Confocal Laser Microscopy Analysis Research Articles

Torsional fatigue behaviour and damage mechanisms of martensitic spring steel in the HCF regime

This study investigates the fatigue behaviour and damage mechanisms of the martensitic spring steel SAE 9254, with emphasis on short crack propagation. Shear stress-controlled torsional fatigue tests are performed at stress ratio R = -1 and test frequency f = 79 Hz using structure-sensitive measurement techniques to detect the relevant damage mechanisms. An in-situ torsional fatigue testing device is applied for shear-controlled fatigue tests at two amplitudes of the torsion angle using R = –1 and f = 5 Hz, and the microstructural-dependent short crack propagation path is documented by means of confocal laser microscopy and electron backscattering analysis. It is found that the early development of fatigue damage is characterised by the formation of slip bands, which serve as crack initiation sites. In this process, crack initiation occurs preferentially at or near prior austenite grain boundaries. Furthermore, prior austenite grain boundaries were identified as obstacles for the propagation of short cracks, because of the change of the crystallographic orientation leading to an oscillating propagation rate of short cracks. The short crack density ρCD, which is an important parameter for fatigue damage, is rapidly growing with increasing shear stress amplitude ΔτT/2 and reveals a stress type specific behaviour.

Evaluation of fish pituitary spheroids to study annual endocrine reproductive control

The pituitary gland is a small endocrine gland located below the hypothalamus. This gland releases several important hormones and controls the function of many other endocrine system glands to release hormones. Fish pituitary hormonal cells are controlled by neuroendocrine and sex steroid feedback. To study the complex pituitary function in vivo, we established an in vitro pituitary spheroid assay and evaluated its suitability for monitoring the annual reproductive physiological conditions in Takifugu rubripes, also known as torafugu, is one of the most economically important species distributed in the northwestern part of the Pacific Ocean, in the western part of the East China Sea, and in more northern areas near Hokkaido, Japan. Fish pituitary spheroids can be easily constructed in liquid or solid plates. The culture medium (L-15) made the aggregation faster than MEM (Hank’s). A Rho-kinase inhibitor (Y-27632, 10 μM) and/or fish serum (2.5 %) also promoted spheroid formation. Laser confocal microscopy analysis of spheroids cultured with annual serum of both sexes revealed that luteinizing hormone (LH) synthesis has the highest peak in the final maturation stage (3 years old, May) in accordance with the highest serum sex steroid levels; in contrast, follicle stimulating hormone (FSH) synthesis has no correlation with the dose of serum or nutrients. Similarly, 3D cell propagation assays using female serum showed that total pituitary cells displayed the highest proliferation at puberty onset (2 years old, October) before half a year of the spawning season. These results indicate that pituitary spheroids are useful in vitro models for monitoring the reproductive physiological status of fish in vivo and may be applicable to the in vitro screening of environmental chemicals and bioactive compounds affecting reproductive efficiency in aquaculture.

Ozone therapy effect in medication-related osteonecrosis of the jaw as prevention or treatment: microtomographic, confocal laser microscopy and histomorphometric analysis.

This study aimed to evaluate the efficacy of ozone therapy in the preoperative (prevention) and/or postoperative (treatment) of MRONJ. Forty male Wistar rats were caudally treated with zoledronic acid (ZOL) and to ozone therapy before extraction (prevention, POG), after extraction (treatment, TOG), or both (prevention and treatment, TPOG), and treated with saline (SAL). The animals received intramuscular fluorochrome (calcein and alizarin), and 28 days postoperatively, they were euthanized, and the tissues were subjected to microtomographic computed tomography (microCT), LASER confocal, and histomorphometric analyses. Micro-CT showed a higher bone volume fraction average in all groups than that in the ZOL group (P < 0.001), the ZOL group showed high porosity (P = 0.03), and trabecular separation was greater in the TOG group than in the POG group (P < 0.05). The mineral apposition rate of the POG group was high (20.46 ± 6.31) (P < 0.001), followed by the TOG group (20.32 ± 7.4). The TOG group presented the highest mean newly formed bone area (68.322 ± 25.296) compared with the ZOL group (P < 0.05), followed by the SAL group (66.039 ± 28.379) and ZOL groups (60.856 ± 28.425). Ozone therapy modulated alveolar bone repair in animals treated with ZOL, mainly after surgery trauma, leading to bone formation as healing tissue. Osteonecrosis has been a challenge in dentistry, and owing to the lack of a consensus regarding therapy, studies presenting new therapies are important, and ozone has been one of the therapies explored empirically.

