Microscopic Visualization Research Articles

Quantification of Polystyrene Uptake by Different Cell Lines Using Fluorescence Microscopy and Label-Free Visualization of Intracellular Polystyrene Particles by Raman Microspectroscopic Imaging.

Environmental pollution caused by plastic is a present problem. Polystyrene is a widely used packaging material (e.g., Styrofoam) that can be broken down into microplastics through abrasion. Once the plastic is released into the environment, it is dispersed by wind and atmospheric dust. In this study, we investigated the uptake of polystyrene particles into human cells using A549 cells as a model of the alveolar epithelial barrier, CaCo-2 cells as a model of the intestinal epithelial barrier, and THP-1 cells as a model of immune cells to simulate a possible uptake of microplastics by inhalation, oral uptake, and interaction with the cellular immune system, respectively. The uptake of fluorescence-labeled beads by the different cell types was investigated by confocal laser scanning microscopy in a semi-quantitative, concentration-dependent manner. Additionally, we used Raman spectroscopy as a complementary method for label-free qualitative detection and the visualization of polystyrene within cells. The uptake of polystyrene beads by all investigated cell types was detected, while the uptake behavior of professional phagocytes (THP-1) differed from that of adherent epithelial cells.

51 A preliminary study of the effect of dietary cottonseed on the follicular reserve of does artificially infected with Haemonchus contortus.

Abstract Numerous studies have reported the negative effect of cottonseed gossypol on the ovarian reserve of the follicles, impairing female fertility. Injuries resulting in the depletion of primordial follicles can lead to permanent infertility. At birth, female mammals possess a fixed number of ovarian primordial follicles, which develop into primary, secondary, and antral follicles in a continuous and irreversible process. In this study, we evaluated the potential effects of dietary cottonseed gossypol in combination with parasitic infection on ovarian follicular reserve in meat goats. A total of 40 Boer x Spanish cross does were randomly subjected to cottonseed (CS) and parasite (P: Haemonchus contortus) in a 2x2 factorial arrangement of does (n = 8) per treatment groups (CS-/P-; CS+/P-; CS-/P+, and CS+/P+). Does were artificially infected with Haemonchus contortus for 2 d, and after 25 d, they began shedding parasite eggs. Thereafter, does were fed a hay diet supplemented with cottonseed (0.5% BW; CS+) or concentrate (CS-), to meet the nutritional requirements. Ten weeks later, animals were slaughtered, and ovaries of each treatment group (n = 4) were fixed with formalin and subjected to a standard procedure of histology staining (Periodic acid–Schiff and Hematoxylin counterstaining). Cuts of one left and one right ovary of two does were placed on microscope slides, and 48 slides were subjected to a microscope visualization (EVOS FL Auto; 20x magnification). Primordial follicles were classified as healthy (stained nucleus), unhealthy (pycnotic), and undetermined. Data were analyzed with ANOVA 1 and expressed as mean ± sem. P < 0.05 indicates a significant difference. A total of 20,562 primordial follicles corresponding to 10,410 (50.6%) healthy; 3,729 (18.1%) unhealthy; and 6,909 (33.6%) undetermined were examined; of which 26%, 20%, 31%, and 23% of total primordial follicles belonged to the CS-/P-, CS-/P+, CS+/P-, and CS+/P+ treatment groups, respectively. A significantly (P < 0.05) greater number of follicles were counted on left vs. right ovary sections (11,584 vs. 8,978) corresponding to 56.3% vs. 43.7% of total follicle counts. Averages of 121 ± 6 vs. 101±6 follicles were calculated per left and right ovary sections. Diets containing cottonseed (CS+/P-) increased the average number of follicles per ovary section, while parasite infections significantly (P < 0.05; CS-/P+ and CS+/P+) decreased these numbers. Regardless of the treatment group, the proportions of healthy follicles remained comparable between treatment groups. We concluded that dietary cottonseed and parasitic infections inversely affect ovarian follicle reserve, while the proportions of healthy follicles were unaffected across treatment groups. Nevertheless, higher microscopic magnification is needed to better characterize the large proportion of undetermined follicles.

