Confocal Microscopy Methods Research Articles

Detailed analysis of nucleation and subsequent δ to γ phase transformation of a high N super austenitic stainless steel

Super austenitic stainless steel (SASS) is widely used in extreme environments due to its excellent corrosion resistance and mechanical properties, and a feature of this grade is its relatively high nitrogen content. As valuable tools to study the solidification process and phase transformation, high temperature confocal microscopy (HTCM) and thermal analysis methods, such as differential thermal analysis (DTA) and differential scanning calorimetry (DSC), have been widely reported in the investigation of SASS. However, the inherent limitations of these two techniques make it difficult to explain the complex solidification behaviors of SASS, which involves multiple phases. Here, a unique design of HTCM-DTA was employed to investigate the nucleation and subsequent phase transformation of SASS. This in-house build apparatus enables contemporaneous in-situ observation of the surface and thermal analysis of the bulk thermal events. Using the novel HTCM-DTA device, we systematically revealed the four solidification modes of SASS and analyzed them in detail through thermodynamic arguments and multiple sets of experimental data under different conditions: (a) bimodal solidification with solid-state phase transformation, (b) bimodal solidification with liquid-state phase transformation, (c) pseudo-bimodal solidification, and (d) unimodal solidification. By comparing with the findings of earlier studies, the limitation of spatiotemporal separation employing HTCM and thermal analysis methods was pointed out. The results suggest that both the shielding gas and the time of retaining in the liquid state affect the N content in the melt, leading to a change of the primary nucleation phase. The cooling rate not only affects the undercooling and microstructure but also the transformation stage of δ-ferrite to γ-austenite, and when the phase transformation occurs in the liquid state it is usually accompanied by recalescence. These insights contribute understanding required for adjusting the continuous casting process of high nitrogen alloys. • In-situ observations on surface and thermal analysis of the bulk in parallel. • Solidification mode and phase transformation highly depend on shielding gas. • Four different solidification modes of S31254 were found and analyzed in detail. • The recalescence phenomenon was observed and analyzed frame by frame. • C.R. and t L influence the solidification behavior.

Novel Chromatic Differential Confocal Matrix Sensor Using Multiband Spectral Images

In this letter, a novel chromatic differential confocal matrix (CDCM) sensor is proposed and demonstrated for accurate and fast three-dimensional (3D) profiling without mechanical scanning. In contrast to conventional chromatic confocal microscopy (CCM) methods that require spectral expansion and a peak-intensity-wavelength detection algorithm for axial positioning, our CDCM employs the intensity difference between a pair of spectral confocal images to accurately discriminate surface height. Due to the elimination of spectral expansion required by a conventional CCM, the CDCM has an at least one order of magnitude higher axial sensitivity and surface profiling efficiency than a conventional CCM. In the CDCM, axial scanning is achieved by residual chromatic aberration of an achromatic objective and by constructing a difference intensity signal between two spectral confocal images. Spectral confocal images are acquired in a parallel manner by using a digital micromirror device as an array of illumination pinholes and digitally applying the virtual detection pinhole concept. Quantitative evaluation experiments with the proof-of-principle sensor system indicate that with a <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$10\times $ </tex-math></inline-formula> NA 0.25 objective, the CDCM can achieve better than <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$0.1~\mu \text{m}$ </tex-math></inline-formula> axial accuracy with an axial measurement range of approximately <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$28~\mu \text{m}$ </tex-math></inline-formula> , a lateral resolution of <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$0.87~\mu \text{m}$ </tex-math></inline-formula> .

Temperature-salt stress increases yield of valuable metabolites and shelf life of microalgae

