3D Confocal Laser Scanning Microscopy Research Articles

Arc Erosion Properties of WCu Material Reinforced with Ti3SiC2under Different Applied Voltages

Introducing reinforcements is a crucial way to modify WCu electric contacts. Herein, the arc erosion performances of WCu/Ti3SiC2under various direct current voltages are investigated. The eroded microstructures are observed by a 3D laser scanning confocal microscopy and a thermal field emission scanning electron microscope. Compositions of the eroded surfaces are analyzed by a Raman spectroscopy. With the elevated voltages, values of breakdown current, arc duration, arc energy, and arc discharge distance increase, while the breakdown strength decreases. Meanwhile, the eroded regions gradually change from circular shape to irregularly curved corrugations under the elevated voltages. Copper and Ti3SiC2can disperse arc during arc discharge, and the liquid and gas generated by the two phases can make differences in protecting WCu/Ti3SiC2.

Multi-Resolution 3D Optical Mapping of Immune Cell Infiltrates in Mouse Asthmatic Lung.

Asthma is a chronic inflammatory airway disease driven by various infiltrating immune cell types into the lung. Optical microscopy has been used to study immune infiltrates in the asthmatic lungs. Confocal laser scanning microscopy (CLSM) identifies the phenotypes and locations of individual immune cells in lung tissue sections by employing high-magnification objectives and multiplex immunofluorescence staining. In contrast, light-sheet fluorescence microscopy (LSFM) can visualize the macroscopic and mesoscopic architecture of whole-mount lung tissues in three dimensions (3D) by adopting an optical tissue clearing method. Despite each microscopy method producing image data with unique resolution from a tissue sample, CLSM and LSFM have not been applied together due to the difference in the tissue preparation procedures. Here we introduce a new approach combining LSFM and CLSM into a sequential imaging pipeline. We built a new optical tissue clearing workflow in which the immersion clearing agent can be switched from an organic solvent to an aqueous sugar solution for sequential 3D LSFM and CLSM of mouse lungs. This sequential combination microscopy offered quantitative 3D spatial analyses of the distribution of immune infiltrates in the same mouse asthmatic lung tissue at the organ, tissue, and cell level. These results show that our method facilitates multi-resolution 3D fluorescence microscopy as a new imaging approach providing comprehensive spatial information for a better understanding of inflammatory lung diseases. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Glyoxal-induced disruption of tumor cell progression in breast cancer.

Breast cancer is the most common malignant tumor in women and remains a major global challenge, with ~1.4 million cases per year, worldwide. Numerous studies have shown that changes in cell metabolism are associated with the regulation of tumor progression. In the present study, the anti-cancer properties of glyoxal (GO), which is the smallest dialdehyde formed in the oxidation-reduction reaction and involved in electron transfer and energy metabolism, in breast cancer was investigated. The biological functions and molecular mechanisms of GO were investigated in breast cancer cell lines using MTT and crystal violet assays, flow cytometry, western blot analysis, 3D laser scanning confocal microscopy and transmission electron microscopy. The results showed that GO strongly inhibited cell proliferation, promoted cell apoptosis and cell cycle G2/M arrest, induced the disappearance of cellular microvilli, and enlarged mitochondria. In addition, the protein expression level of AKT, mTOR and p70-S6K decreased in the AKT-mTOR pathway, accompanied by an increase in p-ERK and p-MEK in the MAPK pathway. The results from the present study indicate that GO suppressed breast cancer progression via the MAPK and AKT-mTOR pathways. Taken together, these results provide the basis for a potential therapeutic strategy for breast cancer.

Quantification using rheological blending-law analysis and verification with 3D confocal laser scanning microscopy of the phase behaviour in agarose-gelatin co-gels

