Confocal Laser Scanning Microscopy Observation Research Articles

3D bioprinting of mature bacterial biofilms for antimicrobial resistance drug testing

The potential to bioprint and study 3D bacterial biofilm constructs could have great clinical significance at a time when antimicrobial resistance is rising to dangerously high levels worldwide. In this study, clinically relevant bacterial species including Escherichia coli, Staphylococcus aureus (MSSA), Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were 3D bioprinted using a double-crosslinked alginate bioink to form mature bacteria biofilms, characterized by confocal laser scanning microscopy (CLSM) and fluorescent staining. Solid and porous bacteria-laden constructs were reproducibly bioprinted with thicknesses ranging from 0.25 to 4 mm. We demonstrated 3D bioprinting of thicker biofilms (>4 mm) than found in currently available in vitro models. Bacterial viability was excellent in the bioprinted constructs, with CLSM observation of bacterial biofilm production and maturation possible for at least 28 d in culture. Importantly, we observed the complete five-step biofilm life cycle in vitro following 3D bioprinting for the first time, suggesting the formation of mature 3D bioprinted biofilms. Bacterial growth was faster in thinner, more porous constructs whilst constructs crosslinked with BaCl2 concentrations of above 10 mM had denser biofilm formation. 3D MRSA and MSSA biofilm constructs were found to show greater resistance to antimicrobials than corresponding two-dimensional (2D) cultures. Thicker 3D E. coli biofilms had greater resistance to tetracycline than thinner constructs over 7 d of treatment. Our methodology allowed for the precise 3D bioprinting of self-supporting 3D bacterial biofilm structures that developed biofilms during extended culture. 3D biofilm constructs containing bacterial biofilms produce a model with much greater clinical relevance compared to 2D culture models and we have demonstrated their use in antimicrobial testing.

Intestine-targeted delivery potency of O-carboxymethyl chitosan-coated layer-by-layer microcapsules: An in vitro and in vivo evaluation.

The intestine-targeted delivery performance of the gum Arabic (GA) - O-carboxymethyl chitosan (OCMC) microcapsules prepared by layer-by-layer (LbL) assembly and genipin crosslinking was evaluated by using an acid-susceptible compound omeprazole as the model. Confocal laser scanning microscope observation revealed that spherical microcapsules with the core-shell structure were successful fabricated. Genipin crosslinking did not affect the microencapsulation yield or drug load, but significantly decreased the particle size and positive charge of the microcapsules, and increased their stability against disintegration in the simulated gastric fluid. Pharmacokinetic analysis indicated that entrapment by GA - OCMC LbL assembly greatly improved the bioavailability of omeprazole and crosslinking by 0.1 mg/mL genipin led to the highest value of 8.76 relative to the control formulation. It was concluded that the GA - OCMC LbL microcapsules could be used for the oral delivery of nutraceuticals and its delivery performance could be tailored by varying the genipin crosslinking degree.

Variety-dependent responses of rice plants with differential cadmium accumulating capacity to cadmium telluride quantum dots (CdTe QDs): Cadmium uptake, antioxidative enzyme activity, and gene expression

The excess release of engineered nanomaterials into farmland poses a serious threat to food security. Although rice varieties exhibit substantial variation in cadmium accumulation, their responses to Cd-based nanoparticles are largely unknown. In this work, we investigated the accumulation of cadmium telluride quantum dots (CdTe QDs at 0.5, 1.0, 2.5, 5.0mg-Cd/L) in two rice varieties with different Cd accumulation capacity. It was found that 5.0mg-Cd/L of CdTe QDs had minor growth inhibition to the high-Cd-accumulating variety (T705) relative to the low-Cd-accumulating variety (X24) after 7-day exposure. The two rice varieties had comparable Cd content in roots; however, T705 exhibited higher Cd content in shoots than X24. Transmission electron and confocal laser scanning microscopic observations demonstrated that more CdTe QDs can be transported and accumulated from roots to shoots in T705. The activities and gene expression of antioxidative enzymes in leaves of T705 increased more significantly than those of X24. Our findings for the first time validated that Cd accumulation divergence exists in different rice varieties when they are exposed to Cd-based QDs, the genetic basis for which needs to be further examined.