Background: Microalgae are very important for production of some chemicals industrially, such as carbohydrates, peptides, lipids, and carotenoids. There are many ways by which the yield of the valuable chemicals can be improved. They may include the reduction of cultivation temperature and change in the composition of growth media. Objectives: study adaptive mechanisms of Dunaliella salina Teodoresco and Chlorococcum dissectum Korshikov to low temperature and to develop the method for their hypothermic storage. Materials and methods: The objects of research were unicellular green microalgae D. salina and Ch. dissectum. Cold adaptation (for 24 hours) and hypothermic storage (for 3–30 days) of cultures were performed at 4 °C without lighting. Light and confocal microscopy methods were used to determine the viability and pigment composition of cells. The Alamar Blue (AB) test was used as an express method for assessing the metabolic activity of cells before and after cold adaptation. Results: The study has showed that lowered cultivation temperature and increased salinity of the growth medium increase the fluorescence of the NR dye in D. salina cells and do not affect this indicator in Ch. dissectum. The 24 h exposition at 4 °C does not lead to a significant decrease in the relative fluorescence units according to the AB test. Storage the algae at 4 °C does not result in their loss of viability and motility for up to 30 days. Conclusions: Incubation of D. salina at 4 °C for 24 hours increase carotenoid production compared to the intact culture, while it has no effect on Ch. dissectum, regardless of the growth medium composition. The short-term effect of low temperatures does not lead to a significant decrease in the metabolic activity of D. salina and Ch. dissectum. Storage of museum collection of D. salina and Ch. dissectum is possible for a period of 30 days at 4 °C without significant loss of metabolic activity, motility and cell concentration. These results also demonstrate that a combination of high salt and low temperature stresses increase the yield of valuable metabolites.

MULTIFUNCTIONAL NANOSYSTEMS BASED ON TWO FLUORESCENT DYES, DOXORUBICIN AND CURCUMIN

The aim of the work was to review the literature data regarding the prospects for the creation and use of multifunctional fluorescent two-dye nanosystems, which enable investigating the distribution of fluorescent components with significant acceleration of the study and introduction of nanomedicines into practice. Special attention is paid to the use of two substances with hydrophobic and hydrophilic properties in one nanoparticle (NP), capable of penetrating a living cell. The method of fluorescence confocal microscopy enables observation of the nanoscale dynamics of distribution and stability of drugs over time. The concomitant use of doxorubicin (DOX) and curcumin (CUR) in single nanoparticle causes synergism in the action of medical drugs, and their own fluorescence makes it possible to use them as multifunctional fluorescent nanosystems. Results. Data from the literature indicate that the use of two or more fluorescent dyes provide an advantage over other, more expensive methods when studying the penetration and distribution of NPs in living samples. The use of nanocarriers is an effective way to significantly increase the bioavailability of those drugs, which are poorly soluble in water. A promising direction of nanomedicine is the creation of complex bio-compatible multifunctional nanomaterials based on several active drugs, with the simultaneous use of their enhancers and the strategy of active targeting. Such recent structures enable targeted and controlled penetration of medicinal compounds into the sites of localization of pathological processes, reducing the toxicity of drugs to normal cells. Conclusions. The use of the fluorescence microscopy method, as exemplified by the two dyes, DOX and CUR, enables to trace the stages of interaction of loaded DOX and CUR nanoparticles with cultured cells, and their release from NPs to determine their amount and localization in organelles cells.

Possibilities of Multiphoton Microscopy in Semiconductor Nanomaterials Research

Multiphoton microscopy is the method using nonlinear optical effect which is two-photon absorption. It occurs when two identical photons are absorbed simultaneously. The photon energy should be around two times smaller than the photon energy for one-photon absorption. Thus, the excitation irradiation wavelength for multiphoton microscopy should be twice that for conventional confocal microscopy. Nowadays, multiphoton microscopy is widely used for biological research, but it is possible to apply this for non-biological materials studying, particularly, for solid-state materials and structures. Low-wavelength laser irradiation deep penetration, focusing and optical signal analysis possibility provides essential new challenges solving of hierarchical "smart" nanoparticles synthesis for target drug delivery, theranostics etc. In this paper we give an answer of some most popular questions about multiphoton microscopy. Nature and aspects of the multiphoton microscopy method were described. Advantages of this method in comparison with confocal microscopy method were shown. Multiphoton microscopy gives the best image contrast and the less photodamage and photobleaching of biological samples, and provides an opportunity of three-dimensional imaging of the biomaterials as well as the solid-state materials. Additionally, capabilities of this method for solid-state materials research were demonstrated by the porous silicon samples example. Also, multiphoton microscopy images of biological objects were shown.

Image analysis workflows to reveal the spatial organization of cell nuclei and chromosomes

ABSTRACT Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images. Abbreviations: 3D FISH: three-dimensional fluorescence in situ hybridization; 3D: three-dimensional; ASY1: ASYNAPTIC 1; CC: chromocenters; CO: Crossover; DAPI: 4',6-diamidino-2-phenylindole; DMC1: DNA MEIOTIC RECOMBINASE 1; DSB: Double-Strand Break; FISH: fluorescence in situ hybridization; GFP: GREEN FLUORESCENT PROTEIN; HEI10: HUMAN ENHANCER OF INVASION 10; NCO: Non-Crossover; NE: Nuclear Envelope; Oligo-FISH: oligonucleotide fluorescence in situ hybridization; RNPII: RNA Polymerase II; SC: Synaptonemal Complex; SIM: structured illumination microscopy; ZMM (ZIP: MSH4: MSH5 and MER3 proteins); ZYP1: ZIPPER-LIKE PROTEIN 1.

