Confocal Scanning Microscopy Research Articles

Tribological Behavior of Ni-Based Coatings Deposited via Spray and Fuse Technique

The tribological behavior of Ni-based coatings was analyzed. The coatings were deposited on grey cast iron substrates in a spray and fuse process using Superjet Eutalloy deposition equipment, varying the oxygen flow conditions in the flame. By means of the X-ray diffraction (XRD) technique, the crystal structure of the coatings was determined. The XRD patterns show the crystalline phases with principal reflections for Ni in the planes (111) and (222). Crystalline properties such as the orientation coefficient, crystallite size, and macrostrain showed the relationship with tribological and mechanical properties such as the dry wear rate and the microhardness. The microhardness was analyzed on the surface and on cross sections of the coatings by means of a Knoop microhardness tester. The topography and the morphological characteristics of the coatings and the tribo-surfaces were exanimated using scanning electron microscopy (SEM) and confocal microscopy, while the chemical composition was measured by means of energy-dispersive X-ray spectroscopy (EDS). The tribological behavior of the coatings was examined via the scratch cohesion–adhesion test, using cross sections of the coatings. Furthermore, adhesion and abrasion wear tests were carried out, using the pin-on-disk method, under the ASTM G99 standard and the ASTM G65 standard, respectively. The wear rate of the coatings showed a strong relation to the porosity in the metal matrix, which was previously determined via electrochemical characterization techniques.

Microbial Community Characterizing Vermiculations from Karst Caves and Its Role in Their Formation

The microbiota associated with vermiculations from karst caves is largely unknown. Vermiculations are enigmatic deposits forming worm-like patterns on cave walls all over the world. They represent a precious focus for geomicrobiological studies aimed at exploring both the microbial life of these ecosystems and the vermiculation genesis. This study comprises the first approach on the microbial communities thriving in Pertosa-Auletta Cave (southern Italy) vermiculations by next-generation sequencing. The most abundant phylum in vermiculations was Proteobacteria, followed by Acidobacteria > Actinobacteria > Nitrospirae > Firmicutes > Planctomycetes > Chloroflexi > Gemmatimonadetes > Bacteroidetes > Latescibacteria. Numerous less-represented taxonomic groups (< 1%), as well as unclassified ones, were also detected. From an ecological point of view, all the groups co-participate in the biogeochemical cycles in these underground environments, mediating oxidation-reduction reactions, promoting host rock dissolution and secondary mineral precipitation, and enriching the matrix in organic matter. Confocal laser scanning microscopy and field emission scanning electron microscopy brought evidence of a strong interaction between the biotic community and the abiotic matrix, supporting the role of microbial communities in the formation process of vermiculations.

A Peptide Found in Human Serum, Derived from the C-Terminus of Albumin, Shows Antifungal Activity In Vitro and In Vivo.

The growing problem of antimicrobial resistance highlights the need for alternative strategies to combat infections. From this perspective, there is a considerable interest in natural molecules obtained from different sources, which are shown to be active against microorganisms, either alone or in association with conventional drugs. In this paper, peptides with the same sequence of fragments, found in human serum, derived from physiological proteins, were evaluated for their antifungal activity. A 13-residue peptide, representing the 597–609 fragment within the albumin C-terminus, was proved to exert a fungicidal activity in vitro against pathogenic yeasts and a therapeutic effect in vivo in the experimental model of candidal infection in Galleria mellonella. Studies by confocal microscopy and transmission and scanning electron microscopy demonstrated that the peptide penetrates and accumulates in Candida albicans cells, causing gross morphological alterations in cellular structure. These findings add albumin to the group of proteins, which already includes hemoglobin and antibodies, that could give rise to cryptic antimicrobial fragments, and could suggest their role in anti-infective homeostasis. The study of bioactive fragments from serum proteins could open interesting perspectives for the development of new antimicrobial molecules derived by natural sources.

