Characteristics and Kinetics of Bainite Transformation Behaviour in a High-Silicon Medium-Carbon Steel above and below the Ms Temperature

This work deals with the kinetic aspects of bainite formation during isothermal holding above and below the martensite start (Ms~275 °C) temperature using a low-alloy, high-silicon DIN 1.5025 steel in a range suitable for achieving ultrafine/nanostructured bainite. Dilatation measurements were conducted to study transformation behaviour and kinetics, while the microstructural features were examined using laser scanning confocal microscopy and electron backscatter diffraction (EBSD) techniques combined with hardness measurements. The results showed that for isothermal holding above the Ms temperature, the maximum bainitic transformation rate decreased with the decrease in isothermal holding temperature between 450 and 300 °C. On the other hand, for isothermal holding below the Ms temperature at 250 and 200 °C, the maximum rate of transformation was achieved corresponding to region I due to the partitioning of carbon and also possibly because of the ledged growth of isothermal martensite soon after the start of isothermal holding. In addition, a second peak was obvious at about 100 and 500 s, respectively, during holding at 250 and 200 °C due to the occurrence of bainitic transformation, marking the beginning of region II.

Semi-automated computational assessment of cancer organoid viability using rapid live-cell microscopy

The creation of patient-derived cancer organoids represents a key advance in preclinical modeling and has recently been applied to a variety of human solid tumor types. However, conventional methods used to assess cellular viability in tissue specimens are poorly suited for the evaluation of cancer organoids because they are time-intensive and involve tissue destruction. To address this issue, we established a suite of 3-dimensional patient-derived glioma organoids, treated them with chemoradiotherapy, stained organoids with non-toxic cell dyes, and imaged them using a rapid laser scanning confocal microscopy method termed 'Apex Imaging'. We then developed and tested a fragmentation algorithm to quantify heterogeneity in the topography of the organoids as a potential surrogate marker of viability. This algorithm, SSDquant, provides a 3-dimensional visual representation of the organoid surface and a numerical measurement of the sum-squared distance (SSD) from the derived mass center of the organoid. We tested whether SSD scores correlate with traditional immunohistochemistry-derived cell viability markers (cellularity and cleaved caspase 3 expression) and observed statistically significant associations between them using linear regression analysis. Our work describes a quantitative, non-invasive approach for the serial measurement of patient-derived cancer organoid viability, thus opening new avenues for the application of these models to studies of cancer biology and therapy.

Morphophysiological Changes in Staphylococcus aureus Biofilms Treated with Plasma-Activated Hydrogen Peroxide Solution

Plasma-activated solution has attracted more attention in the food industry due to no chemical residue and good bacteriostatic properties. This study aimed to evaluate the effects of plasma-activated hydrogen peroxide solution (PAH) on the morphophysiology of Staphylococcus aureus biofilms. PAH was prepared using dielectric-barrier-discharge plasma and incubated with S. aureus biofilms for 0–40 min. Changes in biofilm morphophysiology were evaluated with laser scanning confocal microscopy, electron microscopic images, reactive oxygen species (ROS) content, metabolic capacity, and 1% agarose gel. Results indicated that the population of S. aureus in the biofilms was reduced by 4.04-log after incubation with PAH for 30 min. The thickness and metabolic capacity of biofilms were decreased, the ROS content and DNA fragments of bacteria increased after PAH treatments. Data suggested that PAH treatments significantly destroyed the morphophysiology of S. aureus (ATCC 6538) biofilms and could be considered as a valuable anti-biofilm technology to reduce foodborne pathogens on food and/or in food facilities.