Fermentation of waste water from agar processing with Bacillus subtilis by metabolomic analysis.

Fungal infection has become a major threat to crop loss and affects food safety. The waste water from agar processing industries extraction has a number of active substances, which could be further transformed by microorganisms to synthesize antifungal active substances. In this study, Bacillus subtilis was used to ferment the waste water from agar processing industries extraction to analyze the antifungal activity of the fermentation broth on Alternaria alternata and Alternaria spp. Results showed that 25% of the fermentation broth was the most effective in inhibited A. alternata and Alternaria spp., with fungal inhibition rates of 99.9% and 96.1%, respectively, and a minimum inhibitory concentration (MIC) was 0.156μg/mL. Metabolomic analysis showed that flavonoid polyphenols such as coniferyl aldehyde, glycycoumarin, glycitin, and procyanidin A1 may enhance the inhibitory activity against the two pathogenic fungal strains. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that polyphenols involved in the biosynthesis pathways of isoflavonoid and phenylpropanoid were upregulated after fermentation. The laser confocal microscopy analyses and cell conductivity showed that the cytoplasm of fungi treated with fermentation broth was destroyed. This study provides a research basis for the development of new natural antifungal agents and rational use of seaweed agar waste. KEY POINTS: • Bacillus subtilis fermented waste water has antifungal activity • Bacillus subtilis could transform active substances in waste water • Waste water is a potential raw material for producing antifungal agents.

Healing wounds, defeating biofilms: Lactiplantibacillus plantarum in tackling MRSA infections.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are well-known hospital-borne infections and are a major contributing factor to global health concerns of antimicrobial resistance due to the formation of biofilms. Probiotics are known to assist in the healing of wounds through immunomodulation and also possess anti-pathogen properties via competitive inhibition. The probiotic bacterium, Lactiplantibacillus plantarum MTCC 2621 and its cell-free supernatant (Lp2621) have previously been reported to have antibacterial, excellent antioxidant, and wound healing activity in in vitro conditions and wounds contaminated with S. aureus in mice. In the current study, we evaluated its anti-MRSA, biofilm inhibition and eradication efficacy, immunomodulatory activity in THP-1 cells, and wound healing potential in wounds contaminated with MRSA infection in mice. In agar well diffusion assay, Lp2621 showed anti-MRSA activity and revealed dose-dependent inhibition and eradication of biofilm by crystal violet assay as well as by Confocal Scanning Laser Microscopy (CLSM) analysis. Further, Lp2621 showed immunomodulatory activity at varied concentrations as measured by IL-6 and IL-10 gene expression in THP-1 cells. Similar findings were observed in serum samples of mice after treatment of excision wound contaminated with MRSA infection by Lp2621 gel, as evident by expression of IL-6 (pro-inflammatory) and IL-10 (anti-inflammatory) cytokines. Overall, our results show that Lp2621 has potent anti-MRSA and antioxidant properties and can prevent and eliminate biofilm formation. It also showed promise when applied to mice with MRSA-infected wounds.

Marine-Fungi-Derived Gliotoxin Promotes Autophagy to Suppress Mycobacteria tuberculosis Infection in Macrophage.

The Mycobacterium tuberculosis (MTB) infection causes tuberculosis (TB) and has been a long-standing public-health threat. It is urgent that we discover novel antitubercular agents to manage the increased incidence of multidrug-resistant (MDR) or extensively drug-resistant (XDR) strains of MTB and tackle the adverse effects of the first- and second-line antitubercular drugs. We previously found that gliotoxin (1), 12, 13-dihydroxy-fumitremorgin C (2), and helvolic acid (3) from the cultures of a deep-sea-derived fungus, Aspergillus sp. SCSIO Ind09F01, showed direct anti-TB effects. As macrophages represent the first line of the host defense system against a mycobacteria infection, here we showed that the gliotoxin exerted potent anti-tuberculosis effects in human THP-1-derived macrophages and mouse-macrophage-leukemia cell line RAW 264.7, using CFU assay and laser confocal scanning microscope analysis. Mechanistically, gliotoxin apparently increased the ratio of LC3-II/LC3-I and Atg5 expression, but did not influence macrophage polarization, IL-1β, TNF-a, IL-10 production upon MTB infection, or ROS generation. Further study revealed that 3-MA could suppress gliotoxin-promoted autophagy and restore gliotoxin-inhibited MTB infection, indicating that gliotoxin-inhibited MTB infection can be treated through autophagy in macrophages. Therefore, we propose that marine fungi-derived gliotoxin holds the promise for the development of novel drugs for TB therapy.

