Confocal Laser Scanning Microscopy Images Research Articles

The Toxicity of Polystyrene-Based Nanoparticles in Saccharomyces cerevisiae Is Associated with Nanoparticle Charge and Uptake Mechanism.

Polystyrene latex (PSL) nanoparticles (NPs), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes, and hybrid NPs that have different concentrations, sizes, surface charges, and functional groups were used to determine their toxicity to Saccharomyces cerevisiae cells. The size, charge, and morphology of the nanoparticles were characterized by dynamic light scattering, electrophoretic light scattering, scanning transmission electron microscopy, and transmission electron microscopy analysis. The cell viabilities were determined by colony forming unit analysis and confocal laser scanning microscopy imaging. Uptake inhibition studies were performed to determine the internalization mechanism of PSL NPs. At 50 mg/L, both positively and negatively charged NPs were slightly toxic. With increasing concentration, however, full toxicities were observed with positively charged PSL NPs, while a marginal increase in toxicity was obtained with negatively charged PSL NPs. For negatively charged and carboxyl-functionalized NPs, an increase in size induced toxicity, whereas for positively charged and amine-functionalized NPs, smaller-sized NPs were more toxic to yeast cells. Negatively charged NPs were internalized by the yeast cells, but they showed toxicity when they entered the cell vacuole. Positively charged NPs, however, accumulated on the cell surface and caused toxicity. When coated with DOPC liposomes, positively charged NPs became significantly less toxic. We attribute this reduction to the larger-diameter and/or more-agglomerated NPs in the extracellular environment, which resulted in lower interactions with the cell. In addition to endocytosis, it is possible that the negatively charged NPs (30-C-n) were internalized by the cells, partly via direct permeation, which is preferred for high drug delivery efficiency. Negatively charged PSL NP exposure to the yeast cells at low-to-moderate concentrations resulted in low toxicities in the long term. Our results indicate that negatively charged PSL NPs provide safer alternatives as cargo carriers in drug delivery applications. Moreover, the variations in NP size, concentration, and exposure time, along with the use of hybrid systems, have significant roles in nanoparticle-based drug delivery applications in terms of their effects on living organisms.

Use of a novel 9.3-μm carbon dioxide laser and silver diamine fluoride: Prevention of enamel demineralisation and inhibition of cariogenic bacteria

ObjectiveTo investigate the effects of a 9.3-μm carbon dioxide (CO2) laser and silver diamine fluoride (SDF) on the prevention of enamel demineralisation and inhibition of cariogenic bacteria. MethodsEnamel blocks were applied with Laser (Group-1), SDF (Group-2), Laser + SDF (Group-3) and no treatment (Group-4), and then subjected to an 8-day pH-cycling for cariogenic challenge. Lesion depth and cross-sectional micro-hardness were assessed. Surface morphological and chemical changes were studied using scanning electron microscope (SEM) with energy dispersive spectroscopy (EDS). For the antibacterial activity, treated enamel blocks were incubated with Streptococcus mutans. The biofilm morphology, kinetics and viability were assessed by SEM, colony-forming units (CFUs) and confocal laser scanning microscope (CLSM), respectively. ResultsLesion depths (μm) for Group-1 to Group-4 were 88 ± 21, 26 ± 11, 13 ± 9 and 115 ± 25, respectively (p < 0.001; Group-2 and Group-3 < Group-1 < Group-4). Group-3 had a significantly higher cross-sectional micro-hardness than the other three groups. EDS determined that Group-4 had the lowest calcium-to-phosphorus molar ratio among the groups (p < 0.001). SEM images showed apparent bacteria accumulation on enamel surfaces in Group-4, but not in other groups. Log CFUs for Group-1 to Group-4 were 6.2 ± 0.6, 2.9 ± 0.8, 2.2 ± 1.1 and 7.3 ± 0.3, respectively (p < 0.001; Group-2 and Group-3 < Group-1 < Group-4). CLSM images revealed that live bacteria dominated in Group-4, but not in other groups. SignificanceThe irradiation with a 9.3-μm CO2 laser alone can prevent the demineralisation of enamel and reduce the adhesion of cariogenic bacteria. Moreover, adding SDF can significantly increase the preventive effect and antibacterial ability.

Twisted Intramolecular Charge Transfer—Aggregation-Induced Emission Fluorogen with Polymer Encapsulation-Enhanced Near-Infrared Emission for Bioimaging

Fluorescence probes with strong near-infrared (NIR) emission and water solubility are considered useful visualization tools for localization marking as well as investigating cell migration and tran...

