Fluorescence Microscopy Methods Research Articles

Effect of Syzygium cumini on Oxidative Stress Induced Cardiac Cellular Anomalies.

Doxorubicin (Dox), an antineoplastic agent is used as a primary anticancerous drug against various types of cancers. However, its associated toxicity to the cardiovascular system is major. Literature has recorded the cases of mortality due to poor validation and lack of prediagnosis of Dox-induced cardiotoxicity. Therapeutic interventions using natural products having cardioprotective properties with low toxic outcomes hold therapeutic potential for future cardio-oncological therapies. Syzygium cumini (Black berry), a traditional Indian herbal plant, has been researched and found to exert cardioprotective, anti-inflammatory, and antioxidant activities, which have been credited due to the presence of polyphenols, flavonoids, and tannins. In the current research, we investigated the cardioprotective potential of Syzygium cumini against Doxorubicin-induced cardiotoxicity (DIC) in H9C2 cardiomyocytes. Methanolic seed extract preparation of Syzygium cumini was performed using the Soxhlet apparatus. Cell viability and cell death assays were performed to determine the cardiotoxic doses of Doxorubicin. Furthermore, the cardioprotective potential of Syzygium cumini extract against DIC was studied. Morphological and nuclear alterations in H9C2 cells were studied by microscopic assays using Giemsa, Haematoxylin-Eosin stain, and PI. The intracellular stress level and ROS production were studied using DCFH-DA followed by mitochondrial integrity analysis using fluorescent microscopic methods. In the results, we investigated that Dox exerted a dose and time-dependent cardiotoxicity on H9C2 cardiomyocytes. Moreover, we observed that morphological and nuclear alterations caused by doxorubicin in dose-dependent manner were prevented by supplementing with Syzygium cumini polyphenols and it attenuated the oxidative stress in H9C2 cardiomyocytes effectively. Conclusively, Syzygium cumini possesses cardioprotective potential in H9C2 cardiomyocytes in dox-induced cardiotoxicity.

Optically Imaging In Situ Effects of Electrochemical Cycling on Single Nanoparticle Electrocatalysis.

Single-nanoparticle studies often need one or a series of nanoparticle populations that are designed with differences in a nominally particular structural parameter to clarify the structure-activity relationship (SAR). However, the heterogeneity of various properties within any population would make it rather difficult to approach an ideal one-parameter control. In situ modification ensures the same nanoparticle to be investigated and also avoids complicating effects from the otherwise often needed ex situ operations. Herein, we apply electrochemical cycling to single platinum nanoparticles and optically examine their SAR. An electrocatalytic fluorescent microscopic method is established to evaluate the apparent catalytic activity of a number of single nanoparticles toward the oxygen reduction reaction. Meanwhile, dark-field microscopy with the substrate electrode under a cyclic potential control is found to be able to assess the electrochemically active surface area (ECSA) of single nanoparticles via induced chloride redox electrochemistry. Consequently, nanoparticles with drastically increased catalytic activity are discovered to have larger ECSAs upon potential regulation, and interestingly, there are also a few particles with decreased activity, as opposed to the overall trend, that all develop a smaller ECSA in the process. The deactivated nanoparticles against the overall enhancement effects of potential cycling are revealed for the first time. As such, the SAR of single nanoparticles when subjected to an in situ structural control is optically demonstrated.

An Indirect Fluorescence Microscopy Method to Assess Vaginal Lactobacillus Concentrations.

Lactobacillus species are the main colonizers of the vaginal microbiota in healthy women. Their absolute quantification by culture-based methods is limited due to their fastidious growth. Flow cytometry can quantify the bacterial concentration of these bacteria but requires the acquisition of expensive equipment. More affordable non-culturable methods, such as fluorescence microscopy, are hampered by the small size of the bacteria. Herein, we developed an indirect fluorescence microscopy method to determine vaginal lactobacilli concentration by determining the correlation between surface area bacterial measurement and initial concentration of an easily cultivable bacterium (Escherichia coli) and applying it to lactobacilli fluorescence microscopy counts. In addition, vaginal lactobacilli were quantified by colony-forming units and flow cytometry in order to compare these results with the indirect method results. The colony-forming-unit values were lower than the results obtained from the other two techniques, while flow cytometry and fluorescence microscopy results agreed. Thus, our developed method was able to accurately quantify vaginal lactobacilli.