Variation in tumor pH affects pH-triggered delivery of peptide-modified magnetic nanoparticles

Acidification of the extracellular matrix, an intrinsic characteristic of many solid tumors, is widely exploited for physiologically triggered delivery of contrast agents, drugs, and nanoparticles to tumor. However, pH of tumor microenvironment shows intra- and inter-tumor variation. Herein, we investigate the impact of this variation on pH-triggered delivery of magnetic nanoparticles (MNPs) modified with pH-(low)-insertion peptide (pHLIP). Fluorescent flow cytometry, laser confocal scanning microscopy and transmission electron microscopy data proved that pHLIP-conjugated MNPs interacted with 4T1 cells in two-dimensional culture and in spheroids more effectively at pH 6.4 than at pH 7.2, and entered the cell via clathrin-independent endocytosis. The accumulation efficiency of pHLIP-conjugated MNPs in 4T1 tumors after their intravenous injection, monitored in vivo by magnetic resonance imaging, showed variation. Analysis of the tumor pH profiles recorded with implementation of original nanoprobe pH sensor, revealed obvious correlation between pH measured in the tumor with the amount of accumulated MNPs.

Toward Brain-on-a-Chip: Human Induced Pluripotent Stem Cell-Derived Guided Neuronal Networks in Tailor-Made 3D Nanoprinted Microscaffolds.

Brain-on-a-chip (BoC) concepts should consider three-dimensional (3D) scaffolds to mimic the 3D nature of the human brain not accessible by conventional planar cell culturing. Furthermore, the essential key to adequately address drug development for human pathophysiological diseases of the nervous system, such as Parkinson's or Alzheimer's, is to employ human induced pluripotent stem cell (iPSC)-derived neurons instead of neurons from animal models. To address both issues, we present electrophysiologically mature human iPSC-derived neurons cultured in BoC applicable microscaffolds prepared by direct laser writing. 3D nanoprinted tailor-made elevated cavities interconnected by freestanding microchannels were used to create defined neuronal networks-as a proof of concept-with two-dimensional topology. The neuronal outgrowth in these nonplanar structures was investigated, among others, in terms of neurite length, size of continuous networks, and branching behavior using z-stacks prepared by confocal microscopy and cross-sectional scanning electron microscopy images prepared by focused ion beam milling. Functionality of the human iPSC-derived neurons was demonstrated with patch clamp measurements in both current- and voltage-clamp mode. Action potentials and spontaneous excitatory postsynaptic currents-fundamental prerequisites for proper network signaling-prove full integrity of these artificial neuronal networks. Considering the network formation occurring within only a few days and the versatile nature of direct laser writing to create even more complex scaffolds for 3D network topologies, we believe that our study offers additional approaches in human disease research to mimic the complex interconnectivity of the human brain in BoC studies.

From the roots to the stem: unveiling pear root colonization and infection pathways by Erwinia amylovora.

Fire blight caused by Erwinia amylovora affects pome fruit worldwide, generating serious economic losses. Despite the abundant literature on E. amylovora infection mechanisms of aerial plant organs, root infection routes remain virtually unexplored. Assessing these infection pathways is necessary for a full understanding of the pathogen's ecology. Using the pathosystem Pyrus communis-E. amylovora and different experimental approaches including a green fluorescent protein transformant (GFP1) and epifluorescence microscopy (EFM) and laser confocal scanning microscopy (LCSM), we demonstrated the pathogen's ability to infect, colonize and invade pear roots and cause characteristic fire blight symptoms both in the aerial part and in the root system. Plant infections after soil irrigation with E. amylovora-contaminated water were favored by root damage, which agreed with EFM and LCSM observations. E. amylovora GFP1 cells formed aggregates/biofilms on root surfaces and invaded the cortex through wounds and sites of lateral root emergence. Sugars, sugar-alcohols and amino acids typically secreted by roots, favored the in vitro biofilm development by E. amylovora. Migration of E. amylovora cells to aerial tissues mainly occurred after xylem penetration. Overall, our findings revealed, for the first time, common root infection patterns between E. amylovora and well-known soil borne plant pathogens and endophytes.