Microbial characterization of a natural biofilm associated with Peruvian scallop (Argopecten purpuratus) larvae settlement on artificial collector by confocal imaging, microbiology, and metagenomic analysis

Biofilms strongly influence bivalve settlement patterns on artificial substrates; however, their structure and taxonomic composition remains a black box. We characterized a natural biofilm composition that exhibits a large settlement of larvae of the Peruvian scallop Argopecten purpuratus by culture-dependent and culture-independent methods. Thirty-two different strains, representing six genera (10 strains of Bacillus, 9 of Vibrio, 6 Acinetobacter, 4 Staphylococcus, 2 Photobacterium, and 1 Exiguobacterium) were isolated. Those strains represented only 1.09% of the relative abundance compared with the total microbiota obtained by 16S rRNA high-throughput sequencing. The metagenomic analysis identified 441 species. Prokaryotes were predominant (93.4%) over eukaryotes (6.6%), with Pelobacter (13.4%), Lewinella (5.6%), Marinobacter (5.4%), Hoeflea (4.2%), and Microcystis (3.1%) being the most representative genera. Laser scanning confocal microscopy (LSCM) imaging evidenced an irregular and heterogeneous biofilm with an average thickness of 35 μm, where the heterotrophic prokaryotic community (3.4×106 cell cm-2) dominate the photoautotrophic communities (2.3×105 cell cm-2). For the first time, an A. purpuratus settlement-related biofilm was described by the next generation sequencing tool (NGS) and compared with traditional methodologies.

Antibiofilm Activity of Antarctic Sponge-Associated Bacteria against Pseudomonas aeruginosa and Staphylococcus aureus

Bioprospecting in unusual marine environments provides an innovative approach to search novel biomolecules with antibiofilm activity. Antarctic sponge-associated bacteria belonging to Colwellia, Pseudoalteromonas, Shewanella and Winogradskyella genera were evaluated for their ability to contrast the biofilm formation by Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213, as model organisms. All strains were able to produce biofilm at both 4 and 25 °C, with the highest production being for Colwellia, Shewanella and Winogradskyella strains at 4 °C after 24 h. Antibiofilm activity of cell-free supernatants (CFSs) differed among strains and on the basis of their incubation temperature (CFSs4°C and CFSs25°C). The major activity was observed by CFSs4°C against S. aureus and CFSs25°C against P. aeruginosa, without demonstrating a bactericidal effect on their growth. Furthermore, the antibiofilm activity of crude extracts from Colwellia sp. GW185, Shewanella sp. CAL606, and Winogradskyella sp. CAL396 was also evaluated and visualized by confocal laser scanning microscopic images. Results based on the surface-coating assay and surface tension measurements suggest that CFSs and the crude extracts may act as biosurfactants inhibiting the first adhesion of P. aeruginosa and S. aureus. The CFSs and the novel biopolymers may be useful in applicative perspectives for pharmaceutical and environmental purposes.

Biocompatible and noncytotoxic nucleoside-based AIEgens sensor for lighting-up nucleic acids

Aggregation-induced emission luminogens (AIEgens) have been used in biomacromolecules detection. Herein, TPE-dC and TPE-dU acted as the nucleoside-based AIEgens sensors in the first case, which can be used to detect ctDNA and rRNA in vitro and light up the nucleus in vivo depending on the intermolecular binding affinity. This AIE process enables the quantitative analysis or visualization of nucleic acids in solution or gels state, respectively. Furthermore, confocal laser scanning microscopy (CLSM) images of L929 cells stained with TPE-dC or TPE-dU clearly shows that nucleoside-based AIEgens bio-probes can pass the cell membranes to reach the cell nucleus, without cytotoxicity at the imaging condition (incubation time > 12 h, and 10 μmol/L of concentration). Since the nucleus is rich in DNA/RNA, fluorescence turn-on mode has a great potential in nucleus imaging and clinical diagnosis.