Reduction of nanoparticle size and promotion of cell membrane permeability by LED irradiation

To improve the efficiency of poly (D, L-lactic-co-glycolic acid) (PLGA) nanoparticles (NP) as a gene carrier for the transfection of human mesenchymal stem cells (hMSCs), we developed a light-emitting diode (LED)-induced gene delivery system. The size of NP produced at various LED wavelengths (blue, green, red, and NIR) was controlled in the colloidal state (a mixture of single particles and agglomerates). In addition, physical stimulation of cell membranes using two LED wavelengths (blue and near infrared (NIR)) altered their structure and facilitated the penetration of cells with extracellular substances. The reduction of NP size and increase in cell membrane permeability effectively increased gene delivery to cells and protein expression by improving transfection processes (cellular uptake, endosomal escape, and PEI-gene dissociation). The effects of 10 min irradiation at each LED wavelength were compared with those of the non-irradiated sample. The reduction of NP size by LED irradiation was confirmed using DLS, SEM, and Nanosight, and cell membrane permeability was assessed by confocal laser microscopy and FACS. Transfection efficiency was measured using confocal laser microscopy, FACS, and Western blot analysis. Among the tested LED wavelengths, NIR light irradiation was the most effective in reducing NP size and increasing cell membrane permeability. Conversely, blue light irradiation significantly increased gene delivery to cells by accelerating transfection processes. Thus, the LED-irradiation mediated NP dispersion method improved the delivery efficiency of PLGA NP as gene carrier, and by enhancing cell membrane permeability, extracellular substances that normally cannot easily penetrate the cell membrane were delivered in a timely and effective manner.

Evaluation of Residual Root Canal Sealer Removal Efficacy of Different Irrigation Activation Techniques by Confocal Laser Microscopy Analysis

Objective: The purpose of this study was to use confocal laser microscopy analysis to evaluate the effectiveness of conventional needle irrigation (CNI), EndoActivator (EA), and EDDY during endodontic retreatment.&#x0D; Methods: This study included 45 maxillary incisor teeth with a single root and canal. Root canals were prepared with ProTaper Universal files (Dentsply Sirona, Ballaigues, Switzerland) and obturated with labeled sealer mixed with 0.1% Rhodamine B and gutta percha according to single cone techniques. Initial root canal filling material was removed using ProTaper Universal Retreatment files and F4 files. Teeth randomly were divided into 3 groups (n = 15) depending on the activation technique: CNI, EA (Dentsply, Tulsa Dental Specialties, Tulsa, OK), and EDDY (VDW, Munich, Germany). Confocal laser microscopy was used to evaluate the penetration area, depth, and percentage of the residual sealer in the apical, middle, and coronal sections after irrigation activation.&#x0D; Results: In all sections, the EDDY group had a lower penetration area of residual sealer than the CNI group (P&lt; .05). In comparison to the coronal section, the penetration percentage of the CNI and EA groups was lower in the apical section (P&lt; .05). In the CNI group, the penetration depth was higher at the coronal section than at the apical and middle sections (P&lt; .05), and it was higher at the coronal section than at the apical section.&#x0D; Conclusion: Within the limitations of this study, none of the activation systems tested could completely remove the residual sealer. However, the lowest residual sealer was seen after using EA and EDDY.