Effect of bioactive glass pretreatment on the durability of dentin bonding interface

To study the effect of bioactive glass (BG) on the dentin bond strength and the microleakage of hybrid layer. In the study, 30 dentin planes were prepared from the third molars with no caries and equally assigned to the control group, BG group, and sodium trimetaphosphate (STMP)-polyacrylic acid (PAA)-BG group (S-P-BG group), randomly. After etched with 35% phosphoric acid, the dentin planes of BG group were pretreated with 0.5 g/L BG, and the dentin planes of S-P-BG group were pretreated with 5% STMP, 5% PAA and 0.5 g/L BG. No additional pretreatment was done to the dentin planes of control group. Then the dentin planes were bonded using 3M Single Bond 2 adhesive to 3M Z350XT composite resin, and cut into 0.9 mm×0.9 mm column samples, which were stored at 37 ℃ artificial saliva (AS). After 24 hours, 1 month, and 3 months, the microtensile bond strength test was performed. The data were analyzed using one-way ANOVA and LSD method. The morphology of the bond fracture interface was observed with scanning electron microscope. Other 27 teeth were collected and the enamel layer and roots cut off, with the pulp chamber exposed. 0.1% rhodamine B was added to the 3M Single Bond 2 adhesive, and then the adhesive was applied to complete the bonding procedures as above. The teeth were stored in 37 ℃ AS for 24 hours, 1 month, 3 months, and then 0.1% sodium fluorescein solution was placed in the chambers and stained for 1 hour. Confocal laser scanning microscopy was used to observe the interface morphology and microleakage of the hybrid layer. At the end of 24 hours and 1 month, there was no significant difference in the microtensile bond strength among the three groups (P>0.05). After 3 months of soaking, the S-P-BG group [(36.91±7.07) MPa] had significantly higher microtensile bond strength than the control group [(32.73±8.06) MPa] (P=0.026); For the control group and the BG group, the microtensile bond strength significantly decreased at the end of 3 months compared with 24 hours (control group: P=0.017, BG group: P=0.01); The microtensile bond strength of S-P-BG group af the end of 3 months had no significant difference in compared with 24 hours [(37.99±7.98) MPa] (P>0.05). Observation of the fracture surface at the 24 hours showed no obvious mineralization in all the three groups. After 1 and 3 months, mineral formation was observed in BG group and S-P-BG group, and no obvious collagen exposure was observed in S-P-BG group. Confocal laser scanning microscopy revealed no obvious differences in the morphology and quantity of the resin tag in the control group, BG group and S-P-BG group. At the end of 24 hours, leakage was found in all the three groups. The microleakage of the control group increased at the end of 3 months, while the microleakage of the BG and S-P-BG groups decreased. BG pretreatment of dentin bonding interface can induce mineralization at the bonding interface and reduce the microleakage of the hybrid layer; pretreating the dentin bonding interface with STMP, PAA and BG may enhance the maintaining of the dentin bonding durability.

Comparative cytotoxic and genotoxic potential of benzyl-butyl phthalate and di-n-butyl phthalate using Allium cepa assay

The colossal applications of phthalates make them ubiquitous. Several studies reported toxicity and ecological effects of phthalates. However, toxicological studies using Allium cepa assay are not well explored. Therefore, the current study envisages the morphological toxicity, cytotoxicity, and genotoxicity of two phthalates using Allium cepa assay. To study the effects of phthalates on roots length, the plant was subjected to different concentrations of benzyl-butyl phthalate (BBP) and di-n-butyl phthalate (DBP) for 24 and 120 h. After 120 h treatment, the effect was > 50%; therefore, for further investigations, the treatment durations were set at 24 and 48 h. The roots length was significantly declined with increase in phthalates concentrations. From microscopic studies, it was observed that phthalates significantly decreased mitotic index (MI) after 24 and 48 h exposure. After 24 h exposure, the decline in MI was maximum with BBP treatment, while after 48 h, it was maximum with DBP treatment. On the other hand, a significant increase in percent chromosomal anomalies was induced by phthalates and the maximum increase was with DBP treatment under both exposure durations. The changes in root surface morphology were observed using scanning electron microscope. The loss in cell viability was also observed in treated and untreated root samples using laser confocal scanning microscopy. UHPLC was used to analyze the accumulation of phthalates in the roots. It can be concluded that phthalates cause deleterious effects on A. cepa and extent depends on their concentrations and treatment durations.

Reconstruction of the ocular surface by autologous transplantation of rabbit adipose tissue-derived stem cells on amniotic membrane.