Nuclear-targeted p53 and DOX co-delivery of chitosan derivatives for cancer therapy in vitro and in vivo

The nucleus is one of the most important cellular organelles. Chitosan-grafted poly-(N-3-carbobenzyloxy-lysine) (CCL) decorated with human immunodeficiency virus-1 transactivator of transcription (TAT) can co-deliver p53 and doxorubicin into the nucleus simultaneously, such that their antitumor functions are exerted. However, TAT-CCL has been shown to have an anti-tumor effect only in vitro; the effect in vivo was unsatisfactory. Here, a unique nucleus-targeted delivery system based on amidized TAT (aTAT)-CCL with aTAT functional on the surface was designed to achieve a highly efficient nucleus-targeting gene and drug delivery system for effective cancer cell elimination in vitro and in vivo. In this delivery system, TAT is amidized to inhibit its nonspecific interactions. Confocal laser scanning microscopy observations revealed that if aTAT-CCL was incubated in pH 5.0 acetate buffer solution for 24 h before use (named aTAT-CCL-HB), more aTAT-CCL-HB entered the nucleus compared with aTAT-CCL or CCL. aTAT-CCL-HB can also achieve high gene transfection and drug delivery efficiencies and low viability in HepG2 cells. However, only aTAT-CCL achieved extensive circulation in the blood compartment and high antitumor activity in vivo. Amidization of TAT in vectors may become a promising strategy for nucleus-targeted delivery systems, especially in in vivo applications.

Amphiphilic Janus particles for efficient dispersion of oil contaminants in seawater

Application of dispersants is one of the widely used oil spill response strategies. With the growing concern for particulate emulsifiers, applying amphiphilic Janus particles to emulsify oil in seawater has been examined in this paper. Amphiphilic Janus particles with a combination of colloidal particles and amphiphilic characteristic are found to show a highly effective emulsification for various oils in seawater and overcome several drawbacks of conventional chemical dispersants. We report the synthesis of amphiphilic Janus silica particles (JSPs) as a novel oil spill dispersant and confirm their amphiphilicity by various techniques. The oil-in-seawater emulsion stabilized by JSPs presents small and stable oil droplets in one month, even at a higher volume ratio of oil to seawater and a wide temperature range. It is believed that the small size and high stability of droplets are very important to oil dispersion and eventual biodegradation. Through confocal laser scanning microscopy and scanning electron microscope observations, JSPs were found to anchor tightly at droplet surface and form a dense interfacial particle film to prevent droplets coalescence. The appropriate wettability of JSPs and the reduced interfacial tension contribute to the high interfacial adsorption energy of JSPs. Additionally, JSPs show weak inhibitory effect on oil-degrading bacteria, which is confirmed by bacterial growth curve, resazurin test and oil biodegradation experiment. This study reveals that amphiphilic Janus silica particles hold an impressive capability for environmentally friendly treatment of oil spills.

Swept-source optical coherence tomographic observation on prevalence and variations of cemento-enamel junction morphology.

To investigate the prevalence of different patterns of cemento-enamel junction (CEJ) morphology under swept-source optical coherence tomography (SS-OCT). One hundred extracted human teeth were used consisting of incisors, premolars, and molars. Each sample was observed for every 500μm circumferentially along CEJ and OCT images of the pattern were noted. Microscopic observations were done for the representative sample using confocal laser scanning microscope (CLSM) and transmission electron microscope (TEM). The OCT images exhibited four CEJ patterns: edge-to-edge (type I), exposed dentin (type II), cementum overlapping enamel (type III), and enamel overlapping cementum (type IV). The prevalence of CEJ patterns was further statistically considered for mesial, distal, buccal, and lingual surfaces. The real-time imaging by SS-OCT instantly determined CEJ morphology. CLSM and TEM observation revealed morphological features along CEJ, which corresponded to OCT images of CEJ anatomy. OCT results showed 56.8% of type I pattern predominantly found on proximal surfaces, followed by 36.5% of type II pattern on buccal and lingual surface, 6.4% of type III pattern, and 0.3% of type IV pattern. There was a significant difference in prevalence of CEJ patterns among different types of teeth, but there was no statistically significant difference among the four surfaces in each type of teeth. OCT is a non-invasive diagnostic tool to examine the CEJ patterns along the entire circumference. OCT observation revealed even minor dentin exposure that would need clinical and home procedures to prevent any symptoms.