Ferroelectric Nanodomain Engineering in Bulk Lithium Niobate Crystals in Ultrashort-Pulse Laser Nanopatterning Regime.

Ferroelectric nanodomains were formed in bulk lithium niobate single crystals near nanostructured microtracks laser-inscribed by 1030-nm 0.3-ps ultrashort laser pulses at variable pulse energies in sub- and weakly filamentary laser nanopatterning regimes. The microtracks and related nanodomains were characterized by optical, scanning probe and confocal second-harmonic generation microscopy methods. The nanoscale material sub-structure in the microtracks was visualized in the sample cross-sections by atomic force microscopy (AFM), appearing weakly birefringent in polarimetric microscope images. The piezoresponce force microscopy (PFM) revealed sub-100 nm ferroelectric domains formed in the vicinity of the embedded microtrack seeds, indicating a promising opportunity to arrange nanodomains in the bulk ferroelectric crystal in on-demand positions. These findings open a new modality in direct laser writing technology, which is related to nanoscale writing of ferroelectric nanodomains and prospective three-dimensional micro-electrooptical and nanophotonic devices in nonlinear-optical ferroelectrics.

Huangqin Qingre Qubi Capsule inhibits RA pathology by binding FZD8 and further inhibiting the activity of Wnt/β-catenin signaling pathway

Ethnopharmacological relevanceHuangqin Qingre Qubi Capsule (HQC) is a Chinese herbal compound for the treatment of rheumatoid arthritis (RA), which is made from dry roots of Scutellaria baicalensis Georgi, dry mature seeds of Gardenia jasminoides J.Ellis, dry and mature seeds of Coix lacryma-jobi var. stenocarpa Oliv., dry mature seeds of Amygdalus persica L. and roots and rhizomes of Clematis chinensis Osbeck in the proportion of 10:9:30:5:10. HQC has a significant effect in clinical treatment of RA, which can inhibit RA inflammation, improve oxidative stress state, and effectively relieve symptoms of RA patients. Aim of the studyThe anti-arthritis effect of HQC and its mechanism, especially whether it improves RA through FZD8-Wnt/β-catenin signal axis, were studied using adjuvant arthritis (AA) rats and FLS from RA patients. Materials and methodsReal time qPCR (RT-qPCR), Western blot (WB), confocal microscopy and other molecular biological methods were used to study the anti-RA effect of HQC and its mechanism. ResultsThe expression of FZD8 was significantly up-regulated in synovium and FLS of AA rats and RA FLS. FZD8 significantly activated the Wnt/β-catenin signaling pathway, promoted abnormal proliferation of FLS, increased the levels of inflammatory factors IL-1β, IL-6 and IL-8, and significantly increased the expression of matrix metalloproteinase 3 (MMP3) and fibronectin. HQC has significant therapeutic effect on AA rats. Molecular docking and molecular dynamics showed that HQC had a good binding ability with FZD8. We also confirmed that HQC inhibited Wnt/β-catenin signaling pathway by binding FZD8, and reduced the levels of the above inflammatory factors and pathological genes of RA. ConclusionsThe expression of FZD8 is significantly increased in AA rats and FLS from RA patients. Clarify that HQC improves RA through the FZD8-Wnt/β-catenin signal axis, provide a clear therapeutic mechanism for HQC to improve RA, and also provide a basis for clinical promotion of HQC.

Piperazine Derivative Stabilizes Actin Filaments in Primary Fibroblasts and Binds G-Actin In Silico.