Prototheca spp. induce an inflammatory response via mtROS-mediated activation of NF-κB and NLRP3 inflammasome pathways in bovine mammary epithelial cell cultures

Emergence of bovine mastitis caused by Prototheca algae is the impetus to better understand these infections. Both P. bovis and P. ciferrii belong to Prototheca algae, but they differ in their pathogenicity to induce inflammatory responses. The objective was to characterize and compare pathogenesis of inflammatory responses in bMECs induced by P. bovis versus P. ciferrii. Mitochondrial ultrastructure, activity and mtROS in bMECs were assessed with transmission electron microscopy and laser scanning confocal microscopy. Cytokines, including TNF-α, IL-1β and IL-18, were measured by ELISA and real-time PCR, whereas expressions of various proteins in the NF-κB and NLRP3 inflammasome pathways were detected with immunofluorescence or Western blot. Infection with P. bovis or P. ciferrii damaged mitochondria, including dissolution and vacuolation of cristae, and decreased mitochondrial activity, with P. bovis being more pathogenic and causing greater destruction. There were increases in NADPH production and mtROS accumulation in infected bMECs, with P. bovis causing greater increases and also inducing higher cytokine concentrations. Expressions of NF-κB-p65, p-NF-κB-p65, IκBα and p-IκBα proteins in the NF-κB pathway, as well as NLRP3, Pro Caspase1, Caspase1 p20, ASC, Pro IL-1β, and IL-1β proteins in the NLRP3 inflammasome pathway, were significantly higher in P. bovis-infected bMECs. However, mito-TEMPO significantly inhibited production of cytokines and decreased expression of proteins in NF-κB and NLRP3 inflammasome pathways in bMECs infected with either P. bovis or P. ciferrii. In conclusion, P. bovis or P. ciferrii infections induced inflammatory responses in bMECs, with increased mtROS in damaged mitochondria and activated NF-κB and NLRP3 inflammasome pathways, with P. bovis causing a more severe reaction.

In vivo confocal microscopy qualitative investigation of the relationships between lattice corneal dystrophy deposition and corneal nerves

Background To investigate the corneal neurotropic phenomenon in patients with lattice corneal dystrophy (LCD) with in vivo laser scanning confocal microscopy (IVCM). Methods IVCM was performed on a total of 15 patients (28 eyes) with LCD annually at a follow-up. A collection of the data was acquired to be analyzed. Results As indicated by the analysis, the LCD patients’ normal corneal stromal nerves (Grade 0) presented a decline with the prolongation of the follow-ups, corresponding to a gradual increase in grade I and II involving amyloid-wrapped nerve fibers, which demonstrated that the growing amount of amyloid deposit due to the corneal nerve invasion increased slowly over time. Conclusions The neurotropic phenomenon could increase with its severity in the corneal lesion of the patients with LCD, and also reflect the distribution of the corneal nerves, to some extent. IVCM provides a rapid, noninvasive way to observe the corneal nerves, which can be an efficient means of better understanding the development of LCD.

Multimodal Gold Nanostars as SERS Tags for Optically-Driven Doxorubicin Release Study in Cancer Cells

Surface Enhanced Raman Scattering (SERS) active gold nanostars represent an opportunity in the field of bioimaging and drug delivery. The combination of gold surface chemical versatility with the possibility to tune the optical properties changing the nanoparticles shape constitutes a multimodal approach for the investigation of the behavior of these carriers inside living cells. In this work, SERS active star-shaped nanoparticles were functionalized with doxorubicin molecules and covered with immuno-mimetic thiolated polyethylene glycol (PEG). Doxorubicin-conjugate gold nanoparticles show an intense Raman enhancement, a good stability in physiological conditions, and a low cytotoxicity. The strong adsorption of the anticancer drug doxorubicin in close contact with the gold nanostars surface enables their use as SERS tag imaging probes in vivo. Upon laser irradiation of the nanoparticles, a strong SERS signal is generated by the doxorubicin molecules close to the nanostars surface, enabling the localization of the nanoparticles inside the cells. After long time irradiation, the SERS signal drops, indicating the thermally driven delivery of the drug inside the cell. Therefore, the combination of SERS and laser scanning confocal microscopy is a powerful technique for the real-time analysis of drug release in living cells.