Effect of kansui addition on dough rheology and quality characteristics of chickpea-wheat composite flour-based noodles and the underlying mechanism

The impact of kansui on the dough rheology and quality characteristics of chickpea-wheat composite flour-based noodles was investigated. For noodle dough, both the relative crystallinity and degree of order obtained from X-ray diffraction and Fourier transform infrared spectroscopy increased with the low level of kansui addition (0.5–1.5%), followed by a decreased trend at higher levels of alkali concentration (2.0–3.0%). Such variation was in accordance with the trend observed for G′ and G″. Based on the confocal laser scanning microscopic observation for cooked noodle samples, the low concentration of kansui addition (0.5–1.5%) protected the well-embedded starch granules being disassociate from the gluten network, whereas with the increased alkali addition (2.0–3.0%), the protein network was impaired with a greater amount of ruptured starch granules being disassociated from the gluten network. The observation corresponded well with the electrophoretic profile, and the results of cooking properties, -SH content, and textural properties of cooked noodles.

Rheological and structural characterization of acidified skim milks and infant formulae made from cow and goat milk

In this study, the viscoelastic properties of goat and cow skim milks and infant formula gels induced by acidification using Glucono-δ-Lactone (GDL) were investigated by small and large deformation rheology. The microstructures of the gels were characterized via the combination of confocal laser scanning microscopy (CLSM), ultra-small angle neutron scattering (USANS), small angle neutron scattering (SANS), and small angle X-ray scattering (SAXS). Compared to cow infant formula gels, goat infant formula gels displayed a lower elasticity and breaking stress. Similar differences were also observed in the gels of skim goat milk and skim cow milk. Moreover, for an infant formula from the same animal species, the one with a higher casein to whey ratio exhibited a stronger mechanical gel network. CLSM observations and USANS-SANS analysis indicated that thinner protein strands and more open protein networks with higher porosity were formed in the goat milk gels than in the cow milk gels, confirming the rheological findings. Finally, SAXS revealed that the internal structure of casein micelles from goat and cow milk are different and that as expected the calcium phosphate nanoclusters (CCP) are solubilized upon acidification. The observed differences in mechanical and microstructural properties may impact the digestive behavior, as reported in previous studies.

Shear Stress Affects Biofilm Structure and Consequently Current Generation of Bioanode in Microbial Electrochemical Systems (MESs).

Shear stress is an important factor that affects the formation and structure of anode biofilms, which are strongly related to the extracellular electron transfer phenomena and bioelectric performance of bioanodes. Here, we show that using nitrogen sparging to induce shear stress during anode biofilm formation increases the linear sweep voltammetry peak current density of the mature anode biofilm from 2.37 ± 0.15 to 4.05 ± 0.25 A/m2. Electrochemical impedance spectroscopy results revealed that the shear-stress-enriched anode biofilm had a low charge transfer resistance of 46.34 Ω compared to that of the unperturbed enriched anode biofilm (72.2 Ω). Confocal laser scanning microscopy observations showed that the shear-stress-enriched biofilms were entirely viable, whereas the unperturbed enriched anode biofilm consisted of a live outer layer covering a dead inner-core layer. Based on biomass and community analyses, the shear-stress-enriched biofilm had four times the biofilm density (136.0 vs. 27.50 μg DNA/cm3) and twice the relative abundance of Geobacteraceae (over 80 vs. 40%) in comparison with those of the unperturbed enriched anode biofilm. These results show that applying high shear stress during anode biofilm enrichment can result in an entirely viable and dense biofilm with a high relative abundance of exoelectrogens and, consequently, better performance.

