Study of resveratrol against bone loss by using in-silico and in-vitro methods

By applying the in-silico method, resveratrol was docked on those proteins which are responsible for bone loss. The Molecular docking data between the resveratrol and Receptor activator of nuclear factor-kappa-Β ligand [RANKL] receptors proved that resveratrol binds tightly to the receptors, showed the highest binding affinities of −6.9, −7.6, −7.1, −6.9, −6.7, and −7.1 kcal/mol. According to in-vitro data, Resveratrol reduced the osteoclasts after treating Marrow-Derived Macrophages [BMM] with Macrophage colony-stimulating factor [MCSF] 20ng / ml and RANKL 50ng / ml, with different concentrations of resveratrol (2.5, 10 μg / ml) For 7 days, the cells were treated with MCSF (20 ng / ml) and RANKL (40 ng / ml) together with concentrated trimethyl ether and resveratrol (2.5, 10 μg / ml) within 12 hours. Which, not affect cell survival. After fixing osteoclast cells with formaldehyde fixative on glass coverslip followed by incubation with 0.1% Triton X-100 in PBS for 5 min and after that stain with rhodamine phalloidin staining for actin and Hoechst for nuclei. Fluorescence microscopy was performed to see the distribution of filaments actin [F.actin]. Finally, resveratrol reduced the actin ring formation. Resveratrol is the best bioactive compound for drug preparation against bone loss.

Rhamnolipids as a Tool for Eradication of Trichosporon Cutaneum Biofilm

Microbial biofilms formed by pathogenic and antibiotic-resistant microorganisms represent a serious threat for public health in medicine and many industrial branches. Biofilms are involved in many persistent and chronic infections, the biofouling of water and food contamination. Therefore, current research is involved in the development of new treatment strategies. Biofilm is a complex system, and thus all aspects of the measurement and monitoring of its growth and eradication in various conditions, including static and dynamic flow, are issues of great importance. The antibiofilm character of rhamnolipid mixtures produced by four Pseudomonas aeruginosa strains was studied under different conditions. For this purpose, the biofilm of opportunistic pathogen Trichosporon cutaneum was used and treated under static conditions (microscope glass coverslip in a Petri dish) and under dynamic conditions (a single-channel flow cell). The results show that the biological activity of rhamnolipids depends both on their properties and on the conditions of the biofilm formation. Therefore, this aspect must be taken into account when planning the experimental or application design.

A transparent waveguide chip for versatile total internal reflection fluorescence-based microscopy and nanoscopy

Total internal reflection fluorescence (TIRF) microscopy is an imaging technique that, in comparison to confocal microscopy, does not require a trade-off between resolution, speed, and photodamage. Here, we introduce a waveguide platform for chip-based TIRF imaging based on a transparent substrate, which is fully compatible with sample handling and imaging procedures commonly used with a standard #1.5 glass coverslip. The platform is fabricated using standard complementary metal-oxide-semiconductor techniques which can easily be scaled up for mass production. We demonstrate its performance on synthetic and biological samples using both upright and inverted microscopes, and show how it can be extended to super-resolution applications, achieving a resolution of 116 nm using super resolution radial fluctuations. These transparent chips retain the scalable field of view of opaque chip-based TIRF and the high axial resolution of TIRF, and have the versatility to be used with many different objective lenses, microscopy methods, and handling techniques. We see this as a technology primed for widespread adoption, increasing both TIRF’s accessibility to users and the range of applications that can benefit from it.

Continuous Real-Time Motility Analysis of Acanthamoeba Reveals Sustained Movement in Absence of Nutrients

Acanthamoeba keratitis is a serious ocular infection which is challenging to treat and can lead to blindness. While this pathogen is ubiquitous and can contaminate contact lenses after contact with water, its habits remain elusive. Understanding this organism’s natural behavior will better inform us on how Acanthamoeba colonize contact lens care systems. Acanthamoeba trophozoites were allowed to adhere to either a glass coverslip or non-nutrient agar (NNA) within a flow cell with nutrients (Escherichia coli or an axenic culture medium (AC6)) or without nutrients (Ringer’s solution). Images were taken once every 24 s over 12 h and compiled, and videos were analyzed using ImageJ Trackmate software. Acanthamoeba maintained continuous movement for the entire 12 h period. ATCC 50370 had limited differences between conditions and surfaces throughout the experiment. Nutrient differences had a noticeable impact for ATCC 30461, where E. coli resulted in the highest total distance and speed during the early periods of the experiment but had the lowest total distance and speed by 12 h. The Ringer’s and AC6 conditions were the most similar between strains, while Acanthamoeba in the E. coli and NNA conditions demonstrated significant differences between strains (p < 0.05). These results indicate that quantifiable visual tracking of Acanthamoeba may be a novel and robust method for identifying the movement of Acanthamoeba in relation to contact lens care products. The present study indicates that Acanthamoeba can undertake sustained movement for at least 12 h with and without nutrients, on both rough and smooth surfaces, and that different strains have divergent behavior.

