato starch into oligosaccharides during oro-gastric digestion stabilized at around 75% when boiled compared to 50% when baked. In sweet potato, hydrolysis during this stage represented 30% and 40% of the total starch after boiling or baking, respectively. Overall, the effect can be summarized as boiled white potato > baked white potato > baked sweet potato > boiled sweet potato. Our results show how structural transformations that occur during cooking can drive differences in starch release and hydrolysis during in vitro digestions. This work therefore provides a structural and biochemical basis to better understand the impact of boiling and baking on the glycemic responses to these foods.

Pool-boiling enhancement on periodic micro/nano ripple-structured surfaces fabricated by femtosecond laser

A study was conducted on whether periodic micro/nano ripple structures generated on metal surfaces using femtosecond laser processing could improve pool boiling heat transfer. Depending on the laser irradiation conditions on the metal surface, a surface structure with different geometrical characteristics is created. Changes in sample surface morphology caused by the laser irradiation intensity are examined using scanning electron microscopy and confocal laser microscopy. Pool boiling tests are conducted to compare three laser irradiation conditions fabricated samples and the bare copper surface. The working fluid is deionized water. The pool boiling curves of each sample surface were compared with each other. Visualization of bubble nucleation is performed using a high-speed camera. The superheat, bubble departure diameter, and bubble growth period of each sample are measured and compared at the onset of nucleate boiling (ONB). The most efficient periodic micro/nano ripple-structured surface reduces the superheat at the ONB by 6.13 °C in comparison with that at the bare Cu surface. The enhanced surface structure reduced the bubble departure diameter and renewal period by 60.5% and 55.6% in comparison with that of the smooth surface. Further, the structured surface showed 178.5% enhancement in heat transfer coefficient and 39.1% in critical heat flux, respectively. Thus, the combination of increased nucleate site density, high nucleation site activation, and a wicking effect in the periodic micro/nano ripple structure significantly improves the pool boiling heat transfer.

Effect of Lipopolysaccharide on Development of <i>Aspergillus fumigatus</i> Biofilm on Nasal Epithelial Cells

Background and Objectives <i>Aspergillus fumigatus</i> is one of the common causes of fungal airway inflammatory diseases and lipopolysaccharide (LPS) acts as key regulator of airway inflammation. In addition, bacterial and fungal biofilm commonly coexist in chronic rhinosinusitis. In this study, we evaluated the effect of LPS on the development of <i>A. fumigatus</i> biofilm formation on the nasal epithelial cells.Materials and Method Primary nasal epithelial cells were cultured with <i>A. fumigatus</i> conidia with or without LPS for 5 days. The production of interleukin (IL)-6, IL-8, and transforming growth factor (TGF)-β1 from nasal epithelial cells was determined by enzyme-linked immunosorbent assay. The effects of LPS on <i>A. fumigatus</i> biofilm formation were determined using biofilm dry weight, and crystal violet, concanavalin A, safranin staining, and confocal scanning laser microscopy.Results LPS and <i>A. fumigatus</i> significantly enhanced the production of I L-6, I L-8, and TGF-β1 from nasal epithelial cells. <i.A. fumigatus</i> can form biofilm on primary nasal epithelial cells, and this significantly increased in a time-dependent manner when cocultured with LPS, the dry weight, concanavalin A, and safranin staining.Conclusion The exposure of <i>A. fumigatus</i> to LPS enhanced the formation of biofilms. The coexistence of LPS and <i>A. fumigatus</i> enhanced fungal biofilm formation and this may be associated with the development of recalcitrant airway inflammatory diseases.

Identification of phytochemical, antioxidant, anticancer and antimicrobial potential of Calotropis procera leaf aqueous extract

Since the dawn of civilization, people have turned to plants as a safe and efficient form of treatment for a variety of diseases. It has long been known that Calotropis procera has the potential to treat a number of diseases. In this study, the C. procera leaf aqueous extract was obtained using the maceration method, and p-coumaric was found to be the main compound. The extract was rich in phenols (174.82 mg gallic acid equivalent/g) and flavonoids (1781.7 µg quercetin equivalent/g). The extract had high antioxidant properties, as indicated by the IC50 values obtained for 2,2-diphenyl-1-picrylhydrazyl (DPPH) (366.33 μg/mL) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) (169.04 μg/mL), as well as the ferric ions reducing antioxidant power (FRAP) (1.67 μg ascorbic acid equivalent/g of the extract). The cytotoxicity of the extract was evaluated against the survival of HT 29 cells, and the IC50 was found to be 236.87 μg/mL. The most resistant and sensitive strains to the extract were Escherichia coli and Staphylococcus aureus, respectively. The morphological changes of these strains were demonstrated through scanning electron microscopy and confocal laser scanning microscopy. The C. procera extract could be therefore used as an antioxidant, antimicrobial, and anticancer agent.