In-plane and out-of-plane deformation at the sub-grain scale in polycrystalline materials assessed by confocal microscopy

High-resolution digital image correlation (HR-DIC) techniques have become essential in material mechanics to assess strain measurements at the scale of the elementary mechanisms responsible of the deformation in polycrystalline materials. The purpose of this study is to demonstrate the use of laser scanning confocal microscopy (LSCM) coupled with DIC techniques to deepen knowledge on the deformation process of a polycrystalline nickel-based superalloy at room temperature. The LSCM technique is capable of detecting both in-plane and out-of-plane strain localization within slip bands at the sub-grain level. The LSCM observations are consistent with previous in-situ scanning electron microscopy (SEM) studies: The onset of crystal plasticity occurs primarily near Σ3 twin boundaries with macroscopic loading in the elastic domain (macroscopic stress as low as 80% of the 0.2% offset yield strength (Y.S.0.2%)). This intense irreversible strain localization occurs with either a high Schmid factor (μ > 0.43) or a significant elastic modulus difference between the pair of twins (ΔΕ > 100 GPa). In the plastic deformation domain, transgranular slip activity following slip systems with the highest Schmid factor is mostly responsible for the deformation at the grain level, thus leading to strain percolation. The simultaneous in-plane and out-of-plane deformation assessment via the HR-LSCM-DIC technique was found to be essential for the identification of active slip systems. Finally, the HR-LSCM-DIC technique enabled the quantification of the glide amplitude involved in the three-dimensional shearing process at the grain level that solely in-plane measurements cannot provide.

Fabrication of dual-sensitive poly(β-hydroxyl amine) micelles for controlled drug delivery

Development of multi-responsive biodegradable micellar drug delivery systems for targeted cancer chemotherapy remains a major challenge. Here we demonstrate a kind of pH and reduction dual-responsive cationic polymer via an effective amine–epoxy polymerization, which could self-assemble into the biodegradable micelles in an aqueous solution. On account of the tumor cells or tissues possessing low pH values and high concentrations of reductive agents, these micelles exhibited the specific tumor target ability and maximal drug-release controllability inside tumor cells upon changes in physical and chemical environments, but presented excellent stability at physiological conditions. CCK-8 assay showed that these DOX-loaded micelles had a similar cytotoxicity for MCF-7 tumor cells as the free DOX molecules while blank micelles were low toxic to the cells. Confocal laser scanning microscopy (CLSM) observation revealed that the drug-loaded micelles were quickly internalized by endosomes to effectively restrain the cancer cell growth. These results indicated these biodegradable micelles could be the novel and efficient dual-responsive nanocarriers to improve the drug delivery and enhance the antitumor efficacy.

Three-dimensional assessment of proximal contact enamel using optical coherence tomography

ObjectiveThe purpose of this study was to detect and investigate the association of enamel microcracks with demineralization at proximal contact areas of premolars, using 3D swept-source optical coherence tomography (SS-OCT). MethodsExtracted maxillary and mandibular premolars (n=50 each), without any visible tooth cracks, were examined for demineralization of interproximal contact areas, using the International Caries Detection and Assessment System (ICDAS). SS-OCT was used to evaluate demineralization and detect microcracks. Demineralization progression was divided into 4 types by depth: 0 for sound enamel and Type I, II, and III for enamel demineralization penetrating into the outer third, the middle third, and the inner third of the enamel thickness, respectively. Enamel microcracks were classified according to the predominant crack pattern: Type O, no cracks; Type A, subsurface microdefects; Type B, enamel microcrack running along the direction of the enamel prism; Type C, enamel microcrack running transverse to the enamel prism direction; and Type D, a combination of patterns A, B, and C. SS-OCT findings were confirmed by confocal laser scanning microscopy observation. ResultsMicrocracks confined within the superficial enamel in proximal contact areas were seen as bright lines on SS-OCT. There were significant positive correlations among ICDAS codes, demineralization levels, and microcrack distribution (p≤0.001). The mesial side of maxillary premolars showed significantly more demineralization (n=36) and microcracks (n=27) than that of the mandibular premolars (n=20 and n=14, respectively; p<0.001). SignificanceThe presence of microcracks within the superficial enamel in proximal contact areas could be determined using SS-OCT, and correlated with the level of demineralization. 3D SS-OCT is a valuable diagnostic tool for comprehensive assessment of microstructural changes related to enamel demineralization and crack development.