Stoichiometry of long noncoding RNA interactions with other RNAs: Insights from OIP5-AS1.

Long noncoding (lnc)RNAs modulate gene expression programs in a range of developmental processes in different organs. In skeletal muscle, lncRNAs have been implicated in myogenesis, the process whereby muscle precursor cells form muscle fibers during embryonic development and regenerate muscle fibers in the adult. Here, we discuss OIP5-AS1, a lncRNA that is highly expressed in skeletal muscle and is capable of coordinating protein expression programs during myogenesis. Given that several myogenic functions of OIP5-AS1 involve interactions with MEF2C mRNA and with the microRNA miR-7, it was critical to carefully evaluate the precise levels of OIP5-AS1 during myogenesis. We discuss the approaches used to examine lncRNA copy number using OIP5-AS1 as an example, focusing on quantification by quantitative PCR analysis with reference to nucleic acids of known abundance, by droplet digital (dd)PCR measurement, and by microscopic visualization of individual lncRNAs in cells. We discuss considerations of RNA stoichiometry in light of developmental processes in which lncRNAs are implicated. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

Comparison of cognitive workload and surgical outcomes between a three-dimensional and conventional microscope macular hole surgery

BackgroundPerforming a surgical task subjects the surgeon to multitudinal stressors, especially with the newer 3D technology. The quantum of cognitive workload using this modern surgical system in comparison to the Conventional microscope system remains unexplored. We evaluate the surgeon’s cognitive workload and the surgical outcomes of macular hole(MH) surgery performed on a 3D versus a Conventional microscope operating system.Methods50 eyes of 50 patients with MH undergoing surgery using the 3D or Conventional microscope visualization system. Cognitive workload assessment was done by real-time tools(Surgeons’ heart rate [HR] and oxygen saturation[SPO2]) and self-report tool(Surgery Task Load Index[SURG-TLX] questionnaire) of three Vitreoretinal surgeons. Based on the SURG-TLX questionnaire, an assessment of the workload was performed.ResultsOf the 50 eyes, 30 eyes and 20 eyes underwent surgery with the Conventional microscope and the 3D system, respectively. No difference was noted in the MH basal-diameter(p = 0.128), total surgical-duration(p = 0.299), internal-limiting membrane(ILM) peel time(p = 0.682), and the final visual acuity (VA; p = 0.515) between the two groups. Both groups showed significant improvement in VA(p < 0.001) with a 90% closure rate at one-month post-surgery. Cognitive workload comparison, the intraoperative HR(p = 0.024), total workload score(P = 0.005), and temporal-demand dimension(p = 0.004) were significantly more in Conventional microscope group as compared to 3D group. In both the groups, the HR increased significantly from the baseline while performing ILM peeling and at the end.ConclusionThe surgeon’s cognitive workload is markedly reduced while performing macular hole surgery with a 3D viewing system. Moreover, duration of surgery including ILM peel time, MH closure rates, and visual outcomes remains unaffected irrespective of the operating microscope system.

Using Unity for Scientific Visualization as a Course-based Undergraduate Research Experience.

We have developed a series of course-based undergraduate research experiences for students integrated into course curriculum centered around the use of 3D visualization and virtual reality for science visualization. One project involves the creation and use of a volumetric renderer for hyperstack images, paired with a biology project in confocal microscopy. Students have worked to develop and test VR enabled tools for confocal microscopy visualization across headset based and CAVE based VR platforms. Two applications of the tool are presented: a rendering of Drosophila primordial germ cells coupled with automated detection and counting, and a database in development of 3D renderings of pollen grains. Another project involves the development and testing of point cloud renderers. Student work has focused on performance testing and enhancement across a range of 2D and 3D hardware, including native Quest apps. Through the process of developing these tools, students are introduced to scientific visualization concepts, while gaining practical experience with programming, software engineering, graphics, shader programming, and cross-platform design.

A One-Stone-Two-Birds Strategy of Targeting Microbubbles with "Dual" Anti-Inflammatory and Blood-Brain Barrier "Switch" Function for Ischemic Stroke Treatment.