BackgroundCorneal disease is the second most common cause of blindness in China. Clinically, treatment options for corneal diseases with limbal stem cell deficiency (LSCD) are limited due to a shortage of organ donors and inevitable immune rejection. This study aims to determine the efficacy of reconstructing the ocular surface using autologous cultivated adipose tissue-derived stem cells (ADSCs) and to develop a new clinical treatment for patients with LSCD.MethodsA rabbit LSCD model was first established. Two weeks later, the animals were divided into three groups, including the sham group, the amniotic membrane transplantation group, and the ADSC combined with amniotic membrane transplantation group, and underwent surgery. The efficacy of reconstructing the ocular surface using ADSCs was evaluated using immunofluorescent staining, confocal microscopy (CM) observation, H&E staining, immunohistochemical staining, and scanning transmission electron microscopy observation one, two and four weeks after surgery.ResultsEvaluations of immunofluorescent staining of the cornea pre- and post-surgery yielded significantly lower scores for the corneas in the ADSCs transplantation group than for those in the sham group (F=−7, P=0.002, <0.05) and the amniotic membrane transplantation group (F=−4.67, P=0.018, <0.05) two weeks after surgery. Four weeks after surgery, the corneas of the ADSC combined with amniotic membrane transplantation group were scored significantly lower than those in the sham group (F=−8, P=0.007, <0.05) and the amniotic membrane transplantation group (F=−5.33, P=0.046, <0.05). The data suggest that the use of ADSCs to treat LSCD showed greater efficacy than the other treatment methods. The growth of ADSCs on the corneal surface was examined using confocal and electron microscopes. K3/K12 expression in the corneal epithelium, which was reconstructed by ADSCs, was negative, as shown by immunohistochemical staining.ConclusionsOcular surface reconstruction can be improved by using ADSCs as seed cells and the amniotic membrane as a carrier, thus providing a new therapeutic strategy for patients with LSCD.

Micromorphological analysis and bond strength comparison of two adhesives for different degrees of dental fluorosis

The objective of this study was to analyze morphologically the all-etching bonding system and self-etching bonding system for enamel with different degrees of fluorosis and evaluate the bond strength of each system. Teeth that were indicated for extraction owing to orthodontic or periodontal problems were selected. According to Dean’s index and the Thylstrup-Fejerskov index, 180 extracted teeth were divided into three groups of mild, moderate, and severe dental fluorosis (DF), with 60 teeth in each group. The teeth in each group were randomly divided into two subgroups (n = 30), which were then subjected to the all-etching bonding system (Prime & Bond NT) and self-etching bonding system (SE-Bond). Each group of adhesives was used to bond Z350 universal resin (3 M) to the etched dental enamel. Tensile and shear tests were conducted to determine the bond strength. Subsequently, the fractured specimens were investigated using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The Prime & Bond NT was statistically significant for the tensile and shear strength of enamel with mild fluorosis (P < 0.05) but did not exhibit a significant difference for moderate and severe DF (P > 0.05). The SE-Bond was not statistically significant for the tensile and shear strength of mild, moderate, or severe DF (P > 0.05). The SEM and CLSM results reveal that the mild fluorosis enamel crystals were relatively dense, and a small amount of resin remained. The moderate fluorosis enamel crystals were loosely arranged, and the gaps were widened. The severe fluorosis enamel crystals were irregularly arranged. The disorder was aggravated, and the dentinal orifice was exposed by partial enamel exfoliation. The bonding strength of mild fluorosis enamel with the Prime & Bond NT was better than that with the SE-Bond, and cohesive failure was the most common mode of failure. Because there was no difference in the bonding strength of the SE-Bond for different degrees of DF, we recommend the use of the all-etching adhesive system in the clinical treatment of teeth with mild fluorosis.