Alzheimer's disease (AD) is characterized by synaptic dysfunction, which is expressed through the loss of dendritic spines and changes in their morphology. Pharmacological compounds that are able to protect spines in the AD brain are suggested to be novel drugs that would be able to slow down the disease progression. We have recently shown that a positive modulator of transient receptor potential cation channel subfamily C member 6 (TRPC6), the compound N-(2-chlorophenyl)-2-(4-phenylpiperazine-1-yl) acetamide (51164), causes the upregulation of postsynaptic neuronal store-operated calcium entry, maintains mushroom spine percentage, and recovers synaptic plasticity in amyloidogenic mouse models of Alzheimer's disease. Here, using confocal microscopy and calcium imaging methods, we present the experimental data indicating that 51164 possesses an alternative mechanism of action. We demonstrated that 51164 can increase the mushroom spine percentage in neurons with the downregulated activity of TRPC6-dependent neuronal store-operated calcium entry. Moreover, we report the binding of 51164 to G-actin in silico. We observed that 51164 interacts with Lys 336, Asp157, and Ser14 of G-actin, amino acids involved in the stabilization/polymerization of the G-actin structure. We showed that interactions of 51164 with G-actin are much stronger in comparison to the well-characterized F-actin stabilizing and polymerizing drug, jasplakinolide. The obtained results suggest an alternative protective mechanism of 51164 that is related to the preservation of actin filaments in vitro.

Release Mechanisms of Amorphous Solid Dispersions: Role of Drug-Polymer Phase Separation and Morphology

Formulating poorly soluble molecules as amorphous solid dispersions (ASDs) is an effective strategy to improve drug release. However, drug release rate and extent tend to rapidly diminish with increasing drug loading (DL). The poor release at high DLs has been postulated to be linked to the process of amorphous-amorphous phase separation (AAPS), although the exact connection between phase separation and release properties remains somewhat unclear. Herein, release profiles of ASDs formulated with ritonavir (RTV) and polyvinylpyrrolidone/vinyl acetate (PVPVA) at different DLs were determined using surface normalized dissolution. Surface morphologies of partially dissolved ASD compacts were evaluated with confocal fluorescence microscopy, using Nile red and Alexa Fluor 488 as fluorescence markers to track the hydrophobic and hydrophilic phases respectively. ASD phase behavior during hydration and release of components were also visualized in real time using a newly developed in situ confocal fluorescence microscopy method. RTV-PVPVA ASDs showed complete and rapid drug release below 30% DL, partial drug release at 30% DL and no drug release above 30% DL. It was observed that formation of discrete drug-rich droplets at lower DLs led to rapid and congruent release of both drug and polymer, whereas formation of continuous drug-rich phase at the ASD matrix-solution interface was the cause of poor release above certain DLs. Thus, the domain size and interconnectivity of phase separated drug-rich domains appear to be critical factors impacting drug release from RTV-PVPVPA ASDs.

Fabricating switchable Pickering emulsions by dynamic covalent copolymer amphiphiles

In this work, amphiphilic copolymers D-An based on the dynamic imine bonds were generated in-situ in the aqueous solutions by two water-soluble precursors, the random copolymer poly(N-(2-methacryloylxyethyl)-random-poly(4-(formaldehyde) phenyl methacrylate) (PNMP83-co-PBFMA4, Precursor D) and alkylamines (Precursor An, n represents the carbon number in the alkyl chain that is ranging from 4 to 10). Amphiphiles D-An formed micelles spontaneously in the aqueous solutions, which were studied by the surface tension and cryogen-transmission electron microscopy (cryo-TEM) measurements. Moreover, the CO2/N2 responsive micellization of D-An due to the reversible formation and breaking of the dynamic imine bond (-CH=N-) was confirmed by the nuclear magnetic resonance (NMR) measurements. Accordingly, the emulsification of dynamic covalent amphiphilic copolymers D-An was studied in detail. O/W Pickering emulsions with excellent stability were commonly generated by employing D-An as emulsifiers regardless of n and the nature of oil employed, which was significantly different from the weak emulsification of either precursor D or precursor An. The synergistic emulsification of D-An monomers and micelles was proved to be the major cause as evidenced by the confocal laser scanning microscopy (CLSM) method. Noticeably, the abundant alkylamines were also advantageous in stabilizing emulsions. Attributing to the stimuli-responses of amphiphiles D-An to CO2/N2, the stability of Pickering emulsions could be reversibly controlled by CO2/N2 as well.