Inflammation is considered to be the main target of the development of new stroke therapies. There are three key issues in the treatment of stroke inflammation: the first one is how to overcome the blood-brain barrier (BBB) to achieve drug delivery, the second one is how to select drugs to treat stroke inflammation, and the third one is how to achieve targeted drug delivery. In this study, we constructed hydrocortisone-phosphatidylserine microbubbles and combined them with ultrasound (US)-targeted microbubble destruction technology to successfully open the BBB to achieve targeted drug delivery. Phosphatidylserine on the microbubbles was used for its "eat me" effect to increase the targeting of the microvesicles. In addition, we found that hydrocortisone can accelerate the closure of the BBB, achieving efficient drug delivery while reducing the entry of peripheral toxins into the brain. In the treatment of stroke inflammation, it was found that hydrocortisone itself has anti-inflammatory effects and can also change the polarization of microglia from the harmful pro-inflammatory M1 phenotype to the beneficial anti-inflammatory M2 phenotype, thus achieving dual anti-inflammatory effects and enhancing the anti-inflammatory effects in ischemic areas after stroke, well reducing the cerebellar infarction volume by inhibiting the inflammatory response after cerebral ischemia. A confocal microendoscope was used to directly observe the polarization of microglial cells in living animal models for dynamic microscopic visualization detection showing the advantage of being closer to clinical work. Taken together, this study constructed a multifunctional targeted US contrast agent with the function of "one-stone-two-birds", which can not only "on-off" the BBB but also have "two" anti-inflammatory functions, providing a new strategy of integrated anti-inflammatory targeted delivery and imaging monitoring for ischemic stroke treatment.

Optimal technique for canine mesenchymal stem cells labeling with novel SPIO, MIRB™: for MRI detection of transplanted stem cells canine stroke model

ABSTRACT Background Cell-based therapy has emerged as a promising avenue for post-stroke recovery. A significant challenge lies in tracking the distribution and engraftment of transplanted cells within the target cerebral tissue. To address this, we turn to the potential of Brain MRI detection of mesenchymal stem cells (MSCs), achieved by labeling these cells with superparamagnetic iron oxide (SPIO). This is the first report of a technique to label canine MSCs using a commercially available SPIO, Molday ION Rhodamine B (MIRB), to optimize both viability and labeling efficacy for transplantation purposes.” Method Canine MSCs were incubated with addition of different MIRB concentration from 0, 10, 20, 30 μg Fe/ml. The cellular uptake of MIRB was confirmed through the analysis of fluorescent images and flow cytometry. The morphological characteristics of MSCs were assessed via microscopic visualization. Cellular viability was evaluated using both a cellometer and flow cytometry. Result Fluorescent microscopic images of all MIRB incubated MSCs groups show >70% labeled cells with homogenous signal intensity. Notably, the morphology of MSCs remained unaltered in the 10 μg Fe/ml group compared to the control group. Furthermore, among the labeled groups, the 10 μg Fe/ml concentration exhibited the highest viability when assessed using two different flow cytometry methods (95.3%, p < 0.05). Conclusion This study successfully labels canine MSCs with MIRB. The optimal concentration of 10 μg Fe/ml demonstrates optimal viability, labeling efficacy, and preserved cellular morphology.

Targeted Single Particle Tracking with Upconverting Nanoparticles.

Single particle tracking (SPT) is a powerful technique for real-time microscopic visualization of the movement of individual biomolecules within or on the surface of living cells. However, SPT often suffers from the suboptimal performance of the photon-emitting labels used to tag the biomolecules of interest. For example, fluorescent dyes have poor photostability, while quantum dots suffer from blinking that hampers track acquisition and interpretation. Upconverting nanoparticles (UCNPs) have recently emerged as a promising anti-Stokes luminescent label for SPT. In this work, we demonstrated targeted SPT using UCNPs. For this, we synthesized 30 nm diameter doped UCNPs and coated them with amphiphilic polymers decorated with polyethylene glycol chains to make them water-dispersible and minimize their nonspecific interactions with cells. Coated UCNPs highly homogeneous in brightness (as confirmed by a single particle investigation) were functionalized by immunoglobulin E (IgE) using a biotin-streptavidin strategy. Using these IgE-UCNP SPT labels, we tracked high-affinity IgE receptors (FcεRI) on the membrane of living RBL-2H3 mast cells at 37 °C in the presence and absence of antigen and obtained good agreement with the literature. Moreover, we used the FcεRI-IgE receptor-antibody system to directly compare the performance of UCNP-based SPT labels to organic dyes (AlexaFluor647) and quantum dots (QD655). Due to their photostability as well as their backgroundless and continuous luminescence, SPT trajectories obtained with UCNP labels are no longer limited by the photophysics of the label but only by the dynamics of the system and, in particular, the movement of the label out of the field of view and/or focal plane.