Amplification-free detection of viral RNA by super resolution imaging-based CRISPR/Cas13a System

RNA detection is crucial for biological research and clinical diagnosis. The current methods include both direct and amplification-based RNA detection. These methods require complicated procedures, suffering from low sensitivity, slow turnaround, and amplification bias. The CRISPR/Cas13a system is a direct RNA detection method via target RNA induced collateral cleavage activity. However, to detect low concentration RNA with CRISPR/Cas13a, target amplification is always required. Herein, we optimize the components of the CRISPR/Cas13a assay to enhance the sensitivity of viral RNA detection which improve the detection limit from 1 pM up to 100 fM. In addition, the integration of CRISPR/Cas13a biosensing and single molecule super resolution imaging is a novel strategy for direct and amplification-free RNA detection. After surface modification, fluorescent RNA reporters are immobilized on the glass coverslip surface and fluorescent signals are captured by total internal reflection fluorescence microscopy (TIRFM), shifting the measurement from spectroscopy to imaging. We quantify the fluorescence signal intensity before and after collateral cleavage of the CRISPR system when viral RNA is present and achieve a detection limit of 10 fM. Therefore, we provide a novel TIRFM-based system to visualize the CRISPR trans-cleavage for direct and robust RNA detection.

P–514 RAN-Translation in Fragile X associated Premature Ovarian Insufficiency (FXPOI): FMRpolyG as a predictive tool

Study question Does repeat-associated non-AUG (RAN) translation lead to accumulation of polyglycine- containing protein (FMRpolyG) in human lymphocytes and mural granulosa cells of FMR1 premutation carriers? Summary answer Lymphocytes and granulosa cells from FMR1 premutation carriers contain intracellular inclusions that stain positive for both FMRpolyG and ubiquitin. What is known already: Fragile-X-associated-Primary-Ovarian-Insufficiency (FXPOI) is characterized by oligo/amenorrhea and hypergonadotropic hypogonadism associated with the expansion of CGG-repeats in the 5’UTR of FMR1, called premutation (PM) (n: 55–200). Approximately 20% of women carrying a FMR1-premutation (PM) allele develop FXPOI. RAN-translation dependent on variable CGG-repeat length is hypothesized to cause FXPOI due to the production of a polyglycine-containing FMR1-protein, FMRpolyG. Recently, FMRpolyG inclusions were found in neuronal brain cells of FXTAS patients and stromal cells of the ovary of an FXPOI patient. Study design, size, duration: Lymphocytes and granulosa cells (GCs) from women with PM (6) and women without PM (10) (controls) were analyzed by immunofluorescence (IF) staining for the presence of inclusions positive for ubiquitin and FMRpolyG. Cell lysis and protein extraction samples were subjected to Fluorescent Western Blot (WB) analysis to detect FMRP and FMRpolyG Participants/materials, setting, methods Human GCs were obtained from follicular fluid after oocyte retrieval and lymphocytes were isolated from peripheral blood using Ficoll-Paque. Cells suspended in PBS were adhered to a glass-coverslip placed at the bottom of the 6-well culture plate, via gravity sedimentation. Adhered cells were fixed, IF staining for FMRpolyG and ubiquitin was performed and analyzed by fluorescence microscopy. Fluorescent WB was used to demonstrate the expression of FMRP, FMRpolyG in extracted protein from lymphocytes and GCs. Main results and the role of chance FMRP was successfully detected by fluorescence WB in both lymphocytes and GCs. FMRP is mainly present in cytoplasm and was expressed in greater amount in GCs than in leukocytes. Moreover, FMRP expression was significantly decreased in GCs from FMR1-PM compared with controls. Lymphocytes from PM-carriers and controls were immunostained for FMRpolyG and ubiquitin. In PM-carriers, FMRpolyG was present as aggregates, whereas in controls only a weak signal without inclusions was detectable. The expression pattern of FMRpolyG in GCs was similar to that in lymphocytes with a significant increase in PM-carriers. There, the FMRpolyG-aggregates additionally demonstrated as ubiquitin-positive inclusions. These may resemble the toxic potential of these protein fractions involved the ovarian damage in developing FXPOI. Limitations, reasons for caution More patients are needed to support the present findings. Further investigation into the possible consequences of these FMRpolyG-positive inclusions in PM-carriers is also advisable. Wider implications of the findings: We found for the first time FMRpolyG-accumulation in lymphocytes and GCs from FMR1-PM-carriers in ubiquitin-positive inclusions. Future experiments evaluating consistency in more patients and elucidating the impact on fertility and prospective value for individual ovarian reserve are therefore in preparation. Trial registration number Not applicable