Geobacter sulfurreducens electroactive biofilms on Fe2O3/FTO support-electrodes for developing a sodium acetate electrochemical biosensor

Geobacter sulfurreducens is an essential microorganism in biogeochemical cycles. It is used biotechnologically in bioremediation and electricity production. The importance of G. sulfurreducens lies in its capacity to contact extracellular electron acceptors, including several electrode materials. Taking advantage of the versatility of G. sulfurreducens to grow on different surfaces, it is possible to design and synthesize new electrodes that can expand and improve the applications of these bacteria. In this work, we performed multidisciplinary research studies to demonstrate an innovative application of this well-known microbial system. The interaction between fluorine-doped tin oxide (FTO) and ordinary glass with G. sulfurreducens was enhanced by modifying the surface with Fe2O3 films. As confocal laser microscopy analyses revealed, the enhancement promoted biofilm development, whose parameters were significantly better than the bare support-electrodes. Furthermore, we provided evidence that G. sulfurreducens interacted with and dissolved the Fe2O3 film during incubation. Then, when we added sodium acetate to the system, the microbial metabolism was activated and decreased the electrochemical response of this compound. We benefited from that behavior to develop a voltammetric-based robust biosensor that could measure sodium acetate at high concentrations with an acceptable linear interval, from 10 to 110 mM, with LOD = 5.9 ± mM and LOQ = 18.1 ± 1.5 mM.

High temperature confocal scanning laser microscopy analysis of dead‐burned magnesia aggregates

AbstractDead‐burned magnesia is a commonly used material in the manufacturing of refractories for the steelmaking industry. Aggregates of dead‐burned magnesia contain secondary phases due to the impurities within the magnesite rock used in its production. While these phases can aid in sintering magnesia, they may have some impact on the high‐temperature performance of the refractory product. High‐temperature confocal scanning laser microscopy was utilized to observe the behavior of dead‐burned magnesia aggregates at elevated temperatures (up to 1550°C). Liquid formation was detected even at temperatures below 1350°C. In some cases, this liquid quickly exuded from the aggregate surface. This liquid phase was characterized through microscopy and chemical analysis to determine the impact of impurity content on the formation and behavior of this liquid phase, and conclusions are drawn on its detrimental impact on the material's refractoriness in a steelmaking environment.

Effect of Different Obturation Techniques on Sealer Penetration in Teeth with Artificial Internal Root Resorption: a Confocal Laser Microscope Analysis.

To investigate the efficacy of different obturation techniques on sealer penetration in teeth with internal root resorption using confocal laser microscopy. An artificial internal resorption cavity (3 mm deep and 1.2 mm in diameter) was formed in the round-shaped root canals of 45 single-rooted teeth at a distance of 7 mm from the apex, then roots were instrumented (size 40/.06). The samples were divided into three groups (n = 15) according to the obturation technique: lateral compaction (LC), warm vertical compaction (WVC) and carrier-based (CB). In the resorption regions, the sealer penetration depth in the CB and LC groups was significantly higher than that in the WVC group (P < 0.05). Within the limitations of this study, the penetration depth of the sealer in the resorption region was higher in the CB and LC groups as compared to that in the WVC group.

Pinellia ternata lectin induces inflammation through TLR4 receptor and mediates PI3K/Akt/mTOR axis to regulate NF-κB signaling pathway

Pinellia ternata, a widely used traditional Chinese medicine, contains a strong mucosal irritant that is connected with Pinellia ternata lectin (PTL) in its tubers. The purpose of this study was to explore the mechanisms by which PTL induces inflammation. We found that in RAW264.7 cells, PTL activated the PI3K/Akt/mTOR and NF-κB pathways, which resulted in the release of proinflammatory cytokines. Flow cytometry and laser confocal microscopy analysis showed that FITC-labeled PTL bound to the macrophages’ surface. Based on kinetic analyses and protein-protein docking simulations, PTL was shown to bind toll-like receptor 4 (TLR4).it was demonstrated that PTL binds highly to Toll-like receptor 4 (TLR4). TLR4 knock-down or knockout resulted in a decrease in both cytokine release and PI3K/Akt/mTOR and NF-κB pathway activation in PTL-stimulated macrophages or mice. RNA-seq analysis showed that genes involved in the PI3K/Akt/mTOR signaling pathway were strongly upregulated in response to PTL stimulation, confirming that the PI3K/Akt/mTOR pathway is linked to the inflammatory effect of PTL in RAW264.7 cells. These findings reveal that PTL can mediate inflammation through TLR4 and activating the PI3K/Akt/mTOR to regulate NF-κB signaling pathways.