Experimental investigation of surface wettability on frosting propagation characteristics

The frost propagation process on surfaces plays a crucial role in the initial stages of surface condensation frosting, making it a significant aspect of the entire process. To investigate the relationship between surface wettability and the frost propagation velocity during condensation frosting, a microscopic visualization experiment platform was established. Three high-reflectivity surfaces with varying wettability were prepared to meet the experimental requirements. The frost propagation process can be divided into two stages: the freezing of intra-droplet and the ice bridge propagation between droplets. Experiment results revealed that the primary factor affecting the propagation speed of the ice bridge is the distance between the ice bridge and the droplet. As this distance reduces from approximately 16 μm to 2 μm, the propagation velocity of the ice bridge increases dramatically from around 2 μm/s to about 12 μm/s. Furthermore, the overall frost propagation velocity on surfaces with different wettability properties, were evaluated. The results indicate that the significant difference in droplet distribution characteristics and ice bridge propagation coefficients on superhydrophobic surfaces is the cause of slow frost formation during frosting on cold surfaces. This study identifies the key factor affecting the frost propagation and the inhibition of frost formation, namely, the droplet size distribution characteristics of superhydrophobic surfaces. These insights and findings provide a foundation for the design and optimization of anti-frost surfaces.

Ultrasonic fatigue testing of AISI 304 and 316 stainless steels under environmental and immersion conditions

Ultrasonic fatigue tests were carried out on stainless steel AISI 316 and 304, under two modalities: at room temperature and in immersion (water for 316 and antifreeze for 304 steels); all tests were carried out with a loading ratio R=-1. The results obtained in the tests at room temperature (without immersion), for both materials exhibited a significant increase in temperature, leaving heat marks on the narrow section of the specimens. This phenomenon occurred due to the low coefficient of thermal conductivity of these stainless steels (16.2 W/ (m °K)), and the recorded temperatures were around 200 °C, generating instantaneous failure of material. Analyzes of fracture surfaces on specimens tested at room temperature reveal that crack initiation was related to the high temperature, causing alteration at the granular scale of the material, followed by a typical behavior crack propagation and failure. For specimens tested under immersion conditions, it was possible to reduce the temperature below 100 °C, which solved the problem of failure due to thermal effect. The results for 316 stainless steel immersed in water showed a fatigue life of 1.188×1010 cycles at188 MPa of stress loading in the specimen; while specimens subjected to 263 MPa stress showed a fatigue life of around 7×106 cycles, representing a significant reduction with an approximate factor of 1700. On the other hand, specimens of 304 stainless steel immersed in antifreeze with the lowest loading values of 169 MPa, showing an infinite ultrasonic fatigue life; while tests subjected to 263 MPa loading stress attains 3.62×106 cycles of ultrasonic fatigue life. The scanning electron microscopy visualizations for both cases of immersion tests showed that the initiation and propagation of the crack occurred on the surface of the specimens, exhibiting the typical mechanical fracture behavior without any apparent thermal influence.