Detection of CPSF6 in Biomolecular Condensates as a Reporter of HIV-1 Nuclear Import.

The initial stages of HIV-1 infection involve the transport of the viral core into the nuclear compartment. The presence of the HIV-1 core in the nucleus triggers the translocation of CPSF6/CPSF5 from paraspeckles into nuclear speckles, forming puncta-like structures. While this phenomenon is well-documented, the efficiency of CPSF6 translocation to nuclear speckles upon HIV-1 infection varies depending on the type of cell used. In some human cell lines, only 1-2% of the cells translocate CPSF6 to nuclear speckles when exposed to a 95% infection rate. To address the issue that only 1-2% of cells translocate CPSF6 to nuclear speckles when a 95% infection rate is achieved, we screened several human cell lines and identified a human a cell line in which approximately 85% of the cells translocate CPSF6 to nuclear speckles when 95% infection rate is achieved. This cellular system has enabled the development of a robust fluorescence microscopy method to quantify the translocation of CPSF6 into nuclear speckles following HIV-1 infection. This assay holds the potential to support studies aimed at understanding the role of CPSF6 translocation to nuclear speckles in HIV-1 infection. Additionally, since the translocation of CPSF6 into nuclear speckles depends on the physical presence of the viral core in the nucleus, our method also serves as a reporter of HIV-1 nuclear import.

DNase improves the efficacy of antimicrobial photodynamic therapy in the treatment of candidiasis induced with Candida albicans.

The study evaluated the association of DNase I enzyme with antimicrobial photodynamic therapy (aPDT) in the treatment of oral candidiasis in mice infected with fluconazole-susceptible (CaS) and -resistant (CaR) Candida albicans strains. Mice were inoculated with C. albicans, and after the infection had been established, the tongues were exposed to DNase for 5 min, followed by photosensitizer [Photodithazine®(PDZ)] and light (LED), either singly or combined. The treatments were performed for 5 consecutive days. Treatment efficacy was evaluated by assessing the tongues via fungal viable population, clinical evaluation, histopathological and fluorescence microscopy methods immediately after finishing treatments, and 7 days of follow-up. The combination of DNase with PDZ-aPDT reduced the fungal viability in mice tongues immediately after the treatments by around 4.26 and 2.89 log10 for CaS and CaR, respectively (versus animals only inoculated). In the fluorescence microscopy, the polysaccharides produced by C. albicans and fungal cells were less labeled in animals treated with the combination of DNase with PDZ-aPDT, similar to the healthy animals. After 7 days of the treatment, DNase associated with PDZ-aPDT maintained a lower count, but not as pronounced as immediately after the intervention. For both strains, mice treated with the combination of DNase with PDZ-aPDT showed remission of oral lesions and mild inflammatory infiltrate in both periods assessed, while animals treated only with PDZ-aPDT presented partial remission of oral lesions. DNase I enzyme improved the efficacy of photodynamic treatment.

Expression of human papillomavirus genotype 52 pseudovirus in HEK-293T cells

Human papillomavirus (HPV) is the predominant cause of cervical cancer globally. HPV genotype 52 is categorized as a high-risk type and holds the highest prevalence in Indonesia (23.2 %). The HPV pseudovirus can be used to test the immunogenicity of HPV vaccine candidates and evaluate the neutralization efficiency of the antibodies against the virus. The aim of this study was to express the HPV52 L1 and L2 capsid protein into pseudovirion containing the GFP reporter plasmid (pfwB) in HEK-293T cells. HPV52 L1 and L2 coding plasmid (pVITRO52) along with pfwB were amplified in E. coli. The plasmids were extracted and co-transfected using polyethyleneimine (PEI)-based transfection method into the HEK-293T cells. The expression of HPV52 pseudoviruses were confirmed by fluorescence microscopy and western blot method. Co-transfection of HPV52 L1 and L2 coding plasmid (pVITRO52) along with pfWB into the HEK-293T cells was successfully carried out. The co–transfected HEK-293T cells showed fluorescence. The western blot assay using HPV52 L1 protein primary antibody showed a band around ∼55 kDa. In the future, the results of this study will be used to evaluate the immunogenicity and neutralization assay of HPV vaccine candidates.