Self-Assembly of siRNA/PEG- b-Catiomer at Integer Molar Ratio into 100 nm-Sized Vesicular Polyion Complexes (siRNAsomes) for RNAi and Codelivery of Cargo Macromolecules.

Vesicular polyion complexes (PICs) were fabricated through self-assembly of rigid cylindrical molecules, small interfering RNAs (siRNAs), with flexible block catiomers of poly(ethylene glycol) (2 kDa) and cationic polyaspartamide derivative (70 units) bearing a 5-aminopentyl side chain. 100 nm-sized siRNA-assembled vesicular PICs, termed siRNAsomes, were fabricated in specific mixing ranges between siRNA and block catiomer. The siRNAsome membrane was revealed to consist of PIC units fulfilling a simple molar ratio (1:2 or 2:3) of block catiomer and siRNA. These ratios correspond to the minimal integer molar ratio to maximally compensate the charge imbalance of PIC, because the numbers of charges per block catiomer and siRNA are +70 and -40, respectively. Accordingly, the ζ-potentials of siRNAsomes prepared at 1:2 and 2:3 were negative and positive, respectively. Cross-section transmission electron microscopic observation clarified that the membrane thicknesses of 1:2 and 2:3 siRNAsomes were 11.0 and 17.2 nm, respectively. Considering that a calculated long-axial length of siRNA is 5.9 nm, these thickness values correspond to the membrane models of two (11.8 nm) and three (17.7 nm) tandemly aligned siRNAs associating with one and two block catiomers, respectively. For biological application, siRNAsomes were stabilized through membrane-cross-linking with glutaraldehyde. The positively charged and cross-linked siRNAsome facilitated siRNA internalization into cultured cancer cells, eliciting significant gene silencing with negligible cytotoxicity. The siRNAsome stably encapsulated dextran as a model cargo macromolecule in the cavity by simple vortex mixing. Confocal laser scanning microscopic observation displayed that both of the payloads were internalized together into cultured cells. These results demonstrate the potential of siRNAsomes as a versatile platform for codelivery of siRNA with other cargo macromolecules.

Fabrication of the polyphosphates patched cellulose sulfate-chitosan hydrochloride microcapsules and as vehicles for sustained drug release

Polyphosphates are important polyanionic electrolytes that play a major role in stabilization and consolidation of colloids surface and interior microstructures. In this study, the polyelectrolyte complexes (PEC) microcapsules (sodium cellulose sulfate-chitosan hydrochloride, sample 1), and the patched ones via sodium tripolyphosphate (sample 2), sodium pyrophosphate (sample 3) and sodium hexametaphosphate (sample 4) were fabricated under mild conditions. The effects of polyphosphates on the formation of the PEC microcapsules were investigated systematically. Scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM) observation showed that both of the sample 2 and sample 3 had more compact interior microstructures with higher fluorescence intensity, compared with the sample 4 with macroporous ones and sample 1 with irregular ones. Fourier transform-infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA) showed the electrostatic interactions occurred among the –NH3+ groups, –SO3− groups, HP3O104− groups, P2O74− groups and H2PO4− groups, and the sample 2 and sample 3 had a more thermal stability comparatively. The sample microcapsules showed good capacity of drug loading and encapsulation efficiency (max. 66.9 ± 4.6% and 74.2 ± 5.1%). In the in vitro release studies showed that the sample 2 and sample 3 had a larger accumulative drug release rate of 5-aminosalicylic acid (5-ASA) at the same time point and released completely at 12 h; the drug release mechanisms analysis indicated that the sample 1 and sample 3 were mainly diffusion controlled, while the sample 2 and sample 4 were followed the mechanism of non-Fickian transport. Under the polyphosphate’s consolidation, the PEC microcapsules fabricated with sustained drug release profiles could be used as the promising drug vehicles.