Dissolution Behavior of Different Inclusions in High Al Steel Reacted with Refining Slags

Al2O3, Al2O3·TiN, Al2O3·MgO, and CaO·2Al2O3 are four different types of inclusions in high Al steels. To improve the steel cleanness level, the effective removal of such inclusions during secondary refining is very important, so these inclusions should be removed effectively via inclusion dissolution in the slag. The dissolution behavior of Al2O3, Al2O3·TiN, Al2O3·MgO, and CaO·2Al2O3 in CaO-SiO2-Al2O3-MgO slags, as well as the steel-slag reaction, was investigated using laser scanning confocal microscopy (LSCM) and high-temperature furnace experiments, and thermodynamic calculations for the inclusion in steel were carried out by FactSage 7.1. The results showed that Al2O3·TiN was observed to be completely different from the other oxides. The composite oxides dissolved quickly in the slags, and the dissolution time of the inclusions increased as their melting point increased. SiO2 and B2O3 in the slag were almost completely reacted with [Al] in steel, so the slags without SiO2 showed a positive effect for avoiding the formation of Al2O3 system inclusions and promoting inclusions dissolution as compared with SiO2-rich slags. The steel-slag reaction was also found to influence the inclusion types in steel significantly. Because of the rapid absorption of different inclusions in the slag, it was found that the dissolution time of inclusions mainly depends on the diffusion in the molten slag.

Friction and Wear Behaviors of Fe-19Cr-15Mn-0.66N Steel at High Temperature

The friction and wear behaviors of Fe-19Cr-15Mn-0.66N steel were investigated under applied loads of 5 N and 15 N at the wear-testing temperatures of 300 °C and 500 °C using a ball-on-disc tribometer. The wear tracks were evaluated by scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM) to reveal the variation in morphologies. Energy-dispersive X-ray spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS) were used to determine the components of oxide layers formed on wear surfaces. The results demonstrated that the oxide layers are favorable for obtaining a low friction coefficient under all conditions. The average friction coefficient decreased with increasing load at 300 °C, while it increased with the increase in applied load at 500 °C. At 300 °C, severe abrasive wear characterized by grooves resulted in a high friction coefficient with 5 N applied, whereas the formation of a denser oxide layer consisting of Cr2O3, FeCr2O4, Fe2O3, etc., and the increased hardness caused by work hardening led to a decrease in friction characterized by mild adhesive wear. At 500 °C, the transformation of Fe2O3 to the relatively softer Fe3O4 and the high production of lubricating Mn2O3 resulted in a minimum average friction coefficient (0.34) when 5 N was applied. However, the softening caused by high temperature weakened the hardening effect, and thus the friction coefficient increased with 15 N applied at 500 °C.

Monitoring Tritrophic Biocontrol Interactions Between Bacillus spp., Fusarium oxysporum f. sp. cubense, Tropical Race 4, and Banana Plants in vivo Based on Fluorescent Transformation System

Bacillus spp. is effective biocontrol agents for Fusarium wilt of banana (FWB), tropical race 4 (TR4). This study explores the colonization by Bacillus subtilis, Bacillus velezensis, and Bacillus amyloliquefaciens of host banana plants and elucidates the mechanism of antagonistic TR4 biocontrol. The authors selected one B. subtilis strain, three B. velezensis strains, and three B. amyloliquefaciens strains that are proven to significantly inhibit TR4 in vitro, optimized the genetic transformation conditions and explored their colonization process in banana plants. The results showed that we successfully constructed an optimized fluorescent electro-transformation system (OD600 of bacteria concentration=0.7, plasmid concentration=50ng/μl, plasmid volume=2μl, transformation voltage=1.8kV, and transformation capacitance=400Ω) of TR4-inhibitory Bacillus spp. strains. The red fluorescent protein (RFP)-labeled strains were shown to have high stability with a plasmid-retention frequency above 98%, where bacterial growth rates and TR4 inhibition are unaffected by fluorescent plasmid insertion. In vivo colonizing observation by Laser Scanning Confocal Microscopy (LSCM) and Scanning Electron Microscopy (SEM) showed that Bacillus spp. can colonize the internal cells of banana plantlets roots. Further, fluorescent observation by LSCM showed these RFP-labeled bacteria exhibit chemotaxis (chemotaxis ratio was 1.85±0.04) toward green fluorescent protein (GFP)-labeled TR4 hyphae in banana plants. We conclude that B. subtilis, B. velezensis, and B. amyloliquefaciens can successfully colonize banana plants and interact with TR4. Monitoring its dynamic interaction with TR4 and its biocontrol mechanism is under further study.