Investigation on thickness size effect on ductility of magnetron sputtered Niobium coatings on SS316L substrate for forming of precoated metallic bipolar plates

Metallic bipolar plates (BPPs) are key components of the proton exchange membrane fuel cell (PEMFC). To lower the fabrication cost of metallic BPPs, precoated BPPs have attracted much attention due to the high efficiency of precoating-stamping process. However, precoatings on metallic substrate tend to crack during the forming process, leading to deterioration or even complete loss of corrosion resistance. Therefore, to avoid micro cracks of formed precoated BPPs, development of coatings with high ductility is necessary. In this study, Niobium coatings with different thicknesses on SS316L substrate are prepared with magnetron sputtering process, and uniaxial tensile tests are then conducted for the precoated specimens to evaluate their ductility. The microstructure and fracture behaviour of the Niobium precoatings are characterized by XRD, SEM, TEM, laser confocal microscope analysis. It is found that with the increase of coating thickness, the number of micro cracks at the same strain decreases significantly, and the strain for the first crack to appear also increases. Furthermore, a brittle-to-ductile transition of fracture mechanism is observed. The grain size of Niobium nanocrystalline coating increases with the thickness, which leads to the improvement of plasticity and failure strain. Therefore, the application of precoated metallic BPPs is further advanced.

Antimicrobial potential of new diclofenac hydrogels for disinfection in regenerative endodontics: An in vitro and ex vivo study.

There is a need to explore new alternatives for root canal disinfection in regenerative endodontics, since the current strategies are far from ideal. Currently, the potential use of diclofenac (DC) is being investigated for controlling root canal infections. The objective was to evaluate the antimicrobial efficacy of novel DC-based hydrogels (DCHs) against polymicrobial biofilms grown in radicular dentine and root canals and to compare results with triantibiotic (TAH) and diantibiotic (DAH) hydrogels, and calcium hydroxide (Ca[OH]2 ). The in vitro antimicrobial activity of intracanal medicaments was evaluated against 3-week-old polymicrobial root canal biofilms grown on human radicular dentine. Dentine samples were obtained and randomly divided into the study groups (n=4/group): (1) 1mg/ml TAH; (2) 1mg/ml DAH; (3) 5% diclofenac (DCH); (4) 2.5% DCH; (5) 1.25% DCH; (6) 1mg/ml DAH + 5% DCH; (7) Ca(OH)2 paste; (8) positive control. The microbial viability, in terms of percentage of intact cell membranes, was assessed after 7 days by confocal scanning laser microscopy (CSLM). The ex vivo efficacy of intracanal medications was evaluated in root canals infected with a polymicrobial suspension. Intracanal microbiological samples at baseline (S1) and 7 days post-treatment (S2) were taken; microbial quantification and cell viability were assessed by quantitative polymerase chain reaction (qPCR) and flow cytometry (FC). The mean Log10 of bacterial DNA copies in root canal samples before (S1) and the Log10 reduction of DNA copies S1-S2 in qPCR were recorded. The absolute value of total cells stained, and the percentage reduction of intact membrane cells after treatment (S1-S2), were analysed by FC. Global comparison was done using the Kruskal-Wallis test, whilst the Mann-Whitney U test was used for pair-by-pair comparison. Confocal scanning laser microscopy analysis indicated that the greatest effectiveness was obtained with 5% DCH, showing significant differences with respect to the other groups (p < .001). In root canals, the highest Log10 DNA reduction S1-S2 was obtained with 5% DCH and TAH, with no differences between them. The results of FC showed that only 5% DCH proved significantly superior to the other treatments. Sodium DC hydrogels demonstrate antimicrobial efficacy against endodontic biofilms.