Designing heparan sulfate-based biocompatible polymers and their application for intracellular stimuli-sensitive drug delivery

Heparan sulfate (HS) is a kind of natural polysaccharides with good biocompatibility. And as drug carriers, it has some advantages compared to heparin. However, the preparation of HS is cumbersome and difficult, which limits its application in drug delivery. Here, we use modern separation technique combined with chromatography to establish a new preparation method of HS. The molecular weight and degree of dispersion of HS were (1.03 × 104 ± 107) kDa and 1.106, respectively. HS also showed low anticoagulation activity in comparison with heparin. Subsequently, novel redox-sensitive heparan sulfate-cystamine-vitamin E succinate (HS-SS-VES, HSV) micelles were designed to increase tumor selectivity and improve the therapeutic effect of doxorubicin (DOX). DOX-loaded HSV micelles (DOX/HSV) with spherical morphology had average particle size of 90–120 nm and good redox-triggered release behavior. The cell viabilities of blank micelles were >90% in both human breast cancer (MCF7) cells and African green monkey SV40-transformed kidney fibroblast (COS7) cells. However, the cytotoxicity of DOX/HSV in MCF7 cells was higher than that of COS7 cells. Flow cytometry analyses and confocal laser scanning microscopy observation indicated that DOX/HSV micelles were internalized by endocytosis, and then the drug was released quickly and entered the nuclei of tumor cells. The results demonstrate that high-purity HS can be prepared and has the potential to be further used for drug delivery in antitumor applications.

Comparison of ethyl cellulose–gelatin composite films fabricated by electrospinning versus solvent casting

ABSTRACTIn this study, we fabricated ethyl cellulose (EC)–gelatin (G) composite films by solvent casting and electrospinning. The characterization of the cast and electrospun films was performed by scanning electron microscopy, differential scanning calorimetry, thermogravimetric analysis, X‐ray diffraction, Fourier transform infrared spectroscopy, X‐ray photoelectron spectrometry, and water contact angle measurements. The results showed that the blending of EC with G improved the spinnability of the composite films with higher thermal stability. Compared with solvent casting, electrospinning induced a helix‐to‐coil transition of G and provided more hydrogen‐forming sites for EC to form a compatible network. Interestingly, the electrospun composite film had a hydrophobic surface with a water contact angle of 113.8°, whereas the cast composite film showed a hydrophilic surface at 67.5°. The confocal laser scanning microscopy observations suggested that after water immersion, the cast composite film collapsed into aggregates, whereas the electrospun composite film maintained the nanofibrous structures well; this indicated an improved water resistance. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 135, 46824.

In Situ Observation of the Precipitation, Aggregation, and Dissolution Behaviors of TiN Inclusion on the Surface of Liquid GCr15 Bearing Steel

In this study, the precipitation, aggregation, and dissolution behaviors of TiN inclusions on the surface of liquid GCr15 bearing steel have been investigated by combining the observations of confocal laser scanning microscope (CLSM) and field emission scanning electron microscope (FE-SEM) with those obtained from energy dispersive spectrometer (EDS) and theoretical analysis. The kinetic results show that the initial concentration of Ti and N are 0.0078 and 0.0049, respectively, the precipitation temperature is between 1640 K and 1680 K (1367 °C and 1407 °C), and the local cooling rate is between 0.5 and 10 K/s; TiN inclusion can precipitate only when the solid fraction is higher than 0.847 and its precipitation radius is between 1 and 6 μm. The precipitation radius of a TiN inclusion in the GCr15 bearing steel sheet can be reduced by decreasing the N content and increasing the cooling strength. The aggregation and densification of multi-particle aggregated TiN inclusions are verified by CLSM observation and theoretical analysis. The inclusions are aggregated by the cavity bridge force (CBF), and the aggregated TiN is formed by solid-phase sintering. The results of force analysis show that CBF plays a dominant role in the aggregation process of the inclusions. The atomic ratio of Ti and V obtained by EDS is 18:1, which may melt TiN and form the liquid inclusion at 1688 K (1415 °C) observed by CLSM. The theoretical analysis is conducted for the dissolution of the TiN inclusions observed by CLSM, which shows that the dissolution of the TiN inclusions is related to the size of the inclusions; the larger the size, the greater the dissolution rate. The long-strip TiN inclusion may be formed by the Ostwald ripening of two TiN inclusions. The TiN inclusions smaller than 3 μm in the GCr15 bearing steel may be formed by the dissolved Ti and N generated by the dissolution of TiN.