The efficacy of commercially available image analysis software was evaluated in the quantification of 3D images captured with confocal laser scanning microscopy (CLSM). The focus was on the phase behaviour of a mixed system of agarose and gelatin that was allowed to gel by controlled cooling to refrigeration temperature. Microstructural characteristics of the binary system were first probed with FTIR and microDSC to document the development of phase separated networks. Rheological experiments in the form of small-deformation dynamic oscillation in-shear were then employed to monitor the formation of a gelatin continuous network throughout the range of experimental concentrations tested. Agarose, on the other hand, formed discontinuous inclusions at low concentrations that inverted to another continuous phase, alongside that of gelatin, at the high end of polysaccharide addition in the mixture. Blending law analysis was able to model the phase behaviour of these preparations in relation to the mechanical strength of each polymer phase and its water holding capacity. Depending on the principle of operation, image analysis software sampled phase volumes in the co-gel that were either a close match to the predictions of blending laws or yielded statistically different values. Imaging software outcomes are discussed with a view to developing an alternative tool for estimating phase morphology in composite biopolymer gels. • Blending law can follow the phase behaviour of agarose-gelatin composite gels. • 3D CLSM imaging paired with image analysis generates a Z-stack of the composite. • Volumetric quantification of agarose-gelatin phase volumes matched the predictions of blending law. • A rapid microscopy protocol can replace blending law in determining composite morphology.

From Macro to Micro: Comparison of Imaging Techniques to Detect Vascular Network Formation in Left Ventricle Decellularized Extracellular Matrix Hydrogels.

Background: Angiogenesis is a crucial process in physiological maintenance and tissue regeneration. To understand the contribution of angiogenesis, it is essential to replicate this process in an environment that reproduces the biochemical and physical properties which are largely governed by the extracellular matrix (ECM). We investigated vascularization in cardiac left ventricular ECM hydrogels to mimic post-myocardial repair. We set out to assess and compare different destructive and non-destructive methods, optical as well as non-optical, to visualize angiogenesis and associated matrix remodeling in myocardial ECM hydrogels. Methods: A total of 100,000, 300,000, and 600,000 Human Pulmonary Microvascular Endothelial Cells (HPMEC) were seeded in left ventricular cardiac ECM hydrogel in 48-well plates. After 1, 7, and 14 days of culture, the HPMEC were imaged by inverted fluorescence microscopy and 3D confocal laser scanning microscopy (Zeiss Cell Discoverer 7). In addition, cell-seeded ECM hydrogels were scanned by optical coherence tomography (OCT). Fixed and paraffin-embedded gels were thin-sectioned and assessed for ECM components via H&E, picrosirius red histochemical staining, and immunostaining for collagen type I. ImageJ-based densitometry was used to quantify vascular-like networks and GraphPad was used for statistical analyses. Results: Qualitative analyses were realized through fluoromicrographs obtained by the confocal laser scanning microscope which allowed us to visualize the extensive vascular-like networks that readily appeared at all seeding densities. Quantification of networks was only possible using fluoromicrographs from inverted microscopy. These showed that, after three days, the number of master junctions was seeding density-dependent. The resolution of optical coherence tomography was too low to distinguish between signals caused by the ECM and cells or networks, yet it did show that gels, irrespective of cells, were heterogeneous. Interestingly, (immuno)histochemistry could clearly distinguish between the cast cardiac-derived matrix and newly deposited ECM in the hydrogels. The H&E staining corroborated the presence of vascular-like network structures, albeit that sectioning inevitably led to the loss of 3D structure. Conclusions: Except for OCT, all methods had complementary merit and generated qualitative and quantitative data that allowed us to understand vascular network formation in organ-derived ECM hydrogels.

Allopodocotyle palmi sp. nov. and Prosorhynchus maternus Bray & Justine, 2006 (Digenea: Opecoelidae & Bucephalidae) from the Orange-Spotted Grouper Epinephelus coioides (Hamilton, 1822) off Bali, Indonesia, Described Using Modern Techniques