Selective retention of extracellular polymeric substances induced by adsorption to and coprecipitation with ferrihydrite

Recent work shows that microbially-derived compounds constitute a significant fraction of the soil organic matter (OM) pool. These compounds include extracellular polymeric substances (EPS) whose mass can far exceed total microbial cell biomass. Sorption of EPS to soil minerals occurs via adsorption and coprecipitation and contributes to the preservation of OM in the soil environment. Little is known, however, about the sorption mechanisms of EPS and selective retention of different EPS constituents on iron (oxyhydr)oxides, especially during EPS adsorption versus coprecipitation with these reactive soil phases. This study examines how EPS interacts with the ubiquitous soil iron (oxyhydr)oxide ferrihydrite during EPS adsorption and coprecipitation and whether these different EPS-mineral association pathways affect EPS sorption and selective retention, and thus the mobility and fate of microbially-derived OM in the soil environment. We use several complimentary techniques to i) examine EPS-carbon, EPS-nitrogen and EPS-phosphorus sorption and fractionation, ii) visualize spatial relationships between EPS biomolecular classes and ferrihydrite using confocal laser scanning microscopy (CLSM), iii) determine EPS-C speciation and chemical fractionation with ferrihydrite using X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure spectroscopy (NEXAFS), and iv) determine functional group interactions with ferrihydrite using Fourier transform infrared spectroscopy (FTIR) combined with two-dimensional correlation spectroscopy (2D-COS) analysis. Results show that the coprecipitation of EPS does not change the ferrihydrite mineralogy, as the main mineral phase for EPS association, but it substantially increases the particle size of EPS-ferrihydrite. A substantial difference in the EPS mass fraction associated with the ferrihydrite is observed between the adsorption and coprecipitation experiments at an initial molar C/Fe ratio >1. The EPS-N is relatively enriched during the adsorption process, while more EPS-C and near-complete EPS-P are fixed in the coprecipitation process. XPS results show that the surface of the ferrihydrite formed through EPS adsorption is preferentially enriched with protein-like components, whereas, the surface of the ferrihydrite formed through EPS coprecipitation is enriched with polysaccharide-like components, which is visually confirmed with CLSM images. NEXAFS results reveal that the carboxylic/amide C-containing components are selectively retained during adsorption, with the aliphatic and O-alkyl C-containing components relatively enriched during coprecipitation. 2D-FTIR-COS results indicate that during EPS adsorption on ferrihydrite the PO functional groups are adsorbed faster than the amide and carboxylate functional groups, while during EPS coprecipitation with ferrihydrite the opposite trend is observed. The findings from this study indicate that the formation pathway of EPS-ferrihydrite associations substantially effects the sorption mechanisms and selective retention of EPS and may thus affect the mobility and fate of microbially-derived carbon (C), nitrogen (N) and phosphorus (P) in soils. These new insights on EPS behaviour at the mineral–water interface might be used to evaluate how microbially-derived compounds like EPS are stabilized by iron (oxyhydr)oxides and how EPS-iron (oxyhydr)oxide couplings might affect the reactivity and cycling of OM in natural environments.

Screening of frozen-thawed conditions for keeping nutritive compositions and physicochemical characteristics of goat milk

Frozen milk can help producers overcome the seasonality of goat milk production, low goat production and short lactation periods, and avoid discarding milk during some special periods. We investigated effects of combination between freezing (cryogenic refrigerator of -16 to -20°C or ultra-cryogenic refrigerator of -76 to -80°C) and thawing (homeothermy of 20 to 25°C or refrigeration of 2 to 4°C) on nutritive compositions and physicochemical characteristics of raw goat milk during storage period (80 d). Compared with fresh goat milk, the frozen-thawed milk decreased contents of fat, protein, and lactose, as well as surface tension and stability coefficient, whereas increased effective diameter and polydispersity index. The average values of color values (L*, a*, and b*) in 4 group samples changed from 83.01 to 82.25, -1.40 to -1.54, 3.51 to 3.81, respectively, and the ΔE of most samples did not exceed 2. In contrast to the other 3 frozen-thawed treatments, goat milk treated with ultra-cryogenic freezing-homeothermic thawing (UFHT) possessed higher fat (5.20 g/100 g), smaller effective particle diameter (0.32 µm), and the lowest polydispersity index value (0.26). The color and confocal laser scanning microscopy images of UFHT were similar to those of fresh goat milk, illustrating UFHT was the optimal approach to maintain the natural quality of goat milk. Our finding provides a theoretical basis for producers to freeze surplus milk.