Antibacterial mechanism of vanillin against Escherichia coli O157: H7

Vanillin, a plant-derived antimicrobial volatile substance, has potential microbial control applications in the food industry. However, the effect of vanillin on the food-borne pathogen Escherichia coli (E. coli) O157:H7 has not been well studied. This study aims to explore the antibacterial mechanism of vanillin against E. coli O157:H7. The minimum inhibitory concentration (MIC) and antibacterial effect of vanillin were determined by microdilution. Scanning electron microscopy (SEM) was used to observe the damage of vanillin to the cell membrane, while cell membrane potential and the leakage of nucleic acid protein were measured to explore the effect of vanillin on the membrane system. Confocal laser scanning and intracellular adenosine triphosphate (ATP) concentration determination were utilized to investigate the effects of vanillin on the energy, life, and death of E. coli. Finally, transcriptome sequencing was conducted to investigate the gene expression differences induced by vanillin treatment. The results showed that vanillin treatment effectively controlled E. coli O157:H7 with an MIC of 2 mg/mL. After treatment, damage to the membrane system, depolarization of the membrane, and leakage of nucleic acid and protein were observed. Meanwhile, vanillin treatment caused decreased ATP content and cell death. Transcriptome analysis showed that vanillin treatment significantly affected the expression of genes involved in cell membrane formation, tricarboxylic acid (TCA) cycling pathway, and oxidative phosphorylation pathway in E. coli O157:H7. In conclusion, membrane damage and energy metabolism disruption are important mechanisms of vanillin's inhibitory effect on E. coli O157:H7. This study provides new insights into the molecular reaction mechanism of vanillin against E. coli O157:H7, highlighting its potential as an antibacterial substance for preventing E. coli contamination in the food industry.

Evaluation of a hydroxyapatite-crosslinked fish gelatin membranes

Background/purposePorcine collagen is widely used in regenerative therapies to generate membranes for bone augmentation. However, porcine or bovine gelatin or collagen is often not appropriate for patients with creed and religious beliefs or for allergic reasons. In this study, we evaluated the potential of fish gelatin to generate membranes. Materials and methodsFish gelatin and hydroxyapatite (HAp) were used at three different ratios (2:0, 2:1, 2:1.5, and 2:2) to prepare gelatin-hydroxyapatite (G-HAp) membranes via freeze-drying and heat-crosslinking. The surface morphology and cell attachment of G-HAp membranes were observed using scanning electron microscopy and confocal laser microscopy. G-HAp membrane was placed at the bottom of a well plate, and MC3T3-E1 cells were seeded on it. Cell viability and cytotoxicity were tested after 1 and 3 days of culture. Alkaline phosphatase (ALP) and alizarin red staining was performed at 10 and 21 days, respectively. ResultsViability of cells on G-HAp membrane with the gelatin:HAp ratio of 2:1.5 was significantly higher than that on membranes with other gelatin:HAp ratios. ALP and alizarin red staining showed that ALP-positive areas and calcium deposition were the highest on G-HAp membrane with the gelatin:HAp ratio of 2:1. These membranes showed negligible cytotoxicity. ConclusionFish-derived G-HAp membranes have the potential to promote osteogenic differentiation of MC3T3-E1 cells with negligible cytotoxicity.

Ursolic Acid Inhibits the Growth of Gastric Cancer by Targeting KLF4/YAP1

Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor which has various mechanisms in different tumors. Ursolic acid (UA), a natural compound that exists in many herbs, is known to prevent tumor progression and has anticancer effects on many human cancers. The present study was to evaluate the antitumor effect of UA on gastric cancer (GC) through KLF4 and the Hippo pathway. Our data showed that UA inhibited the growth of GC in vivo and in vitro. UA treatment significantly increased the expression of KLF4 and decreased the expression of CTGF. The overexpression of KLF4 inhibited the proliferation and cell cycle of GC and decreased the expression of CTGF, whereas the knockdown of KLF4 attenuated the effects of UA. Furthermore, the inhibition of CTGF arrested tumor cells in G2/M which blocked proliferation progress. Confocal laser scanning and molecular simulation software MOE showed that KLF4 combined with YAP1 which may block the formation of the TEADs-YAP1 complex to interrupt the expression of CTGF and the downstream oncogenic process. In conclusion, UA can inhibit GC growth both in vivo and in vitro, and it activated KLF4 which may competitively bind with YAP1 against TEADs and block the oncogenic Hippo pathways.