Dually nanocoated planar waveguides towards multi-parameter sensing

The incidence of light on the edge of a glass coverslip for a microscope slide, deposited with a thin film on both faces, permits exciting two resonances in each polarisation state of the input light, TE and TM. This dually nanocoated waveguide can be used for detecting simultaneously two different parameters on the basis of a further deposition of suitable materials on each face. As an example, the possibility of detecting temperature and humidity by using polydimethylsiloxane and agarose coatings, respectively, was demonstrated, which opens the path for the development of other dual-parameter sensors, and for even more parameters in cases in which each face of the coverslip is patterned. Moreover, the device was optimised in order to position two resonances in the near infrared (NIR) and two resonances in the visible region, with sensitivities of 0.34 nm/°C and 0.23 nm/%RH in the visible region and 1.16 nm/°C and 0.34 nm/%RH in the NIR, respectively, demonstrating the possibility of using the device in both spectral ranges and opening the path for the development of sensors based on multiple resonances, each one related to a different parameter to be detected.

Surface Sediment Diatom as A Water Quality Indicator: Case Study: Cilalay and Cibuntu Ponds, Cibinong

Diatom is classified as algae within the Division of Bacillariophyta. They are unicellular eukaryotic organisms characterized by siliceous cell walls that can be long preserved in sediments. Therefore, diatom analysis in sediment records is a potential water quality indicator for present or paleo studies. The current knowledge on the distribution and diversity of diatoms in the sediment in the urban pond is poorly known. This study aimed to identify the distribution and diversity of diatoms from the sediments of the pond. We expect to obtain a primary database of a variety of diatoms. The sediment samples were taken from Cibuntu and Cilalay Ponds in Cibinong Botanical Garden. Sediments were digested using HCL and H2O2. The resulting diatom solution was dried and transferred onto glass coverslip, which subsequently mounted onto microscope slides using Naphrax (Refraction index 1.7). Diatom identification was examined using a light microscope at 1,000x magnification. Diatom communities in Cibuntu Pond were dominated by species Aulacoseria ambigua, Eunotia bilunaris, Cymbopleura sp, Discostella stelligera, and Rossithidium sp with diversity index of 2.4 and species evenness of 0.8. Whereas, species Fragilaria sp, Eunotia monodon, Navicymbula pusilla, Eunotia bilunaris, and Pinnularia viridis were predominant in Cilalay Pond with diversity index of 1.6 and species evenness of 0.5. Based on the diatom community, Cibuntu Pond is eutrophic indicated by the occurrence of Aulacoseria ambigua, whereas Cilalay Pond is meso-eutrophic indicated by the dominance of Fragillaria. This exploratory survey provides the first inventory of diatom assemblage in Cibuntu and Cilalay Ponds for roughly inferring the environmental changes in a shallow lake ecosystem.

Measurement of hindered diffusion in complex geometries for high-speed studies of single-molecule forces

In a high-speed single-molecule experiment with a force probe, a protein is tethered between two substrates that are manipulated to exert force on the system. To avoid nonspecific interactions between the protein and nearby substrates, the protein is usually attached to the substrates through long, flexible linkers. This approach precludes measurements of mechanical properties with high spatial and temporal resolution, for rapidly exerted forces are dissipated into the linkers. Because mammalian hearing operates at frequencies reaching tens to hundreds of kilohertz, the mechanical processes that occur during transduction are of very short duration. Single-molecule experiments on the relevant proteins therefore cannot involve long tethers. We previously characterized the mechanical properties of protocadherin 15 (PCDH15), a protein essential for human hearing, by tethering an individual monomer through very short linkers between a probe bead held in an optical trap and a pedestal bead immobilized on a glass coverslip. Because the two confining surfaces were separated by only the length of the tethered protein, hydrodynamic coupling between those surfaces complicated the interpretation of the data. To facilitate our experiments, we characterize here the anisotropic and position-dependent diffusion coefficient of a probe in the presence of an effectively infinite wall, the coverslip, and of the immobile pedestal.

Stripping Material from a Supported Lipid Bilayer with High Speed Buffer Flow

A microfluidic device was created and used to demonstrate that supported lipid bilayers can be deposited on clean glass slides and removed using high velocity buffer flow (1-4 m/s linear velocity). This was accomplished by forcing the flow through a microfluidic channel covering an annealed glass coverslip bearing a supported lipid bilayer (SLB). The removal of bilayer material was monitored via fluorescence microscopy, and two basic regimes were observed: at 1-2 m/s smaller areas were stripped, while at 3-4 m/s larger areas were stripped. SLB removal was verified by two means. First, lipid vesicles labeled with a different fluorescent dye were added to the device and filled in holes left by the removal of the original SLB, allowing stripping to be verified visually. Second, the solutions obtained from stripping were concentrated and the fluorescence in the concentrates was measured. The ability to strip SLB from glass provides a relatively gentle method of creating spatially inhomogeneous SLB, which could be a useful tool in the continued investigation of membrane properties and components. KEYWORDS: Supported Lipid Bilayer; Membrane Vesicle; Microfluidic Device