BackgroundThe most convincing species of Allopodocotyle Pritchard, 1966 (Digenea: Opecoelidae) are known overwhelmingly from groupers (Serranidae: Epinephelinae). Six species of Allopodocotyle have been reported, collectively, from species of Cromileptes Swainson, 1839, Epinephelus Bloch, 1793 and Plectropomus Oken, 1817. These are A. epinepheli (Yamaguti, 1942), A. heronensis Downie & Cribb, 2011, A. manteri (Saoud & Ramadan, 1984), A. mecopera (Manter, 1940), A. plectropomi (Manter, 1963) and A. serrani (Yamaguti, 1952). In addition, a not yet fully described and unnamed seventh species, morphologically and phylogenetically close to A. epinepheli, was isolated from the orange-spotted grouper Epinephelus coioides (Hamilton, 1822) off Bali, Indonesia in 2016. An eighth species, again from E. coioides off Bali is described herein.MethodsMorphological and phylogenetic analyses justify the recognition of A. palmi sp. nov., which is also genetically different from the as yet unnamed congener from the same host and locality. For the first time, 3D confocal laser scanning microscopy was applied to study and distinguish Digenea taxonomically. We introduce the ‘Palm pattern’, a new simplified way to visualise morphometric differences of related digenean taxa.ResultsAllopodocotyle palmi sp. nov. is distinguished from its congeners that infect groupers by its elongate body with a size > 2.7 mm and diagonal testes. The ovary is located mainly, and the anterior testis completely, in the posterior half of the body; the uterine coils are in the fourth eighth of the body. The cirrus-sac is 0.75–1.4 (1.1) mm long, its posterior extremity is well separated from the anterior extent of the vitelline fields, just reaching the anterior border of uterine coils. In addition, Prosorhynchus maternus Bray & Justine, 2006 (Bucephalidae) was isolated from E. coioides, representing the first record in Indonesia and the third record for this fish species.ConclusionThe biodiversity research in Indonesia is enhanced with a new species description based on modern and newly applied techniques.

Effects of Sulfur on the Surface Crack in Medium Carbon Bloom

The surface cracking problem in medium carbon sulfur‐containing steel is gradually emerging in continuous casting production. Therefore, this article investigates the effect of sulfur content on the surface cracks of medium carbon steel. In addition to macroscopic cracks, network cracks are visible on the surface of medium carbon sulfur‐containing bloom. 3D confocal laser scanning microscopy and industrial computed tomography are used to characterize the 3D realistic morphology of the network and macroscopic cracks. Thermodynamic calculations and hot ductility tests’ results exhibit the higher precipitation of MnS in the sulfur‐containing steel, resulting in the second brittle zone of steel. The microstructure near the cracks revealed a high correlation between cracks and a high proportion of proeutectoid ferrite near the surface of the medium carbon sulfur‐containing bloom. In addition, the sulfur‐containing steel has 0.05% more linear shrinkage than the low‐sulfur steel during the transformation from austenite to ferrite, which increases its crack sensitivity. As a result, this article clarifies the effect of sulfur on the surface crack of medium carbon bloom.

Tribological investigations of Mo films deposited on thin polyimide substrates

In flexible and/or wearable electronics the thin films deposited on compliant substrates are not only subjected to straining and bending but also to tribological loading conditions. A frequently used method to assess tribological properties of materials systems is the ball-on-disc test. In the current work, Mo films were deposited by high power impulse magnetron sputtering on polyimide substrates to serve as a model system. To establish different tribological contact conditions, a series of different counterpart materials was chosen for the performed ball-on-disc tests. A detailed analysis by 3D confocal laser scanning microscopy and scanning electron microscopy revealed details about the wear mechanisms active during sliding where mainly abrasion of the softer one of the material pair was noticed. The obtained results for friction and wear together with the performed simulations of the induced stresses due to the loading conditions show that ball-on-disc tests can contribute to the understanding of the tribological response of metallic films on compliant substrates which in turn may help to improve the lifetime of components in flexible and/or wearable electronic applications.

Solid solubility and charge compensation/exchange mechanisms in Ga- or Mn-Doped CeO2 thin films on 3D printed biomedical titanium alloy

CeO2 films doped with 0–9 mol% Ga/Mn were fabricated by spin coating on 3D-printed Ti6Al4V, calcined at 650 °C for 2 h, and characterised by TEM, FESEM, 3D laser scanning confocal microscopy, GAXRD, and XPS. The results depend on the roles of several factors: (1) Sol-gel precursor viscosity affected pore filling and surface coverage. (2) Lattice contraction and resultant intervalence charge transfer increased the Ce3+ concentration as a minority effect. (3) Substitutional solid solubility and associated redox charge compensation controlled the defect equilibria, which highlight the majority role of this solid solubility mechanism in decreasing the Ce3+ concentration. (4) Electronegativity played a negligible role in affecting the valences but was important in initiating intervalence charge transfer. (5) Multivalence charge transfer combined electron exchanges between film matrix, dopants, and Ti substrate. The present work provides a foundation to interpret the effects of extrinsic effects from both dopant and substrate on the properties of films.