Characterization of Salmonella Thompson-specific bacteriophages and their preventive application against Salmonella Thompson biofilm on eggshell as a promising antimicrobial agent in the food industry

Salmonella Thompson is a foodborne pathogen that has attracted attention in Korea since it was identified as the cause of a large-scale domestic food poisoning outbreak in 2018. The present study aims to isolate and characterize bacteriophages specific to Salmonella Thompson and evaluate their efficacy in reducing Salmonella Thompson biofilm on eggshell. In addition, advanced microscopic analysis using field-emission scanning electron microscopy and confocal laser scanning microscopy was conducted to confirm the anti-biofilm activity. The two isolated bacteriophages CAU-STP-1 and CAU-STP-2 showed very high specificity by targeting only Salmonella Thompson. They were determined to be stable at 60–65 °C and pH 4–11. Bacteriophage particles quickly adsorbed to the host cells, with a latent period and burst size of 30–40 min and 19–100 plaque-forming units per cell, respectively. When comparing the two bacteriophages individually, CAU-STP-1 exhibited the highest in vitro inhibition ability, but treatment with both bacteriophages together in a cocktail was significantly more effective than with CAU-STP-1 alone. This bacteriophage cocktail was consequently employed to treat Salmonella Thompson biofilm on eggshell at different storage temperatures. There was a significant reduction in the biofilm on the eggshell with bacteriophage treatment at 10 and 20 °C after 7 days. For example, After 7 days at 10 °C, the biofilm on eggshell was reduced by 0.26, 0.96, and 2.50 log CFU/cm2 for MOI 103, MOI 104, and MOI 105, respectively. On the other hand, at 20 °C, the biofilm on eggshells was reduced by 0.10, 0.24, and 0.33 log CFU/cm2 for MOI 103, MOI 104, and MOI 105, respectively This study thus provides promising information for the potential development of an effective biocontrol agent against Salmonella Thomson within the food industry.

Self- immobilized bio-nanomaterials based on hybridized green reduced graphene and Burkholderia vietnamiensis C09V for enhanced removal of Sb species from mine wastewater

Elevated levels of antimony (Sb) in the environs of smelters or mines can pose significant human and environmental health risks, where efficient removal of Sb species from mine wastewater is currently a challenge. In this paper, a functional reduced graphene hybrid Burkholderia vietnamiensis C09V biomaterial (RGO-B.V. C09V) was initially synthesized by self-immobilization and subsequently used to remove 100% of Sb(III) within 24 h and 90.0% of Sb(V) within 120 h. This removal efficiency was also much higher than that of free B.V. C09V alone, which removed 100% of Sb(III); but only 26.7% of Sb(V); within 120 h. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) confirmed that Burkholderia vietnamiensis C09V (B.V. C09V) was firmly immobilized on the RGO surface with an abundant distribution of extracellular polymeric substances (EPS). Three-dimensional excitation-emission matrix (3D-EEM) analysis and electrochemical techniques were used to confirm the function of RGO as a potential electron shuttle and the composition of EPS to identify its key role in any redox reactions. Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analysis confirmed that polysaccharides and protein-like substances on the surface of RGO-B.V. C09V participated in the biosorption of Sb via complexation. When RGO-B.V. C09V was practically used to treat a mine wastewater the final Sb concentration decreased by 96% from 0.12 to 0.005 mg·L−1. RGO-B.V. C09V also exhibited high stability, with removal efficiencies of up to 100% for Sb(III), and 72% for for Sb(V) after 4 reuse cycles, with no regeneration. The results showed that RGO-B.V. C09V was a stable, environmentally friendly bionanomaterial suitable for the practical removal of Sb species from mine wastewater over a sustained period.

Evaluation of the constituent compounds, antioxidant, anticancer, and antimicrobial potential of Prangos ferulacea plant extract and its effect on Listeria monocytogenes virulence gene expression.

Prangos ferulacea plant is very popular in Iran due to its unique properties in treating diseases and its special flavor. To check the characteristics of this plant, first, its extract was extracted using the maceration method. Its chemical composition was investigated using high-performance liquid chromatography (HPLC) that p-coumaric was identified as its main compound, and Fourier-transform infrared spectroscopy (FTIR) showed the presence of functional groups related to phenolic, flavonoid, tannins, and carboxylic acids such as caffeic acid and coumaric acid composition. Total phenol content (TPC), total flavonoid content (TFC), and beta-carotene were equal to 202.04 ± 5.46 mg gallic acid equivalent (GAE)/g dry weight, 1,909.46 ± 13 μg quercetin (QE)/g of dry weight, and 2.91 mg/100 g. The antioxidant property of the extract was evaluated using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) free radical scavenging and ferric reducing antioxidant power assay (FRAP). According to the IC50 obtained for DDPH (274 ± 7.2 μg/mL) and ABTS (120.45 ± 9.6 μg/mL) and FRAP values [1.92 ± 0.05 μg ascorbic acid equivalent (AAE)/g of extract], this extract had high antioxidant properties. Cytotoxicity was evaluated against the survival of HT 29 cells that IC50 was 82.15 ± 0.02 μg/mL. The antimicrobial property of the extract was calculated using disk diffusion agar (DDA), well diffusion agar (WDA), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). Listeria monocytogenes has the highest sensitivity to this extract and inhibition zone based on DDA and WDA method and with an MIC and MBC equal to 16 and 128 mg/mL has the least resistance. The morphology change of L. monocytogenes strain was proved through scanning electron microscope (SEM) and confocal laser scanning microscopy (CLSM). The extract caused a significant reduction in the transcription of genes involved in the film formation ability of L. monocytogenes. The obtained results fully prove the very practical and pragmatic characteristics of P. ferulacea.