Exploring plant-microbe interactions of the rhizobacteria Bacillus subtilis and Bacillus mycoides by use of the CRISPR-Cas9 system.

Bacillus subtilis HS3 and Bacillus mycoides EC18 are two rhizosphere-associated bacteria with plant growth-promoting activity. The CRISPR-Cas9 system was implemented to study various aspects of plant-microbe interaction mechanisms of these two environmental isolates. The results show that fengycin and surfactin are involved in the antifungal activity of B. subtilis HS3. Moreover, this strain emits several other volatile organic compounds than 2,3-butanediol, contributing to plant growth promotion. Confocal laser scanning microscopy observations of the GFP-labelled strain showed that HS3 selectively colonizes root hairs of grass (Lolium perenne) in a hydroponic system. For B. mycoides EC18, we found that the wild-type EC18 strain and a ΔasbA (petropectin-deficient) mutant, but not the ΔdhbB (bacillibactin-deficient) and ADKO (asbA and dhbB double knockout) mutants, can increase the plant biomass and total chlorophyll. All the mutant strains have a reduced colonization capability on Chinese cabbage (Brassica rapa) roots, at the root tip and root hair region compared with the wild-type strain. These results indicate that the siderophore, bacillibactin, is involved in the plant growth promoting activity and could affect the root colonization of B. mycoides. Collectively, the CRISPR-Cas9 system we developed for environmental isolates is broadly applicable and will facilitate deciphering the mechanisms of Bacillus-plant interactions. © 2018 The Authors.

In situ research of partial melt in as-cast H13 steel at elevated temperature

ABSTRACTIn situ experimental investigations of partial melt phenomenon in as-cast H13 steel at elevated temperatures between 1150°C and 1300°C was carried out using a confocal laser scanning microscope (CLSM) observation. Stripy primary carbides composed of Cr, Mo, V and Ti were observed in the interdendritic zone of as-cast H13 steel. In the interdendritic zone there existed obvious Cr and Mo segregation and slightly C and V segregation. At temperatures above 1150°C, the liquid phase was observed at the primary carbides-matrix boundary, even though this temperature is much lower than the solidus temperature of H13 steel. A higher heat temperature and longer holding time result in the generation of an increased amount of liquid phase. The maximum fraction of the liquid phase in the present investigation was 6%. Two effects are responsible for the premature appearance of the liquid phase, namely the alloying elements segregation in the interdendritic zone and the decomposition of primary carbides. The presence of the liquid phase accelerates alloying elements diffusion in the interdendritic zone, and cavities will be generated because of the shrinkage of the liquid phase during rapid cooling.

Effect of Icariin on Engineered 3D-Printed Porous Scaffolds for Cartilage Repair.

In recent times, cartilage defects have been the most common athletic injuries, often leading to dreadful consequences such as osteoarthritis, pain, joint deformities, and other symptoms. It is also evident that damage to articular cartilage is often difficult to recover or self-heal because of poor vascular, nervous, and lymphatic supplies. Moreover, cartilage cells have poor regeneration ability and high maturity. Inspired by these facts and the rapid advances in the field of tissue engineering (TE), we fabricated highly porous three-dimensional (3D) scaffold architectures based on cell-responsive polymeric inks, i.e., sodium alginate and gelatin (SA-Gel, 1:3 ratio), by a novel 3D printing method. Moreover, the effect of various processing parameters was systematically investigated. The printed scaffolds of polymer composites gels with excellent transparency, moderate viscosity, and excellent fluid properties showed good surface morphology, better thermal stability and swelling effect, and unique interconnected porous architectures at the optimized operating parameters. In vitro cell proliferation experiments of these cytocompatible scaffolds showed the excellent adhesion rate and growth behavior of chondrocytes. In addition, the porous architectures facilitated the efficient distribution of cells with only a few remaining on the surface, which was confirmed by confocal laser scanning microscopic (CLSM) observations. Icariin (ICA) addition at a concentration of 10 μg/mL further significantly enhanced the proliferation of chondrocytes. We envision that these cell-responsive polymeric inks in the presence of growth regulators like ICA may have potential in engineering complex tissue constructs toward diverse applications in TE.