Abstract 1727: Challenging resistance to temozolomide in glioblastoma by drug encapsulation in apoferritin

Abstract Background: Temozolomide (TMZ) is a DNA alkylating prodrug used for the treatment of malignant, glioblastoma multiforme (GBM) tumors. Yet, TMZ therapy is blighted by resistance systems in GBM that thwart eradication of all cancer cells remaining after surgery and radiotherapy. Resistance mechanisms include drug efflux by P-glycoprotein 1 (Pgp), present on the blood brain barrier (BBB) and on GBM cells, overexpression of O6-methylguanine DNA-methyltransferase (MGMT), which removes cytotoxic O6-methylated guanine (O6-MeG) lesions caused by TMZ, and deficiency in mismatch repair (MMR), which leads to tolerance of O6-MeG lesions. Nanoscale delivery systems are proposed as a strategy to enhance drug targeting and accumulation under systemically tolerable conditions. Apoferritin (AFt), a hollow protein capsule, has been used to deliver anticancer agents. Transferrin receptor 1 (TfR1) binding sites on the AFt cage has reportedly been shown to allow AFt to cross the BBB and accumulate in GBM cells, as both BBB endothelial and GBM (but not glial) cells express TfR1. Thus, we use AFt for targeted delivery, specific uptake and controlled release of TMZ in GBM. Methods: TMZ was encapsulated into AFt via molecular diffusion through the channels in the AFt cage. Encapsulation efficiency and stability for this formulation were assessed by dynamic light scattering (DLS) and UV-vis spectroscopy. In vitro assays were conducted (MTT, live cell count, clonogenic, flow cytometry and ELISA) against isogenic GBM cell lines, U373V (vector control) and U373M (MGMT overexpressing), as well as MMR deficient and Pgp overexpressing HCT116 (colorectal carcinoma) and healthy MRC-5 (lung fibroblasts) cells. Environmental scanning electron microscopy (ESEM) and confocal microscopy were further employed to assess morphological changes to the cells after treatment. Results: Encapsulation of &amp;gt; 500 molecules of TMZ per AFt cage was achieved, with encapsulation efficiency &amp;gt; 70%. MTT assays demonstrated significantly increased activity of AFt-TMZ compared to TMZ alone in all cancer cell lines, with 50% growth inhibition (GI50) in resistant cancer cell lines at &amp;lt; 15 μM, compared to &amp;gt; 200 μM for TMZ alone. Clonogenic and flow cytometric cell cycle and DNA damage analyses further corroborate these findings. Additionally, ELISA quantification demonstrated significantly (P &amp;lt; 0.001) elevated O6-MeG levels following treatment of U373V and U373M cells with AFt-TMZ compared to naked TMZ, correlating with enhanced γ-H2AX burden in cells following exposure to AFt-TMZ. Against MRC-5, lower activity was observed, with GI50 for AFt-TMZ &amp;gt; 70 μM. Furthermore, cell shrinkage and blebbing were observed in GBM cells following 24 h treatment with AFt-TMZ. Conclusions: TMZ delivered by AFt demonstrated enhanced potency. Overcoming resistance by enhancing TMZ intracellular accumulation, may prove to be a turning point in enhancing drug efficacy in GBM. Citation Format: Kaouthar Bouzinab, Neil R. Thomas, Lyudmila Turyanska, Pavel Gershkovich, Nicola Weston, Marianne B. Ashford, Tracey D. Bradshaw. Challenging resistance to temozolomide in glioblastoma by drug encapsulation in apoferritin [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1727.

Enhancement of cartilage extracellular matrix synthesis in Poly(PCL-TMC)urethane scaffolds: a study of oriented dynamic flow in bioreactor.

The development of new technologies to produce three-dimensional and biocompatible scaffolds associated with high-end cell culture techniques have shown to be promising for the regeneration of tissues and organs. Some biomedical devices, as meniscus prosthesis, require high flexibility and tenacity and such features are found in polyurethanes which represent a promising alternative. The Poly(PCL-TMC)urethane here presented, combines the mechanical properties of PCL with the elasticity attributed by TMC and presents great potential as a cellular carrier in cartilage repair. Scanning electron microscopy showed the presence of interconnected pores in the three-dimensional structure of the material. The scaffolds were submitted to proliferation and cell differentiation assays by culturing mesenchymal stem cells in bioreactor. The tests were performed in dynamic flow mode at the rate of 0.4mL/min. Laser scanningconfocal microscopy analysis showed that the flow rate promoted cell growth and cartilage ECM synthesis of aggrecan and type II collagen within the Poly(PCL-TMC)urethane scaffolds. This study demonstrated the applicability of the polymer as a cellular carrier in tissue engineering, as well as the ECM was incremented only when under oriented flow rate stimuli. Therefore, our results may also provide data on how oriented flow rate in dynamic bioreactors culture can influence cell activity towards cartilage ECM synthesis even when specific molecular stimuli are not present. This work addresses new perspectives for future clinical applications in cartilage tissue engineering when the molecular factors resources could be scarce for assorted reasons.