Column bio-oxidation of low-grade refractory gold ore containing high-arsenic and high-sulfur: Insight on change in microbial community structure and sulfide surface corrosion

Currently, there is a lack of research on column bio-oxidation for low-grade refractory gold ore with high arsenic and sulfur content. Therefore, it is essential and significative to investigate column bio-oxidation for this type of gold ore. The column bio-oxidation process of refractory gold ore was characterized by a variety of analytical methods, including X-ray diffraction, 16S rRNA gene analysis, scanning electron microscope coupled with energy-dispersive X-ray spectroscopy and 3D confocal laser scanning microscopy. The evolution of the microbial community structure was analyzed. Simultaneously, the process of passive film formation and the behavior of the dynamic corrosion on the sulfide surfaces were described in detail. The results showed that the cyanidation of gold yielded 81 ± 2% after column bio-oxidation. The oxidation extent for As, Fe and S were 57 ± 1%, 38 ± 1% and 44 ± 1%, respectively. The community structure of attached and planktonic microorganisms was different and changed with the oxidation time. Importantly, the Sulfur film was observed on the arsenopyrite surface under scanning electron microscope, yet it was not found on the pyrite surface. This finding revealed that the inhibitory effect generated by the passive film of arsenopyrite was significantly higher than that of pyrite. Furthermore, the average corrosion pit volume per unit area of pyrite and arsenopyrite were 161 ± 6 μm3/μm2 and 65 ± 3 μm3/μm2. This indicated that arsenopyrite was more easily oxidized during the column bio-oxidation.

Corrosion behavior of Cu/Al casting-rolled clad plates in different alkaline solution

The effect of pH value and different kinds of anions on the corrosion behavior of Cu/Al casting-rolled clad plates in the alkaline solution was evaluated by means of scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), weight loss analysis, 3D confocal laser scanning microscopy (CLSM) and electrochemical test. Results show the corrosion mainly occurs on the aluminum side. The corrosion resistance of the Cu/Al decreases as the pH value increases. When pH≥12, the dissolution of the film layer is faster than the passivation process. The addition of Cl− ions reduces the corrosion resistance of the Cu/Al clad plates, which leads to pitting corrosion. The higher the concentration of Cl− ions, the more prone the pitting to occur. The addition of SO42− ions causes the denudation of the samples. The corrosion resistance of the Cu/Al is better in the alkaline solution containing NO3− ions than that in the solution containing Cl− ions or SO42− ions. When adding SO42−, NO3− and Cl− to the pure alkaline solution, the corrosion resistance of the Cu/Al clad plates decreases.

Regulating the Nb2C nanosheets with different degrees of oxidation in water lubricated sliding toward an excellent tribological performance

Novel two-dimensional (2D) Nb2C nanosheets were successfully prepared through a simple lultrasonic and magnetic stirring treatment from the original accordion-like powder. To further study their water-lubrication properties and deal with common oxidation problems, Nb2C nanosheets with different oxidation degrees were prepared and achieved long-term stability in deionized water. Scanning electron microscope (SEM), transmission electron microscope (TEM), scanning probe microscope (SPM), X-ray powder diffraction (XRD), Raman, and X-ray photoelectron spectrometer (XPS) experiments were utilized to characterize the structure, morphology, and dispersion of Nb2C nanosheets with different degrees of oxidation. The tribological behaviors of Nb2C with different degrees of oxidation as additives for water lubrication were characterized using a UMT-3 friction testing machine. The wear scars formed on the 316 steel surface were measured using three-dimensional (3D) laser scanning confocal microscopy. The tribological results showed that a moderately oxidized Nb2C nanosheet, which owned the composition of Nb2C/Nb2O5/C, displayed excellent tribological performance, with the friction coefficient (COF) decreasing by 90.3% and a decrease in the wear rate by 73.1% compared with pure water. Combining the TEM and Raman spectra, it was shown that Nb2O5 nanoparticles filled in the worn zone, and the layered Nb2C and C were adsorbed into the surface of the friction pair to form a protective lubricating film. This combined action resulted in an excellent lubricating performance.

Influence of Elemental Carbon (EC) Coating Covering nc-(Ti,Mo)C Particles on the Microstructure and Properties of Titanium Matrix Composites Prepared by Reactive Spark Plasma Sintering.