Microscopic experiment on efficient construction of underground gas storages converted from water-invaded gas reservoirs

Based on the microfluidic technology, a microscopic visualization model was used to simulate the gas injection process in the initial construction stage and the bottom water invasion/gas injection process in the cyclical injection-production stage of the underground gas storage (UGS) rebuilt from water-invaded gas reservoirs. Through analysis of the gas-liquid contact stabilization mechanism, flow and occurrence, the optimal control method for lifecycle efficient operation of UGS was explored. The results show that in the initial construction stage of UGS, the action of gravity should be fully utilized by regulating the gas injection rate, so as to ensure the macroscopically stable migration of the gas-liquid contact, and greatly improve the gas sweeping capacity, providing a large pore space for gas storage in the subsequent cyclical injection-production stage. In the cyclical injection-production stage of UGS, a constant gas storage and production rate leads to a low pore space utilization. Gradually increasing the gas storage and production rate, that is, transitioning from small volume to large volume, can continuously break the hydraulic equilibrium of the remaining fluid in the porous media, which then expands the pore space and flow channels. This is conducive to the expansion of UGS capacity and efficiency for purpose of peak shaving and supply guarantee.

Toxic effects of polystyrene nanoplastics and polycyclic aromatic hydrocarbons (chrysene and fluoranthene) on the growth and physiological characteristics of Chlamydomonas reinhardtii

While the toxicity of nano-microplastics and polycyclic aromatic hydrocarbons (PAHs) to aquatic organisms is well-studied, their joint impact on microalgae is less explored. This study focused on single and combined effects of PS-NPs (30 nm; concentrations: 2, 5, 10, and 25 mg/L) and two PAHs (chrysene and fluoranthene at 10, 100 µg/L) for 96 h on the accumulation, growth, photosynthetic parameters, and oxidative stress in the Chlamydomonas reinhardtii. The findings revealed that exposure to increasing concentrations of PS-NPs significantly reduced the growth inhibition ratio and chlorophyll-a content after 96 h. Both PAHs (100 µg/L) + PS-NPs (25 mg/L), significantly reduced the growth inhibition ratio and chlorophyll-a levels. Individual and combined exposures of PS-NPs and PAHs can prompt antioxidant responses like SOD, GPx, and GST, as well as an unaffected level of non-enzymatic antioxidant GSH and diminished CAT activity. Furthermore, both PAHs + PS-NPs triggered ROS levels, resulting in cell membrane damage. However, the reduced oxidative effect of LPO of combined exposures can be attributed to the activation of antioxidant defenses. In addition, the microscopic visualization data shows that PS-NPs adhered to the surface of microalgae. Also, PS-NPs reduced the adsorption of PAHs on the surface of C. reinhardtii. Altogether, this study implied that the influence of coexistent PS-NPs should be considered in the environmental risk assessment of PAHs in aquatic environments.

Simulated outcomes for durotomy repair in minimally invasive spine surgery

Minimally invasive spine surgery (MISS) is increasingly performed using endoscopic and microscopic visualization, and the captured video can be used for surgical education and development of predictive artificial intelligence (AI) models. Video datasets depicting adverse event management are also valuable, as predictive models not exposed to adverse events may exhibit poor performance when these occur. Given that no dedicated spine surgery video datasets for AI model development are publicly available, we introduce Simulated Outcomes for Durotomy Repair in Minimally Invasive Spine Surgery (SOSpine). A validated MISS cadaveric dural repair simulator was used to educate neurosurgery residents, and surgical microscope video recordings were paired with outcome data. Objects including durotomy, needle, grasper, needle driver, and nerve hook were then annotated. Altogether, SOSpine contains 15,698 frames with 53,238 annotations and associated durotomy repair outcomes. For validation, an AI model was fine-tuned on SOSpine video and detected surgical instruments with a mean average precision of 0.77. In summary, SOSpine depicts spine surgeons managing a common complication, providing opportunities to develop surgical AI models.