Lysosome Targeting Bis-terpyridine Ruthenium(II) Complexes: Photophysical Properties and In Vitro Photodynamic Therapy.

Three heteroleptic bis-terpyridine ruthenium(II) complexes (Ru1-Ru3) [Ru(tpy-R1)(tpy-R2)]2+ (tpy = 2,2':6',2″-terpyridine, R1/R2 = phenyl, 4-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}phenyl, pyren-1-yl, or 4-phenyl-BODIPY (boron dipyrromethene)) were synthesized and investigated for their potential applications as photosensitizers (PSs) for photodynamic therapy. All complexes displayed broad and intense absorption band in the green spectral regions (450-600 nm), which arose from the spin-allowed charge-transfer transitions mixed with ligand-localized 1π,π* transitions. All complexes show weak green emission at 513-549 nm and/or even weaker red emission at 646-674 nm at room temperature depending on the excitation wavelength and the solvent used. Incorporating the BODIPY motif to the 4'-position of one of the tpy ligands in Ru2 and Ru3 drastically prolonged the lifetimes of the lowest triplet excited states (T1) of Ru2 and Ru3 to tens of microseconds. This promoted the singlet oxygen formation sensitized by Ru2 and Ru3 upon green light activation, which in turn induced significant photocytotoxicity toward the A549 human lung cancer cell line with an EC50 value of 1.50 μM for Ru2 and 7.41 μM for Ru3 under 0.48 J·cm-2 500 nm light irradiation. Laser confocal scanning microscopy imaging revealed that Ru2 mainly distributed to lysosomes upon cell uptake. Upon 500 nm light activation, Ru2 induced lysosomal damage and subsequent mitochondrial membrane potential decrease. The dominant cell death pathway was apoptosis. These results demonstrated the potential utilization of [Ru(tpy-R1)(tpy-R2)]2+ complexes as PSs for PDT.

Biotite reactivity in nitric and oxalic acid at low temperature and acid pH from surface and bulk dissolution measurements

The dissolution of biotite, a trioctahedral mica, was investigated for a temperature range of 25–70 °C and a pH range of 1–3 in the presence of HNO3 (nitric acid) and H2C2O4 (oxalic acid) solutions. Single millimetric, cleaved flakes of biotite were reacted in batch and flow-through experiments to obtain kinetic information and elucidate the mechanisms that control the overall dissolution reaction under these conditions. The reacting basal surface was explored using in-situ laser confocal microscopy (LCM-DIM) and ex-situ scanning and phase shifting interferometry (VSI-PSI), while the release of tetrahedral (Al and Si), octahedral (Mg and Fe) and exchange (K) cations in solution was monitored over time. This experimental approach allowed us to calculate far-from-equilibrium dissolution rates associated with changes in topography of the (001) surface (horizontal retreat (Rstep) and vertical retreat (Rvertical)), etch pit formation and growth (Rpit) and the release of Si in solution, i.e., bulk dissolution (Rbulk,Si). In HNO3 solutions, a proton promoted reaction mechanism (PPRM), through proton adsorption on the biotite surface, was dominant. The variation of Rstep, Rvertical and Rbulk,Si accounting for the dissolution of (hk0) surfaces at different temperature and pH was used to calculate the apparent activation energy (Ea) and the proton reaction order (nH+). The respective values decreased from 63 kJ mol−1 at pH 1 to 31 kJ mol−1 at pH 3 and increased from 0.46 at 25 °C to 0.77 at 70 °C. This variability was related to the variation of proton consumption with temperature. In H2C2O4 solutions, formation and growth of etch pits on the (001) surface occurred, yielding an increase in surface roughening. Rpit accounted for the rate of etch pit development which was observed to increase, as temperature increased. Based on Rpit, relatively high Ea values (121 kJ mol−1 and 162 kJ mol−1) were associated with an early evolution of the basal etch pits, whereas lower values (36 kJ mol−1 and 56 kJ mol−1) were calculated using Rbulk,Si. The present study contributes to the interpretation of the mechanisms involved in the biotite dissolution process at highly acidic pH, in the presence of inorganic and organic acid, and low temperature (T ≤ 80 °C). Furthermore, our results highlight the importance of combining surface and bulk analyses to assess the balance between the different reactions involving proton consumption during the dissolution and its dependency on temperature, pH and organic acids concentration.