This paper describes the microstructure and properties of titanium-based composites obtained as a result of a reactive spark plasma sintering of a mixture of titanium and nanostructured (Ti,Mo)C-type carbide in a carbon shell. Composites with different ceramic addition mass percentage (10 and 20 wt %) were produced. Effect of content of elemental carbon covering nc-(Ti,Mo)C reinforcing phase particles on the microstructure, mechanical, tribological, and corrosion properties of the titanium-based composites was investigated. The microstructural evolution, mechanical properties, and tribological behavior of the Ti + (Ti,Mo)C/C composites were evaluated using X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), electron backscatter diffraction analysis (EBSD), X-ray photoelectron spectroscopy (XPS), 3D confocal laser scanning microscopy, nanoindentation, and ball-on-disk wear test. Moreover, corrosion resistance in a 3.5 wt % NaCl solution at RT were also investigated. It was found that the carbon content affected the tested properties. With the increase of carbon content from ca. 3 to 40 wt % in the (Ti,Mo)C/C reinforcing phase, an increase in the Young’s modulus, hardness, and fracture toughness of spark plasma sintered composites was observed. The results of abrasive and corrosive resistance tests were presented and compared with experimental data obtained for cp-Ti and Ti-6Al-4V alloy without the reinforcing phase. Moreover, it was found that an increase in the percentage of carbon increased the resistance to abrasive wear and to electrochemical corrosion of composites, measured by the relatively lower values of the friction coefficient and volume of wear and higher values of resistance polarization. This resistance results from the fact that a stable of TiO2 layer doped with MoO3 is formed on the surface of the composites. The results of experimental studies on the composites were compared with those obtained for cp-Ti and Ti-6Al-4V alloy without the reinforcing phase.

3D fluorescence confocal microscopy of InGaN/GaN multiple quantum well nanorods from a light absorption perspective.

A nanostructure of In0.18Ga0.82N/GaN multiple quantum well (MQW) nanorods (NRs) was fabricated using top-down etching with self-organized nickel (Ni) nanoparticles as masks on the wafer. The optical properties of In0.18Ga0.82N/GaN MQW NRs were discussed by experiment and theory from a light absorption perspective. Three-dimensional (3D) optical images of NRs were successfully obtained by confocal laser scanning microscopy (CLSM) for physical observation of the optical phenomenon of InGaN/GaN MQW NRs. Moreover, optical simulations were performed by COMSOL Multiphysics via the three-dimensional finite-element method to explore the influences of NR geometrical parameters on optical absorption. The simulated results demonstrate that the absorption of NRs is higher than that of the film due to the waveguide properties of NRs resulting from their higher refractive index than embedding medium and higher aspect ratio than bulk. In addition, an increase in the diameter results in a red-shift of the absorption peak position of In0.18Ga0.82N/GaN MQW NRs. The smaller pitch enhances the near-field coupling of the nanorods and broadens the absorption peak. These results clearly illustrate the optical properties of In0.18Ga0.82N/GaN MQW NRs from the perspective of 3D confocal laser scanning microscopy. This work is promising for the applications of III–V optoelectronic devices.

A novel type of amyloid‐beta plaques discovered in early‐onset AD patients

AbstractBackgroundThe clinical presentation of Alzheimer’s disease (AD), including age at onset, is remarkably heterogeneous. Besides clinical heterogeneity, we recognize distinct Aβ deposits in AD pathology. These deposits are called plaques when located in brain parenchyma or cerebral amyloid angiopathy (CAA) when located in cerebral vessels. Recently, we described a novel Aβ plaque‐type, referred to as ‘the coarse‐grained plaque’. In this study, we evaluate its clinical relevance and perform in‐depth morphological characterization.MethodThe coarse‐grained plaque’s presence was investigated in the frontal cortex of Aβ‐pathology‐positive cases (N = 74), including non‐demented controls (n = 15), early‐onset (EO)AD (n = 38), and late‐onset (LO)AD (n = 21), using a semi‐quantitative approach. Main plaque characteristics were compared to other well‐defined Aβ deposits; the classic cored plaque, the cotton wool plaque, and CAA. In‐depth characterization was done by studying the coarse‐grained plaque’s Aβ isoform (Aβ40, Aβ42, AβN3pE, and pSer8Aβ) composition, neuroinflammatory component, and vascular association using immunohistochemistry and 3D confocal laser scanning microscopy imaging.ResultThe plaque was only observed in cases with clinical symptoms, it was more prominent in EOAD (95%) than in LOAD (66%) and was associated with a homozygous ApoE4 status (Figure 1). The coarse‐grained plaque showed a multi‐cored Aβ morphology and was seen in the proximity of CAA‐affected sulci. Its predominant Aβ40 composition was distinct from other plaques, but showed similarities to that of CAA (Figure 2A). 3D imaging revealed a unique hollow Aβ40‐shell structure (Figure 2B) with microglia in Aβ‐devoid holes (Figure 3). Furthermore, the plaque’s immunoreactivity for the CAA‐Type 1 specific marker norrin suggests – although having a different morphology ‐ a close etiological relation with CAA.ConclusionThe coarse‐grained plaque is a clinically relevant Aβ deposit as it occurs only in clinical AD and is more frequent in EOAD. The plaque is strongly associated with neuroinflammatory and vascular changes. Differences in presence of coarse‐grained plaque among AD subtypes, contributes to the compiling evidence that also pathophysiology in AD is heterogeneous.