Preparation of mcl-polyhydroxyalkanoate nanoparticles stabilized by the Tween 80 nonionic surfactant

Nanoparticles based on biodegradable polymers find numerous applications in medicine as substances for intracellular drug delivery. Biosynthetically produced poly-3-hydroxyalkanoates (P3HAs) are among the most promising polymers of a lipid nature. In particular, polyhydroxybutyrate and polyhydroxyvalerate (scl-poly-3-hydroxyalkanoates) are widely used compounds, which are soluble only in organochlorine solvents. The use of organochlorine solvents faces several obstacles, since such chemicals may exhibit carcinogenic effects on the human body. However, the P3Has compounds consisting of hydroxybutyric acid residues with 6–14 carbon atoms in the main chain (mcl-poly-3-hydroxyalkanoates) are soluble not only in CHCl3 or CH2Cl2 but also in paraffins. These hydrocarbons, such as n-hexane, can be easily separated from aqueous solutions and are not known to exhibit high cytotoxicity. Consequently, the application of mcl-poly-3-hydroxyalkanoates can prevent the contamination of prepared dosage forms with organochlorine compounds. To this end, a methodology for the synthesis of mcl-P3HA nanoparticles stabilized with the Tween 80 nonionic surfactant was proposed. The ratio between the concentration of P3HA and the detergent was optimized. The present study revealed that the obtained particles have an average size of 200±90 nm and a zeta potential of -17±5 mV. Upon investigating the stability of the particle solution at 4 °C, it was found that the nanoparticles did not alter their size and zeta potential for 90 days. The fluorescence microscopy method showed that they could be delivered into BHK-21 cells within 2 h. In addition, the nanoparticles synthesized had no toxic effect on BHK-21 cells at a concentration of up to 200 μg/mL.

EFFECT OF USING MULTIPOTENT MESENCHYMAL STROMAL CELLS IN BONE GRAFTING WITH XENOGENIC BIOMATERIALS

Currently, there is a significant frequency of degenerative diseases of the spine after surgical interventions with the use of bone grafting, especially in elderly patients. The results of the research indicate that the use of stem cells is one of the promising areas to increase the efficiency of osseointegration. The aim of the study was to evaluate the effectiveness of osteogenesis in the conditions of using xenografts loaded with stem cells, as well as morphological features of osseointegration. Materials and methods. An experimental study was conducted on 22 male rabbits. 2 animals were used to obtain stem cells, the remaining animals were implanted with xenogenic bone–substituting material "Osteomatrix" in the iliac wing, of which 10 animals were implanted with a xenograft not populated with cells, and the other 10 animals were implanted with an identical xenograft populated with mesenchymal stromal cells. Withdrawal from the experiment was carried out on the 60th day after implantation. The material's examination was carried out using the methods of fluorescence and light microscopy. To assess the statistical significance of observed differences (evaluation of proliferation, neoangiogenesis and osseointegration) in the experimental and control groups, the Mann–Whitney U test was used with a critical significance of p ≤ 0.05. Results. When using an osteoplastic material that was not loaded with multipotent mesenchymal stromal cells within 60 days after surgery, osseointegration passes through indirect osteogenesis with formation of full-fledged bone tissue in the future, which increases the time of complete defect healing. In the case of using an osteomatrix loaded with multipotent mesenchymal stromal cells of the bone marrow, osteogenesis proceeds in a direct way with the formation of a full-fledged bone tissue. Osteomatrix loading with multipotent mesenchymal stromal cells stimulates neoangiogenesis and proliferative activity of the tissue, which promotes activation of bone tissue repair processes and stimulates the processes of xenograft osseointegration. Conclusions. The use of multipotent mesenchymal stromal cells in bone grafting using xenografts increases the efficiency of osseointegration by stimulating direct osteogenesis, increasing the activity of proliferation and angiogenesis.

Cerium Oxide Nanoparticles Protect Cortical Astrocytes from Oxygen-Glucose Deprivation through Activation of the Ca2+ Signaling System.