Fine Particle Migration in a Gas Hydrate Sand: Single- and Two-Phase Fluid Using a Device for Observation at the Pore Scale

The production of natural gas hydrates will change the cementation strength, porosity, and effective stress in the stratum, which may lead to engineering and geological disasters. Sand production is a phenomenon where sand particles are carried out of the reservoir along with fluids during gas extraction, posing challenges to safe and sustainable production. This study explored the mechanism of fine particle migration in multiphase flow by a microscopic visualization test device. The device can inject a gas–liquid–solid phase at the same time and allow real-time observation. Experimental tests on fine particle migration of single- and two-phase fluid flow were carried out considering different conditions, i.e., fine particle concentration, fine particle size, fluid flow rate, and gas–liquid ratio. The results show that in single-phase fluid flow, the original gas will gradually dissolve in the liquid phase, and finally stay in the test device as bubbles, which can change the pore structures, resulting in the accumulation of fine particles at the gas–liquid interface. In two-phase fluid flow with mixed gas–water fluids, there are two flow modes of gas–liquid flow: mixed flow and separated flow. The interfacial tension at the gas–liquid interface can effectively migrate fine particles when the gas–liquid flows alternately and the sand production rate further increases as the gas–liquid ratio increases. In addition, changes in the concentration of fine particles, particle size, fluid flow rate, and the gas–liquid ratio will affect the migration of fine particles, leading to differences in the final sand production.

Research on the Influence of Heterogeneity and Viscosity on the Fluid Intrusion Mechanism of the Water Flooding Process Based on the Microscopic Visualization Experiment.

The flow law of immiscible fluids in porous media plays an important role in the development of oil and gas fields. In the process of water flooding reservoir development, when the water phase displaces the oil phase, a fluid with higher viscosity, as a fluid with lower viscosity, the oil-water interface will always be unstable, resulting in different fingering effects. After water flooding, the distribution law of oil and water in the reservoir is mainly affected by the fluid intrusion mechanism. Due to the difference of capillary force, viscous force, and other microscopic forces, the fluid intrusion mechanism is mainly divided into two types: viscous fingering and capillary fingering. At the same time, due to the influence of reservoir heterogeneity, the fingering effect in the process of water displacement in porous media will be influenced to a certain extent. Based on the two-dimensional microscopic visualization experiment, this paper extracted the variance of the static parameter G in the capillary number calculation method of the two-dimensional microscopic model to represent the heterogeneity and conducted displacement experiments with different viscosities and flow rates to study the influence of the flow rate, viscosity, and heterogeneity on the results of water flooding. The experiments found that as for the influence of flow velocity, with the increase of flow velocity, that is, with the increase of capillary number, the recovery degree decreases first and then increases. As for the influence of viscosity, from a numerical point of view, the displacement efficiency and conformance coefficient of the low-viscosity group are higher than those of the high-viscosity group. From the trend, with the increase of the capillary number, the displacement efficiency of both the low-viscosity and high-viscosity groups increases, while the conformance coefficient decreases first and then increases, indicating that capillary fingering and viscous fingering can occur in different viscosity reservoirs. As for the influence of heterogeneity, the conformance coefficient of the water flooding decreases with the increase of heterogeneity, and the viscous pointing trend caused by heterogeneity is stronger, resulting in an uneven water injection sweep and higher oil displacement efficiency within the swept area. It can be seen from the fluid intrusion mechanism diagram that with the increase of heterogeneity, the viscous fingering trend becomes more obvious; with the increase of viscosity, the fluid intrusion mechanism boundary moves down and the viscous fingering trend becomes more obvious.

Entomological indicators and food sources of triatomines in the Brazilian semi-arid region.

Triatomines are biological vectors of Trypanosoma cruzi, the etiological agent of Chagas Disease (CD) and have various mammalian hosts. This study evaluated the entomological indicators and food sources of triatomines in Petrolina in the semi-arid region of Brazil, where CD is endemic. Triatomines were captured indoors and outdoors through an active search and entomological indices (household and natural infections) were calculated. Parasitological analyses were performed through microscopic visualization using Giemsa-stained insect feces, and DNA sequencing was employed to identify food sources from the gut contents of 82 insects (9.05%) that were better preserved. We captured triatomines (906) in peridomicile (807) and intradomicile (99): Triatoma brasiliensis (84.7%, 767 specimens), Triatoma spp. (8.2%, 74 specimens), T. pseudomaculata (6.5%, 59 specimens), Rhodnius spp. (0.4%, four specimens), R. nasutus (0.1%, one specimen), and T. sordida (0.1%, one specimen). The household infestation index is 11.8%. Thirty-five triatomines were infected (33 T. brasiliensis and two T. pseudomaculata), corresponding to a natural infection index of 3.8%. The identified food sources were human T. pseudomaculata and T. brasiliensis, dogs for T. brasiliensis and rodents (Mus musculus) for T. brasiliensis. The results reinforce the need to intensify CD diagnosis, surveillance, and control actions, as an increase in entomological indices was recorded. Blood from humans and domestic and synanthropic animals was detected in the infected triatomines, suggesting a risk of CD vector transmission in Petrolina. As CD is a zoonosis, multidisciplinary and intersectoral CD surveillance must be conducted in the context of the One Health.