Mitochondrial networks of cumulus cells and oocyte quality

BACKGROUND: Mitochondria play vital roles in oocyte functions and they are critical indicators of oocyte quality which is important for fertilization and development into viable offspring. Quality-compromised oocytes in which mitochondrial dysfunction plays a significant role are correlated with infertility, developmental disorders and embryo loss. A significant role in the oocytes of cytoplasmic accumulation and a sufficient amount of the mitochondrial pool of oocytes is played by the cumulus cells surrounding it.&#x0D; AIM: to conduct a comparative analysis between the state of mitochondrial networks of cumulus cells and the quality of oocytes.&#x0D; MATERIALS AND METHODS: The study included 22 women aged 28 to 37 years inclusive (mean age 32.3 1.2 years) with tubal infertility. During the assisted reproductive technology procedures, 74 oocytes were obtained. 39 good quality oocytes and their cumulus cells were compared with 35 bad quality oocytes and their cumulus cells respectively. After puncture of ovarian follicles cumulus cells were isolated and stained with fluorescent dye for cell tracing and mitochondrial imaging in vivo. The method of confocal microscopy was used to analyze the three-dimensional organization of mitochondria in 2030 cells of one pool of follicular fluid. The following parameters were used to evaluate the mitochondrial network: fluorescence decay time and fluorescence intensity.&#x0D; RESULTS: The decay time of fluorescence in cumulus cells associated with a good quality oocyte was significantly higher (p = 0.032) than in the case of identification of oocytes with negative signs.&#x0D; CONCLUSION: The structure of mitochondrial networks in cumulus cells correlates (r = 0.76) with the quality of oocytes.

Synthesis, Anticancer Activity and Molecular Docking Studies of Novel N-Mannich Bases of 1,3,4-Oxadiazole Based on 4,6-Dimethylpyridine Scaffold.

Cancer is one of the greatest challenges in modern medicine today. Difficult and long-term treatment, the many side effects of the drugs used and the growing resistance to treatment of neoplastic cells necessitate new approaches to therapy. A very promising targeted therapy is based on direct impact only on cancer cells. As a continuation of our research on new biologically active molecules, we report herein the design, synthesis and anticancer evaluation of a new series of N-Mannich-base-type hybrid compounds containing morfoline or different substituted piperazines moieties, a 1,3,4-oxadiazole ring and a 4,6-dimethylpyridine core. All compounds were tested for their potential cytotoxicity against five human cancer cell lines, A375, C32, SNB-19, MCF-7/WT and MCF-7/DX. Two of the active N-Mannich bases (compounds 5 and 6) were further evaluated for growth inhibition effects in melanoma (A375 and C32), and normal (HaCaT) cell lines using clonogenic assay and a population doubling time test. The apoptosis was determined with the neutral version of comet assay. The confocal microscopy method enabled the visualization of F-actin reorganization. The obtained results demonstrated that compounds 5 and 6 have cytotoxic and proapoptotic effects on melanoma cells and are capable of inducing F-actin depolarization in a dose-dependent manner. Moreover, computational chemistry approaches, molecular docking and electrostatic potential were employed to study non-covalent interactions of the investigated compounds with four receptors. It was found that all the examined molecules exhibit a similar binding affinity with respect to the chosen reference drugs.

Intracytoplasmic Trafficking of Nanoparticles based on Hyaluronic Acid and Chitosan for Targeted Delivery of Anticancer Drugs

Cancer diseases are characterized by high incidence and mortality worldwide. The main problem in cancer therapy is the lack of specificity of anti-cancer drugs. Therefore, the development of new methods of targeted delivery of anti-cancer drugs is an urgent task in oncology. Nanoparticles from hyaluronic acid and chitosan (HA:CS) were obtained using ionotropic gelation technology. The size of the nanoparticles was investigated using dynamic light scattering. Nanoparticles were obtained of a size of 100-400 nm. A physical association method has been developed for encapsulating nanoparticles with doxorubicin, a well-known antitumor drug, and dinitrosyl iron complex (DNIC; donor of nitric oxide). Using the method of dynamic light scattering, the surface potential of nanoparticles was measured. It was found that the resulting nanoparticles (HA-DOX:CS) were stable and had a surface potential of -45.6 meV. Using the method of confocal and FLIM microscopy, the localization of nanoparticles in cancer cells was studied. These methods have shown that nanoparticles pass through the cytoplasmic membrane and are localized inside the cells. It was also shown that nanoparticles (HA:CS-Rhod) were localized in the cytoplasm of African green monkey renal epithelial cells. It was found that the incorporation of DNIC into the composition of nanoparticles significantly increased the stability of DNIC, while prolonging the formation time and increasing the yield of nitric oxide. Thus, we have developed unique nanoparticles for the targeted delivery of antitumor drugs into cells.