Effects of Salinity and pH of Seawater on the Reproduction of the Sea Urchin Paracentrotus lividus.

AbstractFertilization and early development are usually the most vulnerable stages in the life of marine animals, and the biological processes during this period are highly sensitive to the environment. In nature, sea urchin gametes are shed in seawater, where they undergo external fertilization and embryonic development. In a laboratory, it is possible to follow the exact morphological and biochemical changes taking place in the fertilized eggs and the developing embryos. Thus, observation of successful fertilization and the subsequent embryonic development of sea urchin eggs can be used as a convenient biosensor to assess the quality of the marine environment. In this paper, we have examined how salinity and pH changes affect the normal fertilization process and the following development of Paracentrotus lividus. The results of our studies using confocal microscopy, scanning and transmission electron microscopy, and time-lapse Ca2+ image recording indicated that both dilution and acidification of seawater have subtle but detrimental effects on many aspects of the fertilization process. They include Ca2+ signaling and coordinated actin cytoskeletal changes, leading to a significantly reduced rate of successful fertilization and, eventually, to abnormal or delayed embryonic development.

The effect of laser surface treatment on the adhesive bonding performance of aluminum alloy

For the purpose of convenient treatment before gluing and improving the adhesive bonding performance, we compared the tensile shear strengths of epoxy adhesive bonding to 2A12 aluminum with preferred pre-treatments. Taking advantages on both convenience and pre-treatment quality, we investigated the influences of duration of laser treatment on the adhesive bonding strength to obtain the optimized extents of laser treatment. With the analysis of scanning electron microscopy, laser confocal scanning microscopy and contact angle measurements adopted to characterize the surface morphology, roughness and wettability, respectively, we discussed the effects of pre-treatments on the surface status and adhesive bonding. As a result, we proposed a pre-treatment with laser treatment on aluminum to improve adhesive bonding performance, by which the removal of oxide layer, surface roughening and cleaning were easily accomplished before gluing.

Characteristics delineation of piscidin 5 like from Larimichthys crocea with evidence for the potent antiparasitic activity

Several researches reported that piscidin members of teleosts owned strong antiparasitic activity. Cryptocaryon irritans, a type of ectoparasite, could infect most of the marine teleosts. Larimichthys crocea could severely suffer from marine white spot disease caused by C. irritans, and their mortality rate was significantly high. Concentrating on this problem, we have done many related works. Piscidin 5 like (termed Lc-P5L) was another piscidin member isolated from a comparative transcriptome of C. irritans-immuned L. crocea. In the paper, quantitative Real-time PCR (qRT-PCR) showed Lc-P5L was upregulated in examined tissues, including gill, head kidney, muscle, liver, spleen and intestine after challenged by C. irritans, the significant upregulation time was in accordance to key developmental stages of C. irritans, which implied different infection stages could result in host immune response. Furthermore, using microscope techniques, we observed theronts or trophonts became weakly motile, cilia became detached, cells were out of shape, membranes eventually lysed in different cell positions and cytoplasmic contents leaked. Laser confocal scanning microscope (LCSM) observed theronts macronucleus grew swell and depolymerized after treated by recombinant Lc-P5L (rLc-P5L). Data suggested rLc-P5L was significantly lethal to C. irritans, and the death state of the parasite incubated with rLc-P5L was remarkably similar to other piscidin members or other antiparasitic peptides (APPs). Thus, these data provided new insights into L. crocea immunity against C. irritans and potential of rLc-P5L as a therapeutic agent against pathogen invasion.