Most of the works aimed at studying the cytoprotective properties of nanocerium are usually focused on the mechanisms of regulation of the redox status in cells while the complex effects of nanocerium on calcium homeostasis, the expression of pro-apoptotic and protective proteins are generally overlooked. There is a problem of a strong dependence of the effects of cerium oxide nanoparticles on their size, method of preparation and origin, which significantly limits their use in medicine. In this study, using the methods of molecular biology, immunocytochemistry, fluorescence microscopy and inhibitory analysis, the cytoprotective effect of cerium oxide nanoparticles obtained by laser ablation on cultured astrocytes of the cerebral cortex under oxygen-glucose deprivation (OGD) and reoxygenation (ischemia-like conditions) are shown. The concentration effects of cerium oxide nanoparticles on ROS production by astrocytes in an acute experiment and the effects of cell pre-incubation with nanocerium on ROS production under OGD conditions were studied. The dose dependence for nanocerium protection of cortical astrocytes from a global increase in calcium ions during oxygen-glucose deprivation and cell death were demonstrated. The concentration range of cerium oxide nanoparticles at which they have a pro-oxidant effect on cells has been identified. The effect of nanocerium concentrations on astrocyte preconditioning, accompanied by increased expression of protective proteins and limited ROS production induced by oxygen-glucose deprivation, has been investigated. In particular, a correlation was found between an increase in the concentration of cytosolic calcium under the action of nanocerium and the suppression of cell death. As a result, the positive and negative effects of nanocerium under oxygen-glucose deprivation and reoxygenation in astrocytes were revealed at the molecular level. Nanocerium was found to act as a "double-edged sword" and to have a strictly defined concentration therapeutic "window".

Super-Resolution Imaging of Neuronal Structures with Structured Illumination Microscopy.

Super-resolution structured illumination microscopy (SR-SIM) is an optical fluorescence microscopy method which is suitable for imaging a wide variety of cells and tissues in biological and biomedical research. Typically, SIM methods use high spatial frequency illumination patterns generated by laser interference. This approach provides high resolution but is limited to thin samples such as cultured cells. Using a different strategy for processing raw data and coarser illumination patterns, we imaged through a 150-micrometer-thick coronal section of a mouse brain expressing GFP in a subset of neurons. The resolution reached 144 nm, an improvement of 1.7-fold beyond conventional widefield imaging.

Resolution enhancement with a task-assisted GAN to guide optical nanoscopy image analysis and acquisition

Super-resolution fluorescence microscopy methods enable the characterization of nanostructures in living and fixed biological tissues. However, they require the adjustment of multiple imaging parameters while attempting to satisfy conflicting objectives, such as maximizing spatial and temporal resolution while minimizing light exposure. To overcome the limitations imposed by these trade-offs, post-acquisition algorithmic approaches have been proposed for resolution enhancement and image-quality improvement. Here we introduce the task-assisted generative adversarial network (TA-GAN), which incorporates an auxiliary task (for example, segmentation, localization) closely related to the observed biological nanostructure characterization. We evaluate how the TA-GAN improves generative accuracy over unassisted methods, using images acquired with different modalities such as confocal, bright-field, stimulated emission depletion and structured illumination microscopy. The TA-GAN is incorporated directly into the acquisition pipeline of the microscope to predict the nanometric content of the field of view without requiring the acquisition of a super-resolved image. This information is used to automatically select the imaging modality and regions of interest, optimizing the acquisition sequence by reducing light exposure. Data-driven microscopy methods like the TA-GAN will enable the observation of dynamic molecular processes with spatial and temporal resolutions that surpass the limits currently imposed by the trade-offs constraining super-resolution microscopy.