On the quantitative assessment of corrosion damages of aluminum at the early stages using confocal laser scanning microscopy

Despite being widely used in such industries as chemical, aviation and food ones, aluminum and its alloys are known to be prone to localized corrosion, and this remains a problem to be solved, especially when it comes to pitting corrosion. Therefore, there is a necessity to detect traces of corrosion at the earliest stages and to quantify the extent of corrosion damage. The simplest solution for assessing the overall corrosion rate is to use the gravimetric method, which, however, does not provide information for assessment of localized corrosion. This paper is devoted to the consideration of the possibilities of using the method of confocal laser scanning microscopy (CLSM) for assessing corrosion resistance using high-purity aluminum. The CLSM method, due to its high resolution (especially along the vertical axis), enables to obtain quantitative data on the volume of corroded metal, determine the depth of corrosion damage including building their profilograms, and analyze the morphology of the surface damaged by corrosion. Owing to the high sensitivity of the CLSM method, corrosion losses were detected within 21 days, contrary to the standard gravimetric method, which failed to determine the loss of metal even after 160 days of corrosion testing.

Bioenergetic and Metabolic Impairments in Induced Pluripotent Stem Cell-Derived Cardiomyocytes Generated from Duchenne Muscular Dystrophy Patients.

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene and dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality in DMD patients. We tested the hypothesis that DCM is caused by metabolic impairments by employing induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from four DMD patients; an adult male, an adult female, a 7-year-old (7y) male and a 13-year-old (13y) male, all compared to two healthy volunteers. To test the hypothesis, we measured the bioenergetics, metabolomics, electrophysiology, mitochondrial morphology and mitochondrial activity of CMs, using respirometry, LC–MS, patch clamp, electron microscopy (EM) and confocal microscopy methods. We found that: (1) adult DMD CMs exhibited impaired energy metabolism and abnormal mitochondrial structure and function. (2) The 7y CMs demonstrated arrhythmia-free spontaneous firing along with “healthy-like” metabolic status, normal mitochondrial morphology and activity. In contrast, the 13y CMs were mildly arrhythmogenic and showed adult DMD-like bioenergetics deficiencies. (3) In DMD adult CMs, mitochondrial activities were attenuated by 45–48%, whereas the 7y CM activity was similar to that of healthy CMs. (4) In DMD CMs, but not in 7y CMs, there was a 75% decrease in the mitochondrial ATP production rate compared to healthy iPSC-CMs. In summary, DMD iPSC-CMs exhibit bioenergetic and metabolic impairments that are associated with rhythm disturbances corresponding to the patient’s phenotype, thereby constituting novel targets for alleviating cardiomyopathy in DMD patients.

Luminescence confocal microscopy of 3D components of photonic integrated circuits fabricated by two-photon photopolymerization

Direct laser writing (DLW) lithography has been growing rapidly and is widely used for photonic integrated circuit (PIC) fabrication manufacturing in micro optics and photonics to fabricate 2D, 2.5D, and 3D components due to low cost, high precision and no need for mask exposure. There is a huge demand for PIC for instruments, systems and complexes of optoelectronic navigation instrumentation, as well as for use in a wide range of areas: biophotonics, signal detection and processing, robotics, telecommunications. The technique of DLW is based on nonlinear interaction between laser radiation and the photoresist medium (two photon absorption and consequent photopolymerization). The traditional method for studying the morphology of obtained PIC elements is the method of scanning electron microscopy. An alternative method of morphological analysis of PICs obtained by two photon absorption-direct laser writing (TPA-DLW) is proposed here - scanning luminescence confocal microscopy method. It is an effective method that enables obtaining conclusive information on the geometrical parameters as the morphology properties as well as the luminescence information.

Are nanohedgehogs thirsty? Toward new superhydrophobic and anti-icing carbon nanohorn-polymer hybrid surfaces