A novel type of amyloid‐beta plaques identified in early‐onset AD

AbstractBackgroundThe clinical presentation of Alzheimer’s disease (AD) including age at onset, is remarkably heterogeneous. In previous research, we showed that inflammation is differently distributed between typical and atypical AD (Boon et al., 2018). In addition, distinct Aβ deposits in AD pathology are recognized. These deposits are called plaques when located in brain parenchyma or cerebral amyloid angiopathy (CAA) when located in the cerebral vessels. Recently, we described a novel Aβ plaque‐type, referred to as ‘the coarse‐grained plaque’. In this study, we evaluate its clinical relevance and perform in‐depth morphological characterization.MethodThe coarse‐grained plaque’s presence was investigated in the frontal cortex of Aβ‐pathology‐positive cases (N=74), including non‐demented controls (n=15), early‐onset (EO)AD (n=38), and late‐onset (LO)AD (n=21), using a semi‐quantitative approach. Main plaque characteristics were compared to other well‐defined Aβ deposits; the classic cored plaque, the cotton wool plaque, and CAA. In‐depth characterization was done by studying the coarse‐grained plaque’s Aβ isoform (Aβ40, Aβ42, AβN3pE, and pSer8Aβ) composition, neuroinflammatory component, and vascular association using immunohistochemistry and 3D confocal laser scanning microscopy imaging.ResultThe plaque was only observed in cases with clinical symptoms, it was more prominent in EOAD (95%) than in LOAD (66%) and was associated with a homozygous ApoE4 status (Figure 1). The coarse‐grained plaque showed a multi‐cored Aβ morphology and was seen in the proximity of CAA‐affected sulci. Its predominant Aβ40 composition was distinct from other plaques, but showed similarities to that of CAA (Figure 2A). 3D imaging revealed a unique hollow Aβ40‐shell structure (Figure 2B) with microglia in Aβ‐devoid holes (Figure 3). Furthermore, the plaque’s immunoreactivity for the CAA‐Type 1 specific marker norrin suggests – although having a different morphology ‐ a close etiological relation with CAA.ConclusionThe coarse‐grained plaque is a clinically relevant Aβ deposit as it occurs only in clinical AD and is more frequent in early‐onset cases. The plaque is strongly associated with neuroinflammatory and vascular changes. Differences in presence of coarse‐grained plaque among AD subtypes, contributes to the compiling evidence that also pathophysiology in AD is heterogeneous.

3D Confocal Laser Scanning Microscopy for Quantification of the Phase Behaviour in Agarose-MCC co-gels in Comparison to the Rheological Blending-law Analysis

The need for a rapid and direct alternative to the rheology-based blending laws in quantifying phase behaviour in biopolymer composite gels is explored in this study. In doing so, the efficacy of confocal laser scanning microscopy (CLSM) paired with image analysis software – FIJI and Imaris - in quantifying phase volume was studied. That was carried out in a model system of agarose with varying concentrations of microcrystalline cellulose (MCC) in comparison to the rheological blending laws. Structural studies performed using SEM, FTIR, differential scanning calorimetry and dynamic oscillation in-shear unveiled a continuous, weak agarose network supporting the hard, rod-shaped MCC inclusions where the composite gel strength increased with higher ‘filler’ concentration. The phase volumes of MCC, estimated with the microscopic protocol, matched the predictions obtained from computerized modelling using the Lewis-Nielsen blending laws. Results highlight the suitability of the microscopic protocol in estimating the water partition and effective phase volumes in the agarose-MCC composite gel.