Fabrication and stability of Pickering emulsions using moringa seed residue protein: Effect of pH and ionic strength

AbstractFood‐grade Pickering emulsions stabilised by plant protein have low toxicity. In this study, to explore the potential of Moringa seed residue protein (MSRP) as Pickering particles, MSRP was obtained from Moringa seed residue and used to fabricate emulsions at different pH and ionic strength. The molecular weight of MSRP ranged between 5 and 25 kDa. The particle diameter of MSRP with positive charge was &gt; 233 nm. The highest MSRP solubility was achieved at pH 5 (206.89 mg g−1) and 0.2 M NaCl (202.55 mg g−1). MSRP generated an excellent interfacial layer as verified by confocal laser scanning microscope imaging, and had lower droplet size, and formed well‐developed elastic networks at pH 5 and 0.2 M NaCl. MSRP‐stabilised emulsion through steric hindrance, possessing excellent tolerance in salt (&lt; 0.2M) and weakly acid (pH 5–7) environment. Our study finds MSRP can be used as potential Pickering particles applying in food and beverages industries.

Effect of Propolis Nanoparticles against Enterococcus faecalis Biofilm in the Root Canal.

To determine the antibacterial effect of propolis nanoparticles (PNs) as an endodontic irrigant against Enterococcus faecalis biofilm inside the endodontic root canal system. Two-hundred-ten extracted human teeth were sectioned to obtain 6 mm of the middle third of the root. The root canal was enlarged to an internal diameter of 0.9 mm. The specimens were inoculated with E. faecalis for 21 days. Following this, specimens were randomly divided into seven groups, with 30 dentinal blocks in each group including: group I—saline; group II—propolis 100 µg/mL; group III—propolis 300 µg/mL; group IV—propolis nanoparticle 100 µg/mL; group V—propolis nanoparticle 300µg/mL; group VI—6% sodium hypochlorite; group VII—2% chlorhexidine. Dentin shavings were collected at 200 and 400 μm depths, and total numbers of CFUs were determined at the end of one, five, and ten minutes. The non-parametric Kruskal–Wallis and Mann–Whitney tests were used to compare the differences in reduction in CFUs between all groups, and probability values of p < 0.05 were set as the reference for statistically significant results. The antibacterial effect of PNs as an endodontic irrigant was also assessed against E. faecalis isolates from patients with failed root canal treatment. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were also performed after exposure to PNs. A Raman spectroscope, equipped with a Leica microscope and lenses with curve-fitting Raman software, was used for analysis. The molecular interactions between bioactive compounds of propolis (Pinocembrin, Kaempferol, and Quercetin) and the proteins Sortase A and β-galactosidase were also understood by computational molecular docking studies. PN300 was significantly more effective in reducing CFUs compared to all other groups (p < 0.05) except 6% NaOCl and 2% CHX (p > 0.05) at all time intervals and both depths. At five minutes, 6% NaOCl and 2% CHX were the most effective in reducing CFUs (p < 0.05). However, no significant difference was found between PN300, 6% NaOCl, and 2% CHX at 10 min (p > 0.05). SEM images also showed the maximum reduction in E. faecalis with PN300, 6% NaOCl, and 2% CHX at five and ten minutes. CLSM images showed the number of dead cells in dentin were highest with PN300 compared to PN100 and saline. There was a reduction in the 484 cm−1 band and an increase in the 870 cm−1 band in the PN300 group. The detailed observations of the docking poses of bioactive compounds and their interactions with key residues of the binding site in all the three docking protocols revealed that the interactions were consistent with reasonable docking and IFD docking scores. PN300 was equally as effective as 6% NaOCl and 2% CHX in reducing the E. faecalis biofilms.

Targeting effect of berberine on type I fimbriae of Salmonella Typhimurium and its effective inhibition of biofilm.

As a primary cause of food contamination and human diseases, Salmonella Typhimurium can easily form a biofilm that is difficult to remove from food surfaces, and often causes significant invisible threats to food safety. Although berberine has been widely used as an anti-infective drug in traditional medicine, some basic principles underlying its mechanism, especially the interaction between berberine and type I fimbriae genes, has not been verified yet. In this study, two strains of major fimbrial gene mutants (ΔfimA and ΔfimH) were constructed to demonstrate the possible action of berberine on type I fimbriae genes. The broth microdilution method was used to determine the antibacterial activity of berberine against selected strains (WT, ΔfimA, and ΔfimH). Cell agglutination experiments revealed that the number of S. Typhimurium type I fimbriae reduced after berberine treatment, which was consistent with transmission electron microscopy results. Quantitative real-time PCR experiments also confirmed that berberine reduced fimA gene expression, indicating a certain interaction between berberine and fimA gene. Furthermore, confocal laser scanning microscopy imaging of biofilm clearly revealed that berberine prevents biofilm formation by reducing the number of type I fimbriae. Overall, it is well speculated for us that berberine could be an excellent combating-biofilm drug in clinical microbiology and food preservation. KEY POINTS: • Reduce the number of fimbriae. • Berberine targeting fimA. • Effective biofilm inhibitor.