85% of the world’s polymers production is made up of thermoplastics, and among them, one can find: polyethylene (PE), polypropylene (PP) and polyethylene terephthalate (PET). In this study, these three polymers were applied for the first time as supports for superhydrophobic and anti-icing surfaces created using single walled carbon nanohorns (SWCNH). A facile method of thermal “feathering” was used and the obtained materials were deeply characterized using thermal (thermogravimetry, differential scanning calorimetry) and microscopic (scanning electron microscopy, atomic force microscopy, confocal microscopy) methods as well as tribological and nanoindentation tests. We show that, depending on the polymer, the process of thermal “bulk” or “surface feathering” occurs. The results supported by Hansen Solubility Parameters calculation combined with Molecular Dynamics Simulations allow to explain the stability of surfaces as well as the wetting behavior revealing that all new materials are superhydrophobic. Moreover, some of them exhibit ice-phobic properties, while droplet freezing process is well described by the dynamic growth angle model. Finally, by discussing the mechanical stability, superhydrophobic and anti-icing properties two optimal surfaces (PP + SWCNH 5 min, PE + SWCNH 10 min) are chosen. Both of them possess intermediate values of Young’s modulus and show intermediate values of static contact angle hysteresis. Since the PE-based solids show enhanced tribological performance, tests, PE + SWCNH 10 min sample emerges as the most perspective in practical hydrophobic and anti-icing applications.

Gliadin induced oxidative stress and altered cellular responses in human intestinal cells: An in-vitro study to understand the cross-talk between the transcription factor Nrf-2 and multifunctional APE1 enzyme.

The present study examined the wheat protein gliadin-induced oxidative and nitrosative stress and its downstream responses in human intestinal HCT-116 and HT-29 cells. The beneficial role of dietary phytochemical curcumin and role of multifunctional enzyme Apurinic/aprymidinic endonuclease 1 (APE1) a major player involved in the base excision repair (BER)-pathway in gliadin intolerant intestinal HCT-116 and HT-29 cell lines were evaluated as an in vitro model study. The cultured cells were exposed to gliadin protein, H2 O2 , and curcumin followed by the assessment of oxidative stress and the consequences were measured using spectrophotometric, PCR, flow cytometer, Western blotting, confocal microscopy, and other methods. Results demonstrate that a 3 h pretreatment of curcumin, followed by the treatment of gliadin protein for 24 h time period protected both the HCT-116 and HT-29 cells via: (i) decreasing the ROS/RNS, restoring the mitochondrial transmembrane potential; (ii) re-establishing the cellular antioxidant defense system (superoxide dismutase, catalase, and GSH); (iii) enhancing the functions of APE1 viz. endonuclease activity and redox activation of transcription factor Nrf-2, the later binds with the antioxidant response elements (ARE) and activates downstream targets involved in cell survival. The cross-talk between APE1 and Nrf-2 was also established using immunofluorescence imaging and co-immunoprecipitation assays. In conclusion, gliadin protein induces oxidative/nitrosative stress, mitochondrial dysfunction and it damages cellular biomolecules in the intestinal cells. Hence it can be attributed to the tissue damage and disease pathogenesis in wheat intolerance-associated intestinal diseases. The gliadin-induced stress and its consequences are significantly reduced by the pretreatment of curcumin via BER-pathway and ARE-pathway; which is evident through the interaction between these two essential proteins. Hence suggesting for the intervention of curcumin and other natural dietary phytochemicals-based disease management and treatment of gliadin intolerance associated intestinal diseases like celiac disease.

Pollution from Transport: Detection of Tyre Particles in Environmental Samples

Transport is one of the most important sources of environmental pollution. More and more information has shown that one of the greatest sources of emissions from transport are emissions related to the release of microplastics from tyres. This is one of the most underestimated sources of emissions into the environment. In this study, environmental samples are analysed for the presence of these particles. For this purpose, optical methods and spectroscopic methods are used. Fourier transform infrared (FTIR) spectroscopy is used to identify synthetic rubber, most likely derived from car tyres. A complementary confocal microscopy method is used to confirm the FTIR results. The soil samples and road dust from the areas with heavy traffic are tested. An average of 372 ± 50 fragments per kilogram dry weight are detected in the soil samples. In the case of samples from the road, this number is 515 ± 20 fragments per kilogram of dry matter. In the samples, most of the microplastics come from tyres, which confirms the scientists’ assumptions about the amount of emissions in the environment. More than 90% of the black fragments later identified as tyre-derived synthetic rubber are found in the samples. A greater number of microplastics are found in road dust samples than in the soil. This may be due to the direct influence of braking, which causes greater accumulation of samples at the emission source than at a short distance into the soil. There is also a noticeable difference in the size of the fraction. In the case of soils, a fraction below 50 µm accounts for the majority of cases. When one analyses road dust samples, one may observe that most of the fractions are between 50 and 200 µm. This may be due to the possibility of smaller emission particles over longer distances and the greater degradation process that occurs in soils. The microplastics from the road dust are less degraded than the microplastics from the soil.