Enhanced Antibiofilm Effects of N2 Plasma-Treated Buffer Combined with Antimicrobial Hexapeptides Against Plant Pathogens.

Suppression of pathogenic bacterial growth to increase food and agricultural productivity is important. We previously developed novel hexapeptides (KCM12 and KCM21) with antimicrobial activities against various phytopathogenic bacteria and N2 plasma-treated buffer (NPB) as an alternative method for bacterial inactivation and as an antibiofilm agent of crops. Here, we developed an enhanced antibiofilm method based on antimicrobial hexapeptides with N2 plasma-treated buffer against plant pathogens. Our results demonstrated that hexapeptides effectively inhibited the growth of Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and the biofilm it formed. Potent biofilm formation-inhibiting effects of hexapeptides were observed at concentrations of above 20 µM, and samples treated with hexapeptide above 100 µM reduced the ability of the bacteria to produce biofilm by 80%. 3D confocal laser scanning microscopy imaging data revealed that the antimicrobial activity of hexapeptides was enough to affect the cells embedded inside the biofilm. Finally, combination treatment with NPB and antimicrobial hexapeptides increased the antibiofilm effect compared with the effect of single processing against multilayered plant pathogen biofilms. These findings show that the combination of hexapeptides and NPB can be potentially applied for improving crop production.

A-to-I RNA editing in bacteria increases pathogenicity and tolerance to oxidative stress.

Adenosine-to-inosine (A-to-I) RNA editing is an important posttranscriptional event in eukaryotes; however, many features remain largely unexplored in prokaryotes. This study focuses on a serine-to-proline recoding event (S128P) that originated in the mRNA of fliC, which encodes a flagellar filament protein; the editing event was observed in RNA-seq samples exposed to oxidative stress. Using Sanger sequencing, we show that the S128P editing event is induced by H2O2. To investigate the in vivo interaction between RNAs and TadA, which is the principal enzyme for A-to-I editing, genome-wide RNA immunoprecipitation–coupled high-throughput sequencing (iRIP-Seq) analysis was performed using HA-tagged TadA from Xanthomonas oryzae pv. oryzicola. We found that TadA can bind to the mRNA of fliC and the binding motif is identical to that previously reported by Bar-Yaacov and colleagues. This editing event increased motility and enhanced tolerance to oxidative stress due to changes in flagellar filament structure, which was modelled in 3D and measured by TEM. The change in filament structure due to the S128P mutant increased biofilm formation, which was measured by the 3D laser scanning confocal microscopy. RNA-seq revealed that a gene cluster that contributes to siderophore biosynthesis and Fe3+ uptake was upregulated in S128P compared with WT. Based on intracellular levels of reactive oxygen species and an oxidative stress survival assay, we found that this gene cluster can contribute to the reduction of the Fenton reaction and increases biofilm formation and bacterial virulence. This oxidative stress response was also confirmed in Pseudomonas putida. Overall, our work demonstrates that A-to-I RNA editing plays a role in bacterial pathogenicity and adaptation to oxidative stress.

Cx43 in Neural Progenitors Promotes Glioma Invasion in a 3D Culture System.

The environment that envelops the cancer cells intimately affects the malignancy of human cancers. In the case of glioma, an aggressive adult brain cancer, its high rate of recurrence after total resection is responsible for a poor prognosis. Connexin43 (Cx43) is a gap junction protein with a prominent presence in glioma-associated normal brain cells, specifically in the reactive astrocytes. We previously demonstrated that elimination of Cx43 in these astrocytes reduces glioma invasion in a syngeneic mouse model. To further our investigation in human glioma cells, we developed a scaffold-free 3D platform that takes into account both the tumor and its interaction with the surrounding tissue. Using cell-tracking dyes and 3D laser scanning confocal microscopy, we now report that the elimination of Cx43 protein in neural progenitor spheroids reduced the invasiveness of human brain tumor-initiating cells, confirming our earlier observation in an intact mouse brain. By investigating the glioma invasion in a defined multicellular system with a tumor boundary that mimics the intact brain environment, our findings strengthen Cx43 as a candidate target for glioma control.