Rejection and fouling of track-etched microfiltration membranes by Acholeplasma laidlawii: Clues to mycoplasma behavior during “sterile” dead-end filtration

Mycoplasma contamination is commonplace during processing of biopharmaceuticals necessitating its sterile filtration. Consequently, we measured the passage of Acholeplasma laidlawii (a small bacterium lacking cell wall like mycoplasma) across track-etched membranes rated at 100 nm and 220 nm that are designated as “sterile filters” and analyzed the entire progression of fouling beginning with pore occlusion before transitioning to a cake. Initial biofouling was quantified to be intermediate blocking before bacteria formed highly compressible cakes. Under identical experimental conditions, phase inversion membranes of the same nominal pore rating rejected A. laidlawii to a significantly greater extent suggesting depth filtration and capture within the polymer matrix in contrast to surface capture by their track-etched counterparts. A facile fluorescence microscopy method to rapidly detect slow-growing A. laidlawii cells was demonstrated under conditions of low retention. Evidence of cake relaxation by depressurizing the filter and subsequent mobilization of individual cells upon re-pressurization is given. Multiple lines of evidence demonstrated the pressure-induced deformation of individual cells resulting in their incomplete rejection by membranes with pore ratings smaller than the bacterium's unstressed diameter: (i) complete retention of rigid silica colloids of similar size, shape, and zeta potential regardless of pressure in contrast to incomplete A. laidlawii removals, (ii) reduced bacterial removal by both membranes with increasing filtration pressure, (iii) 150–600 fold less permeable bacterial cakes at any given pressure compared with silica cakes, and (iv) presence of flattened and irregularly shaped A. laidlawii cells in electron micrographs whereas they were near perfect cocci at atmospheric pressure.

Nondestructive 3D Imaging of Microscale Damage inside Polymers-Based on the Discovery of Self-Excited Fluorescence Effect Induced by Electrical Field.

The development of high-precision, non-destructive, and three-dimensional (3D) in situ imaging of micro-scale damage inside polymers is extremely challenging. Recent reports suggest that 3D imaging technology based on micro-CT technology causes irreversible damage to materials and is ineffective for many elastomeric materials. In this study, it is discovered that electrical trees inside silicone gel induced by an applied electric field can induce a self-excited fluorescence effect. Based on this, high-precision, non-destructive, and 3D in situ fluorescence imaging of polymer damages is successfully achieved. Compared with the current methods, the fluorescence microscopic imaging method enables slicing of the sample in vivo with high-precision operation, realizing the precise positioning of the damaged area. This pioneering discovery paves the way for high-precision, non-destructive, and 3D in situ imaging of polymer internal damage, which can solve the problem of internal damage imaging in insulating materials and precision instruments.

Detection of fine asbestos fibers using fluorescently labeled asbestos-binding proteins in talc

Asbestos contamination in common materials, including powdered talc used in manufacturing cosmetics, pharmaceuticals, and baby powder, is of great public concern. The use of polarized light microscopy (PLM) for asbestos detection is limited because it cannot detect fibers with diameters in the nano range. Furthermore, the use of electron microscopy (EM), the preferred method for determining asbestos contamination in talc, is hindered by a rather complicated and laborious process. Fluorescence microscopy (FM) is a simple analytical tool in modern life sciences, especially given its superior sensitivity and selectivity; however, it is rarely used for inorganic materials. Previously, we demonstrated that airborne asbestos fibers were fluorescently visualized under FM using fluorescently labeled asbestos-binding proteins. Here, we evaluated the FM method's feasibility in detecting fine asbestos fibers in powdered talc. The limit of detection of the fiber diameter of asbestos was approximately 0.06 µm using FM. The FM method's sensitivity surpassed that of PLM and approached that of EM, and thus, it can detect nano-scale fibers. Notably, the FM method enabled the selective visualization of asbestos fibers, including those hidden beneath numerous talc particles. A point counting method using FM images enabled a semi-quantitative analysis of 0.01–1.0% (w/w) asbestos contamination in talc.

Front Cover: Chemical and Biological Strategies for Profiling Protein‐Protein Interactions in Living Cells (Chem. Asian J. 14/2023)

Elucidation of Protein-Protein Interactions (PPIs) in living cells/animals is very important for the study of basic biology and the development of novel therapeutics. Recent advances in the techniques for profiling PPIs, such as fluorescence microscopy and various proximity labeling-based methods coupled with mass spectrometry (MS), have provided such a great opportunity to study PPIs in their native states with spatiotemporal resolution, especially the ones that are inaccessible to conventional co-immunoprecipitation (co-IP) and pull-down assays. More information can be found in the Review by You-Yu Wang, Wenyi Li, Bang-Ce Ye, and Xiao-Bao Bi.