Tannin extracted from Penthorum chinense Pursh, a potential drug with antimicrobial and antibiofilm effects against methicillin-sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen. Due to the widespread use and abuse of antibiotics, various drug-resistant strains of S. aureus have emerged, with methicillin-resistant Staphylococcus aureus (MRSA) being the most prevalent. Bacterial biofilm is a significant contributor to bacterial infection and drug resistance. Consequently, bacterial biofilm formation has emerged as a therapeutic strategy. In this study, the chemical constituents, antimicrobial and antibiofilm properties of tannins isolated from Penthorum chinense Pursh (TPCP) were investigated. In vitro, TPCP exhibited antimicrobial properties. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) for methicillin-sensitive Staphylococcus aureus (MSSA) and MRSA were 156.25 and 312.5 μg/mL, and 312.5 and 625 μg/mL, respectively. According to the growth curves, TPCP significantly inhibited the growth of MSSA and MRSA. The results of the crystal violet biofilm assay in conjunction with confocal laser scanning and scanning electron microscopy demonstrated that TPCP destroyed preformed MSSA and MRSA biofilms. TPCP significantly decreased the secretion of exopolysaccharides and extracellular DNA. Subsequently, the mechanism was investigated using RT-PCR. Examining the expression of icaA, cidA, sigB, agrA, and sarA genes in MRSA, we discovered that TPCP inhibited biofilm formation by affecting the quorum-sensing system in bacteria. Our study demonstrates that TPCP exerts antibacterial effects by disrupting the formation of bacterial biofilms, suggesting that TPCP has clinical potential as a novel antibacterial agent for the prevention and treatment of MSSA and MRSA infections.

Inhibition of biofilm formation and virulence factors of cariogenic oral pathogen Streptococcus mutans by natural flavonoid phloretin

ABSTRACT Objectives To evaluate the effect and mechanism of action of the flavonoid phloretin on the growth and sucrose-dependent biofilm formation of Streptococcus mutans. Methods Minimum inhibitory concentration, viability, and biofilm susceptibility assays were conducted to assess antimicrobial and antibiofilm effect of phloretin. Biofilm composition and structure were analysed with scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Water-soluble (WSG) and water-insoluble glucan (WIG) were determined using anthrone method. Lactic acid measurements and acid tolerance assay were performed to assess acidogenicity and aciduricity. Reverse transcription quantitative PCR (RT-qPCR) was used to measure the expression of virulence genes essential for surface attachment, biofilm formation, and quorum sensing. Results Phloretin inhibited S. mutans growth and viability in a dose-dependent manner. Furthermore, it reduced gtfB and gtfC gene expression, correlating with the reduction of extracellular polysaccharides (EPS)/bacteria and WIG/WSG ratio. Inhibition of comED and luxS gene expression, involved in stress tolerance, was associated with compromised acidogenicity and aciduricity of S. mutans. Conclusions Phloretin exhibits antibacterial properties against S. mutans, modulates acid production and tolerance, and reduces biofilm formation. Clinical significance Phloretin is a promising natural compound with pronounced inhibitory effect on key virulence factors of the cariogenic pathogen, S. mutans.

Exploring 3D leaf anatomical traits for C4 photosynthesis: chloroplast and plasmodesmata pit field size in maize and sugarcane.

Volume and surface area of chloroplasts and surface area of plasmodesmata pit fields are presented for two C4 species, maize and sugarcane, with respect to cell surface area and cell volume. Serial block face scanning electron microscopy (SBF-SEM) and confocal laser scanning microscopy with the Airyscan system (LSM) were used. Chloroplast size estimates were much faster and easier using LSM than with SBF-SEM; however, the results were more variable than SBF-SEM. Mesophyll cells were lobed where chloroplasts were located, facilitating cell-to-cell connections while allowing for greater intercellular airspace exposure. Bundle sheath cells were cylindrical with chloroplasts arranged centrifugally. Chloroplasts occupied c. 30-50% of mesophyll cell volume, and 60-70% of bundle sheath cell volume. Roughly 2-3% of each cell surface area was covered by plasmodesmata pit fields for both bundle sheath and mesophyll cells. This work will aid future research to develop SBF-SEM methodologies with the aim to better understand the effect of cell structure on C4 photosynthesis.