Identification of Antifungal Compounds from Avocado Rhizobacteria ( Bacillus spp.) against Fusarium spp., by a Bioassay‐Guided Fractionation Approach

Antimicrobial compounds produced by bacteria have been increasingly acknowledged as an important resource for the control of phytopathogens. We used a bioassay-guided fractionation approach to identify antifungal metabolites produced by two avocado rhizobacteria (INECOL-4742 and INECOL-5927), both members of the Bacillus subtilis/B. amyloliquefaciens species complex, against Fusarium solani and F. kuroshium, causal agent of Fusarium dieback in avocado and other hosts. The butanol (BuOH) organic extract from INECOL-4742 (B1-Bu) exhibited the highest percentage of inhibition (PI) against F. solani (78.76 %), also inhibiting F. kuroshium by up to 44.30 %. Primary fractions, Bu-F3, Bu-F12 and Bu-F15, obtained by silica gel open column chromatography, exhibited the highest PI against F. solani (28.57 % to 33.50 %) and F. kuroshium (38.78 % to 45.00 %). The presence of cyclic lipopeptides from the iturin, surfactin and fengycin families in B1-Bu extracts and primary fractions was determined by UPLC-ESI-HRMS. The Confocal Laser Microscopy analysis revealed deformations in the hyphae of F. kuroshium exposed to extracts, primary fractions and C-13 surfactin chemical standard. These results emphasize the potential of natural products from Bacillus for the control of the emerging phytopathogenic fungus F. kuroshium.

The applicability of Calcofluor White (CWS) and Fluorescent Brightener (CFB) dyes for confocal laser microscopic analysis (CLSM) of various β-glucans in selected dairy products and water

The aim of this study was to determine the applicability of commonly used dyes (CWS and CFB) to stain different types of β-glucans in water and selected, most popular, dairy products for CLSM imaging analysis. Structurally different pure β-glucan preparations: curdlan-CU, oat β-glucan-OG and scleroglucan-SC were tested. Our study showed for the first time that CWS is a dye with a specific affinity for all analyzed β-glucans, while CFB is suitable for curdlan labeling only. Despite the technological processes, β-glucans structures in dairy products are similar to those in aqueous suspension forms; each of the β-glucans forms a different and characteristic structure, ranging from spindle-shaped and branched till granular. The presented results for the first time systematize the knowledge on CWS and CFB applicability in β-glucan CLSM staining and allow future researchers to correctly analyze simultaneously stained β-glucan, fat, and protein in a complex matrix as dairy products.

Strontium-loaded titanium-15molybdenum surface improves physicochemical and biological properties in vitro

The titanium alloy composition and microdesign affect the dynamic interplay between the bone cells and titanium surface in the osseointegration process. The current study aimed to evaluate the surface physicochemical properties, electrochemical stability, and the metabolic response of the MC3T3-E1 cells (pre-osteoblast cell line) cultured onto titanium-15molybdenum (Ti-15Mo) discs treated with phosphoric acid (H3PO4) and sodium hydroxide (NaOH) and/or strontium-loading by the hydrothermal method. The x-ray dispersive energy spectroscopy (EDS) and x-ray diffraction (XRD) analysis showed no trace of impurities and the possible formation of hydrated strontium oxide (H2O2Sr), respectively. The confocal laser microscopy (CLSM) analysis indicated that titanium samples treated with strontium (Sr) showed greater surface roughness. The acid/alkali treatment prior to the hydrothermal Sr deposition improved the surface free energy and resistance to corrosion of the Ti-15Mo alloy. The acid/alkali treatment also provided greater retention of the Sr particles on the Ti-15Mo surfaces accordingly with inductively coupled plasma optical emission spectrometry (ICP-OES) analysis. The AlamarBlue and fluorescence analysis indicated noncytotoxic effects against the MC3T3-E1 cells, which allowed cells’ adhesion and proliferation, with greater cells’ spreading in the Sr-loaded Ti-15Mo samples. These findings suggest that Sr deposition by the hydrothermal method has the potential to enhance the physicochemical properties of the Ti-15Mo previously etched with H3PO4 and NaOH, and also improve the initial events related to cell-mediated bone deposition.

Increase of intracellular Ca2+ concentration in Listeria monocytogenes under pulsed magnetic field