Terahertz confocal imaging: Polarization and sectioning characteristics

ABSTRACT The development of high-resolution THz imaging systems is currently of great practical significance, (e.g. in the areas of security, agri-food, biomedicine etc.). Implementing and calibrating such systems is essential, as is the verification and appropriate use of models. Confocal microscopes are well known to improve resolution and provide 3D images. In this paper a novel terahertz (THz) confocal transmission scanning microscopy is implemented using a commercial 300 GHz (~1 mm) electronic THz source and sensor array. In order to calibrate the system, two objects, a high contrast amplitude metallic object and a low contrast surface relief phase pattern, are produced. These simple samples are imaged to quantify the polarization and sectioning properties of the imaging system. A scalar model is shown to be appropriate and is shown to predict the measured lateral and axial resolutions. Experimental results using two more complex objects are then presented, confirming confocal operation. This paper details the implementation and calibration of a 3D THz confocal imaging system.

Mapping of index of refraction profile for polymer gradient index optics using confocal Raman spectroscopy

Scanning confocal Raman microscopy is proposed to measure a gradient index (GRIN) profile at an optical surface. The Raman microscope is calibrated to index of refraction for a binary copolymer GRIN material, and then the index of refraction is mapped on the plano surface of a GRIN polymer lens. The measurement deduces axial shift of 680 μm and identifies lateral tilt or decenter with respect to the nominal position of the GRIN profile. Results suggest that the mapping method is a nondestructive way to measure the GRIN profile of a GRIN lens and its positioning within the lens geometry, to within the sampling precision of the Raman microscope.

Skeletal Muscle Cells Derived From Old Donors Show Mitochondrial Fragmentation And Decreased Oxygen Consumption Rates

With age, skeletal muscles lose their oxidative capacity and have a reduced mitochondrial fusion leading to fragmentation. These phenomena can lead to a reduction in oxygen consumption, atrophy, and an increased risk of developing age-related diseases such as sarcopenia. Skeletal muscle cells derived from humans can be used to investigate these physiological capacities in primary culture. PURPOSE: Investigate mitochondrial morphology and maximal oxygen consumption rates (OCR) of skeletal muscle cells derived from healthy young and old men. METHODS: Primary skeletal muscle cells derived from the Rectus abdominis muscle of healthy active eighteen and sixty-nine year old men (SKM18M and SKM69M, respectively) were obtained from Cook MyoSite Inc. (Pittsburgh, PA). Cells were stained with MitoTracker Red (Cell Signaling; Danvers, MA) and mitochondria morphology was observed using a Zeiss LSM 710 AxioObserver confocal scanning microscope (Carl Zeiss; White Plains, NY). The mitochondrial network was analyzed using the Mitochondrial Network Analysis tool in ImageJ (MiNA, FIJI) to estimate mitochondrial footprint from a binarized image. Oxygen consumption rates were measured in intact cells using Seahorse Cell Mito Stress Tests on a XFp extracellular flux analyzer (Agilent Technologies; Santa Clara, CA). RESULTS: Primary cells derived from the young donor (SKM18M) had a larger mitochondrial footprint, longer branch length, and a greater number of network branches compared to SKM69M (Footprint: 34.65 ± 25.30 vs. 11.64 ± 9.53 μm2; Branch Length: 20.59 ± 7.23 vs. 12.10 ± 6.84 μm; Network: 17.25 ± 0.16 vs. 7.67 ± 4.97 counts). SKM18M also showed higher Basal and Maximal OCR compared to SKM69M (Basal: 38.78 ± 8.34 vs. 12.82 ± 2.07; Maximal: 60.09 ±10.84 vs. 20.52 ± 2.36 pmol/min/protein). CONCLUSIONS: We observed differences morphologically and metabolically between the primary skeletal muscle cells derived from young and old donors. These preliminary results give us an insight into human skeletal muscle-derived cellular physiological capacity. Technology to observe human muscle mitochondrial fragmentation in vitro will help us elucidate the effects of aging on skeletal muscle mitochondrial fragmentation and loss of metabolic flexibility in aging.