Copper-free click chemistry assisted antibodies for immunodetection of interleukin-10 in saliva

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that is secreted in response to an acute phase inflammation in patients who are suffering from heart failure (HF). The aim of this work was to develop an electrochemical biosensor for determining salivary IL-10 levels. Biofunctionalization strategy was improved through the use of copper-free click chemistry for the developed sensor due to its advantages, leading to high quantitative yields of stable triazoles, rapid reaction, no cytotoxic Cu(I) catalyst requirement, and high specificity of cyclooctynes toward azides. The approach involved in binding of dibenzocyclooctyne acid (DBCO-COOH) to thiol-azide assembled gold microelectrodes, later capturing the monoclonal IL-10 antibody (IL-10 mAb), and ultimately allowing direct detection of IL-10 antigen. Fourier transform infrared spectroscopy (FTIR) and nanoplotter associated with fluorescence microscopy methods have been employed to analyze and prove the biofunctionalization of the gold microelectrodes. Moreover, the electrochemical impedance spectroscopy (EIS) technique was used for detecting IL-10 antigen. The developed immunosensor showed a semi-logarithmic linear range, from 0.1 pg/mL to 5 pg/mL with R2 = 0.9815 and a limit of quantitation (LOQ) of 0.1 pg/mL with relative standard deviation (RSD) of 10.67%. The specificity of the immunosensor was evaluated using an inflammatory cytokine, and none of it generated detectable EIS signals. Finally, the successful analysis of saliva samples from a healthy volunteer without Coronavirus (COVID-19) infection demonstrated the usefulness of the developed immunosensor.

Structure of Microbial Communities and Biological Activity in Tundra Soils of the Euro-Arctic Region (Rybachy Peninsula, Russia).

The relevance of the Arctic regions' study is rapidly increasing due to the sensitive response of fragile ecosystems to climate change and anthropogenic pressure. The microbiome is an important component that determines the soils' functioning and an indicator of changes occurring in ecosystems. Rybachy Peninsula is the northernmost part of the continental European Russia and is almost completely surrounded by Barents Sea water. For the first time, the microbial communities of the Entic Podzol, Albic Podzol, Rheic Histosol and Folic Histosol as well as anthropogenically disturbed soils (chemical pollution and human impact, growing crops) on the Rybachy Peninsula were characterized using plating and fluorescence microscopy methods, in parallel with the enzymatic activity of soils. The amount and structure of soil microbial biomass, such as the total biomass of fungi and prokaryote, the length and diameter of fungal and actinomycete mycelium, the proportion of spores and mycelium in the fungal biomass, the number of spores and prokaryotic cells, the proportion of small and large fungal spores and their morphology were determined. In the soils of the peninsula, the fungal biomass varied from 0.121 to 0.669 mg/g soil. The biomass of prokaryotes in soils ranged from 9.22 to 55.45 μg/g of soil. Fungi predominated, the proportion of which in the total microbial biomass varied from 78.5 to 97.7%. The number of culturable microfungi ranged from 0.53 to 13.93 × 103 CFU/g in the topsoil horizons, with a maximum in Entic Podzol and Albic Podzol soils and a minimum in anthropogenically disturbed soil. The number of culturable copiotrophic bacteria varied from 41.8 × 103 cells/g in a cryogenic spot to 5551.3 × 103 cells /g in anthropogenically disturbed soils. The number of culturable oligotrophic bacteria ranged from 77.9 to 12,059.6 × 103 cells/g. Changes in natural soils because of anthropogenic impact and a change in vegetation types have led to a change in the structure of the community of soil microorganisms. Investigated tundra soils had high enzymatic activity in native and anthropogenic conditions. The β-glucosidase and urease activity were comparable or even higher than in the soils of more southern natural zone, and the activity of dehydrogenase was 2-5 times lower. Thus, despite the subarctic climatic conditions, local soils have a significant biological activity upon which the productivity of ecosystems largely depends. The soils of the Rybachy Peninsula have a powerful enzyme pool due to the high adaptive potential of soil microorganisms to the extreme conditions of the Arctic, which allows them to perform their functions even under conditions of anthropogenic interference.