Stability, temperature dependence, and microstructure of high pressure jet-treated dairy foams

The objective of this work was to study the effects of high pressure jet (HPJ) processing on the foaming of whole milk at different temperatures. High pressure jet-treated whole milk (200 and 400 MPa) was foamed at 5, 20, 40, and 50 °C using a custom-made air injection device and the foam expansion and foam stability was compared to a non-HPJ-treated control. Aliquots of the foams (formed at 5 and 50 °C) were extracted for proximate analysis and visualization using confocal scanning laser microscopy (CSLM). The relative foam expansion values for the non-HPJ-treated sample at 5, 20, 40, and 50 °C were 45.5 ± 11.3%, 0 ± 0%, 79.6 ± 9.7%, and 88.2 ± 2.8%, respectively while the 400 MPa-treated sample foam expansion values were 36.4 ± 10.8%, 60.8 ± 5.7%, 50.0 ± 5.3%, and 71.7 ± 3.0%. Notably the HPJ-treated sample foamed at 20 °C while the control milk did not foam at all. Furthermore, the 400 MPa-treated foam was stable for >2 h at all foaming temperatures, unlike the non-HPJ-treated whole milk foam, which completely collapsed in 60 s and 1490 s after being foamed at 5 °C and 40 °C, respectively. CSLM and proximate analysis of the non-HPJ-treated sample revealed differences in foam composition based on foaming temperature with more fat present in the foam formed at 50 °C (3.2 ± 0.3%) than at 5 °C (2.5 ± 0.1%), likely due to the ability of liquid oil (at 50 °C) to migrate to the air-water interface during air incorporation. In contrast, there were no compositional differences in the foams from 400 MPa-treated milk based on foaming temperature, reflecting that there are structural differences, particularly in the fat, compared to the non-HPJ-treated foams. This was supported by evidence of fat-protein complexation and micron-size bubbles in CSLM images of the 400 MPa-treated foam.

Optical comparison between micro-CT and OCT in imaging of marginal composite adaptation: Observational study.

Dental composite is the most used aesthetic restorative biomaterial worldwide. However, it undergoes polymerisation shrinkage that could lead to loss of the interfacial seal between tooth and resin in some circumstances. This demands high-resolution imaging technologies to detect these defects. This study carried out a comparison between microcomputed tomography (micro-CT; Shimadzu, Japan) and swept-source optical coherence tomography (SS-OCT; Santec, Japan) in the detection of marginal adaptation defects at the tooth-resin interface. Unlike in micro-CT, it was possible to outline interfacial gaps along with tooth-resin interfaces with SS-OCT, which was attributed to the Fresnel diffraction of light. This in vitro comparison demonstrates SS-OCT has great potential in dental imaging to effectively assess dental composite adaptation and marginal defects when high resolution is desired in real time. LAY DESCRIPTION: Detection of tooth-colored restoration defects had been assessed by different radiographic methods. However, most of these methods are either invasive or suffer from low-resolution. In this study, a comparison has been carried out between two different high-resolution imaging systems; microcomputed tomography and optical coherence tomography, to explore their potentials in detecting restorations defects. The results showed optical coherence tomography has a great accuracy in locating the underlying defects when the obtained images were validated against confocal laser scanning microscopy images.

Formation of disinfection by-products during sodium hypochlorite cleaning of fouled membranes from membrane bioreactors.