Pulsed magnetic field (PMF) has been proposed as a non-thermal technology for inactivation of microorganisms in food. The changes in intracellular Ca2+ concentration in microorganisms may contribute to explain the inactivation mechanism by PMF. A fluorescence method for evaluating the intracellular Ca2+ concentration in Listeria monocytogenes was established to reveal the effect of PMF on the intracellular Ca2+ level in cell. Fluo-4 AM, a fluorescent calcium assay reagent, was used to determine the intracellular Ca2+ concentration in L. monocytogenes. The results showed that Fluo-4 AM was successfully loaded into L. monocytogenes and could be used to determine the concentration of intracellular free Ca2+ level. The optimal loading temperature and time were 37 °C and 120 min, respectively. After exposure to PMF, intracellular Ca2+ concentration in L. monocytogenes increased by amounts varying between 27.5 and 45.2%. In addition, laser confocal scanning microscope (LCSM) analysis conformed that the Ca2+ fluorescence intensity in L. monocytogenes increased after PMF treatment, indicating that large amounts of exocellular Ca2+ permeated into the cells. Therefore, it can be inferred that PMF may increase the permeability of the cell membrane and promote Ca2+ transmembrane behavior. These results help to explore the inactivation mechanism of microorganism treated by PMF from the angle of the intracellular Ca2+ concentration.

Evaluation of Clean-in-Place Agents on the Removal of Thermophilic Biofilms Formed under Partially Simulated Conditions Associated with the Dairy Industry

In this study, various clean-in-place agents against thermophilic biofilms were evaluated. Dairy contaminants such as Geobacillus thermodenitrificans, Geobacillus thermoglucosidans, Anoxybacillus flavithermus, and Anoxybacillus kamchatkensis subsp. asaccharedens were sampled under simulated conditions (whole milk, static and dynamic conditions). Biofilm removal strategies were conducted, and agents acting on proteins such as trichloroacetic acid (TCA) and sodium dodecyl sulfate (SDS) were found to be most effective on thermophilic biofilms. It was found that thermophilic strains, especially under dynamic conditions, can form intense biofilms at an early stage (&lt;5 h) and that these biofilms cannot be removed by the previously recommended routine sanitation procedures. In the current study, tandem treatment (SDS after TCA) with chemicals such as TCA and SDS for 30 min gave clear results in removing thermophilic biofilms. Moreover, enzymes such as trypsin and protease were highly effective in removing thermophilic biofilms. It was also found that surface materials used in the dairy industry, such as stainless steel, polypropylene, polyvinyl chloride, and polycarbonate, are not critical for the removal of thermophilic biofilms. Thermophilic biofilms sampled on surfaces under simulated conditions (whole milk, temperature, steady-state, and dynamic conditions) were also evaluated using confocal laser microscopy analysis after sanitary treatment.

Layered Double Hydroxide Modified with Deoxycholic and Hyaluronic Acids for Efficient Oral Insulin Absorption.

IntroductionThis study aimed to construct a layered double hydroxide (LDH) nanoparticle delivery system that was modified by deoxycholic acid (DCA) and hyaluronic acid (HA) to increase the bioavailability of oral insulin.MethodsLDH-DCA-HA was synthesized by the hybridization of DCA and HA with LDH. Subsequently, insulin was loaded onto LDH-DCA-HA, resulting in the formation of INS@LDH-DCA-HA. The in vivo and in vitro mechanisms of insulin release, as well as the efficiency of insulin absorption, were analyzed before and after DCA-HA modification.ResultsMTT assay showed that there was satisfactory biocompatibility between LDH-DCA-HA and Caco-2 cells at a concentration below 1000 μg/mL. Flow cytometry analysis revealed that Caco-2 cells absorbed INS@LDH-DCA-HA more readily than insulin. Measurement of transepithelial electrical resistance indicated that INS@LDH-DCA-HA induced the reversible opening of tight cell junctions, thereby facilitating its absorption. This was confirmed via laser confocal microscopy analysis, revealing that a large amount of zonula occludens-1 tight junction (TJ) protein was utilized for the paracellular pathway of nanoparticles. We also measured the blood glucose levels of type I diabetic mice and found that oral INS@LDH-DCA-HA exerted a steady hypoglycemic effect lasting 12 h, with a small range of postprandial blood glucose fluctuation. Immunofluorescence analysis showed that the strong penetration ability of INS@LDH-DCA-HA allowed insulin to enter epithelial cells more readily than free insulin. Finally, immunohistochemical analysis of anti-SLC10A1 protein confirmed that the cholic acid transporter receptor protein played a key role in the functioning of INS@LDH-DCA-HA.ConclusionLDH nanoparticles modified by DCA and HA improved the absorption efficiency of insulin by opening the TJs of cells and interacting with the cholic acid transporter receptor protein.