Graphene Quantum Dot‐PEI‐Cyclodextrin Nanocarrier for Simultaneous miR21a Delivery and Cell Imaging in Cancer Therapy

AbstractOver‐expression of miR21 plays an important role in several cancers by promoting cancer cell proliferation, migration, invasion, and metastasis. Here, we attempted to prepare a beta cyclodextrin‐polyethyleneimine‐graphene quantum dot (βCD‐PEI‐GQD) nanocarrier for cellular delivery of miR21a. For this purpose, tosylated‐βCD and GQD were conjugated to branched PEI. The product was characterized by FTIR, 1H‐NMR, and fluorescence spectroscopy. The morphology, particle size distribution, and ζ‐potential of miR21a were examined by TEM and DLS following overnight incubation with βCD‐PEI‐GQD in aqueous media. The miR21 silencing was measured by stem‐loop RT‐PCR in HepG2 human hepatoma cell line. Cellular uptake and cell toxicity assays were determined by fluorescence microscopy and Trypan blue staining method, respectively. The formation of miR21a/CD‐PEI‐GQD Nanoplex with a decreased average size of 114 nm and a ζ‐potential (+36.1 mV) lower than CD‐PEI‐GQD nanocarrier by adding miR21a was confirmed at optimum C/P ratio =8.7. RT‐PCR revealed that miR21a/βCD‐PEI‐GQD Nanoplex significantly downregulated miR21 expression levels effectively. Overall, miR21a delivery using CD‐PEI‐GQD is presented as a novel trackable nanocarrier for cancer therapy.

Detection of Apical Dental Resorption Caused by Endodontic Infection in Mice Using Fluorescence and Bright-Field Microscopy

The aim of this study was to evaluate the sensitivity, specificity, and predictive values of the fluorescence microscopy method in the detection of apical dental reabsorption after induction of apical periodontitis in animal models. Forty-first molars of mice, aged 6 to 8 weeks, had their root canals exposed to the oral environment or were maintained healthy as controls (n = 20). After 14 and 42 days, mice were euthanized and tissues were collected for histological evaluation by means of bright field and fluorescence microscopy. The accuracy of fluorescence microscopy in identifying apical external dental resorption was investigated using a diagnostic validation test based on the sensitivity (S) and specificity (E) properties. Bright-field microscopy revealed a higher number of specimens with scores of 1 to 3 - absence of apical dental resorption (n = 29; 52%), while fluorescence microscopy revealed a higher number of specimens with scores of 4 to 6 - presence of apical dental resorption (n = 37; 66%). Out of 56 specimens, 26 were TP, 11 were FP, and 19 were TN. No FN result was observed. Fluorescence microscopy presented a sensitivity value of 1, similar to the bright-field method, while specificity was lower (0.633). The accuracy of the fluorescent method to detect apical dental resorption was 0.804. Fluorescence microscopy revealed a higher number of false positive apical dental resorption than bright-field microscopy. The detection of apical dental resorption was not impacted by the sensitivity of the method but by its specificity.

Research on advanced asbestos testing using asbestos-binding proteins and fluorescence

It was discovered in the 1970s that asbestos fibre inhalation can cause lung cancer and other lethal lung conditions, and its use is therefore banned in many countries. However, large quantities of asbestos containing materials remain in old buildings and it is also found in natural materials such as talc. Professor Akio Kuroda, Graduate School of Integrated Sciences for Life, Hiroshima University, is focused on the importance of fast and accurate asbestos testing. He and his team have been developing testing techniques to accurately pinpoint the presence of asbestos, including a fluorescent microscopy (FM) method that offers increased sensitivity, accuracy and convenience, and an asbestos-specific protein probe combined with a fluorescent marker that allows users to easily visualise asbestos fibres under a fluorescent microscope. Kuroda has also used engineered peptides, to develop a highly specific probe that binds to asbestos fibres and flags the presence of even minute fibres of the material. He and the team have used proteins based on samples from the Escherichia coli cellular protein library to develop probes that cover different types of asbestos.