Periodic chemical cleaning with sodium hypochlorite (NaClO) is essential to restore the membrane permeability in a membrane bioreactor (MBR). However, the chlorination of membrane foulants results in the formation of disinfection by-products (DBPs), which will cause the deterioration of the MBR effluent and increase the antibiotic resistance in bacteria in the MBR tank. In this study, the formation of 14 DBPs during chemical cleaning offouled MBR membrane modules was investigated. Together with the effects of biofilm extracellular polymeric substances (EPS), influences of reaction time, NaClO dosage, initial pH, and cleaning temperature on the DBP formation were investigated. Haloacetic acids (HAAs) and trichloromethane (TCM), composed over 90% of the DBPs, were increasingly accumulated as the NaClO cleaning time extended. By increasing the chlorine dosage, temperature, and pH, the yield of TCM and dichloroacetic acid (DCAA) was increased by up to a factor of 1–14, whereas the yields of haloacetonitriles (HANs) and haloketones (HKs) were decreased. Either decreasing in the chlorine dosage and cleaning temperature or adjusting the pH of cleaning reagents toward acidic or alkaline could effectively reduce the toxic risks caused by DBPs. After the EPS extraction pretreatment, the formation of DBPs was accelerated in the first 12 h due to the damage of biofilm structure. Confocal laser scanning microscopy (CLSM) images showed that EPS, particularly polysaccharides, were highly resistant to chlorine and might be able to protect the cells exposed to chlorination.Electronic Supplementary MaterialSupplementary material is available in the online version of this article at 10.1007/s11783-021-1389-3 and is accessible for authorized users.

In vivo MRI detection of intraplaque macrophages with biocompatible silica-coated iron oxide nanoparticles in murine atherosclerosis.

Identification of a vulnerable atherosclerotic plaque before rupture is an unmet clinical need. Integrating nanomedicine with multimodal imaging has the potential to precisely detect biological processes in atherosclerosis. We synthesized silica-coated iron oxide nanoparticles (SIONs) coated with rhodamine B isothiocyanate and polyethylene glycol and investigated their feasibility in the detection of macrophages in inflamed atherosclerotic plaques of apolipoprotein E-deficient (ApoE-/-) mice via magnetic resonance (MR) and fluorescence reflectance (FR) imaging. In vitro cellular uptake of SIONs was assessed in macrophages using confocal laser scanning microscopy (CLSM). In vivo MR imaging was performed 24 h after SION injection via the tail vein in 26-week-old ApoE-/- mice fed a high-cholesterol diet (HCD). We also performed FR imaging of the extracted aortas from four different mice: two normal-diet-fed C57BL/6 mice injected with saline or 10 mg/kg SIONs and two HCD-fed ApoE-/- mice injected with 5 or 10 mg/kg SIONs. The harvested aortas were cryosectioned and stained with immunohistochemical staining. The CLSM images at 24 h after incubation showed efficient uptake of SIONs by macrophages, with no evidence of cytotoxicity. The in vivo and ex vivo MR and FR images demonstrated SION deposition in the atheroma. Upon immunohistochemical staining of the aorta, CLSM images revealed colocalization of macrophages and SIONs in the atherosclerotic plaque. These results demonstrate that polyethylene glycosylated SIONs could be a highly effective method to identify macrophage activity in atherosclerotic plaques as a multimodal imaging agent.

Method for fast staining and obtaining high-magnification and high-resolution cell images of Nicotiana benthamiana.

The online version contains supplementary material available at 10.1007/s12298-021-00931-5.

Biopolymer segmentation from CLSM microscopy images using a convolutional neural network

AbstractConfocal microscopy allows visualization of biopolymer networks at the nano scale. Analyzing the structure and assembly of protein networks from images requires a segmentation process. This has proven to be challenging due to multiple possible sources of noise in images as well as exhibition of out‐of‐focus planes. Here, we present a deep learning‐based segmentation procedure for confocal laser scanning microscopy images of biopolymer networks. Utilizing an encoder‐decoder network architecture, our deep neural network achieved a dice score of 0.88 in segmenting images of filamentous temperature sensitive Z proteins from chloroplasts of Physcomitrella patens, a moss.

Insight into probiotic properties of lactic acid bacterial endosymbionts of Apis mellifera L. derived from the Polish apiary.

Taking into account that fructophilic lactic acid bacteria (FLAB) can play an important role in the health of honey bees and can be used as probiotics, phenotypic properties of probiotic interest of Lactobacillus kunkeei (12 strains) and Fructobacillus fructossus bacteria (2 strains), isolated from Apis mellifera gastrointestinal tract, have been studied. We have evaluated survival of tested FLAB in honey bee gut, their susceptibility to antibiotics (ampicillin, erythromycin, tylosin), cell surface hydrophobicity, auto-aggregation ability, co-aggregation with model pathogenic bacteria, biofilm formation capacity, and effect of studied FLAB, added to sucrose syrup bee diet, on longevity of honey bees. The tested FLAB exhibited good gastrointestinal tract tolerance and high antibiotic susceptibility, which are important criteria in the screening of probiotic candidates. It was also found that all FLAB studied have high cell surface hydrophobicity and fulfil next selection criterion for their use as probiotics. Symbionts of A. mellifera showed also auto- and co-aggregation capacities regarded as valuable features for biofilm formation and inhibition of pathogens adhesion to the bee gut cells. Biofilm-development ability is a desired characteristic of probiotic lactic acid bacteria. As indicated by quantitative crystal violet-stained microplate assay and confocal laser scanning microscopy imaging, all studied A. mellifera gut isolates exhibit a biofilm positive phenotype. Moreover, it was also documented, on honey bees kept in cages, that supplementation of A. mellifera sucrose diet with FLAB decreases mortality and improves significantly longevity of honey bees. Presented research showed that A. mellifera FLAB symbionts are good candidates for application as probiotics.

High-internal-phase emulsions (HIPEs) for co-encapsulation of probiotics and curcumin: enhanced survivability and controlled release.

Synergistic biological activities of probiotics and curcumin can be achieved based on the gut-brain axis. However, it is still a challenge for utilizing both of them in actual food products due to their high sensitivity to environmental conditions. In the present study, high-internal-phase emulsions (HIPEs) were fabricated to co-encapsulate the probiotics and curcumin in response to the customer demand for convenience. β-Lactoglobulin-propylene glycol alginate composite hydrogel particles (β-lgPPs) with proper size and intermediate wettability were prepared at β-lg to PGA mass ratio of 2 : 1 and employed as particulate emulsifiers. Stable HIPEs with a fixed oil fraction (φ = 0.8) could be formed within a wide range of β-lgPPs concentrations, ranging from 0.1 to 2.0 wt%. Confocal laser scanning microscopy (CLSM) images indicated that the interfacial structure of the oil droplets was composed of both β-lg nanoparticles and a PGA network, which jointly contributed to the gel-like structures in HIPEs. An increase in elasticity and gel strength, as well as centrifugal stability, could be achieved by elevating the particle concentration as determined by diffusing wave spectroscopy and Lumisizer analysis. HIPEs with high particle concentrations showed a high resistance against pasteurization since no obvious flocculation or coalescence could be observed in these emulsions. HIPEs also provoked a significant reduction in the death of LGG as well as the chemical degradation of curcumin: up to 7.91 log CFU cm-3 of LGG and 93.0% of curcumin were retained after pasteurization treatment. Moreover, the HIPEs could also retard the release of curcumin and protect the LGG in simulated gastrointestinal tract conditions. The results from this work provide useful information for developing a promising delivery system for the co-encapsulation of curcumin and probiotics.

Features of oral squamous cell carcinoma in ex vivo fluorescence confocal microscopy.

Real-time microscopic imaging of freshly excised oral squamous cell carcinomas (OSCCs) would be potentially supportive in rapid recognition of oral malignancy and an optimal and time-saving management of patients' surgical treatment. The aim of this study was to examine oral squamous cell cancer tissue in regards to the commonly known and well-described histomorphologic criteria for the diagnosis of OSCC in ex vivo confocal fluorescent microscopy and to analyze its correlation with grade of differentiation and level of invasiveness. Ex vivo confocal laser scanning microscopy (CLSM) images of 38 OSCCs were evaluated immediately after excision for presence or absence of various cytological and architectural features based on the histopathological background. Next, these features were compared to the grade of differentiation as elaborated via gold standard histologic examination. Of 38 invasive OSCCs, 14 were well differentiated, while three moderately and 19 were poorly differentiated. The presence of the commonly known cytologic and histopathologic criteria for the diagnosis of oral squamous cell carcinoma such as the destruction of the basal cell membrane, cellular and nuclear pleomorphism, anisocytosis, intraepithelial keratinization, nuclear hyperchromasia, atypical mitotic figures as well as the presence of necrosis, and mixed inflammation could be observed in ex vivo fluorescence confocal microscopy (FCM). In ex vivo fluorescence confocal microscopy pictures, cellular pleomorphism and anisocytosis were observed more often in poorly differentiated OSCCs. Intraepithelial keratinization was associated with well differentiated and moderately differentiated OSCCs. The results demonstrate the high potential of ex vivo fluorescence confocal microscopy in fresh tissue for rapid real-time diagnosis of OSCC.