Isolation and Characterization of Extracellular Vesicles Derived from Ex Vivo Culture of Visceral Adipose Tissue.

Extracellular vesicles (EVs) are a heterogeneous group of nanoparticles possessing a lipid bilayer membrane that plays a significant role in intercellular communication by transferring their cargoes, consisting of peptides, proteins, fatty acids, DNA, and RNA, to receiver cells. Isolation of EVs is cumbersome and time-consuming due to their nano size and the co-isolation of small molecules along with EVs. This is why current protocols for the isolation of EVs are unable to provide high purity. So far, studies have focused on EVs derived from cell supernatants or body fluids but are associated with a number of limitations. Cell lines with a high passage number cannot be considered as representative of the original cell type, and EVs isolated from those can present distinct properties and characteristics. Additionally, cultured cells only have a single cell type and do not possess any cellular interactions with other types of cells, which normally exist in the tissue microenvironment. Therefore, studies involving the direct EVs isolation from whole tissues can provide a better understanding of intercellular communication in vivo. This underscores the critical need to standardize and optimize protocols for isolating and characterizing EVs from tissues. We have developed a differential centrifugation-based technique to isolate and characterize EVs from whole adipose tissue, which can be potentially applied to other types of tissues. This may help us to better understand the role of EVs in the tissue microenvironment in both diseased and normal conditions. Key features • Isolation of tissue-derived extracellular vesicles from ex vivo culture of visceral adipose tissue or any whole tissue. • Microscopic visualization of extracellular vesicles' morphology without dehydration steps, with minimum effect on their shape. • Flow cytometry approach to characterize the extracellular vesicles using specific protein markers, as an alternative to the time-consuming western blot.

Microscopic visualization of heterogeneous condensation of water vapor on hydrophilic and hydrophobic particles

Heterogeneous condensation of water vapor on particles is widely used in the fields of atmospheric cloud physics and industrial particulate abatement. Particles surface wettability plays a fundamental role in water vapor condensation, but a microscopic insight of the impact of particles surface wettability on vapor condensation is lacked. Therefore, the Environmental Scanning Electron Microscope (ESEM) is adopted to directly visualize vapor condensation on particles with different surface wettability at a microscopic scale. Firstly heterogeneous condensation of water vapor on the hydrophilic and hydrophobic particles is obtained by the ESEM. The results show that when water vapor condenses on the hydrophilic particles, a spherical cap-shaped embryo first appears and subsequently develops into a spherical droplet to finally wrap the single particle. While on the hydrophobic particles, a spherical cap-shaped embryo will first appear and continue to grow into a spherical droplet to finally detach from the particle. Then the wetting coefficient is introduced to characterize the wetting degree of the droplet on the particle. The wetting coefficient will increase with the decrease of the contact angle, so the wettability of water vapor on the hydrophilic particles is better than that on the hydrophobic particles. Meanwhile based on classical nucleation theory, the geometrical factor and the critical supersaturation will decrease when the contact angle decreases, making it easier for water vapor to condense on the hydrophilic particles. Finally the line tension is investigated to explain the condensation mechanism. On the hydrophilic particles, when the droplet has crossed the equatorial line of the particle, the positive line tension promotes the spreading movement of the droplet over the particle to form a wrapped spherical droplet. Therefore, there is a transition from a spherical cap-shaped embryo before crossing the equatorial line of the particle to a wrapped spherical droplet after crossing the equatorial line. While on the hydrophobic particles, the negative line tension suppresses the spreading movement of the droplet over the particle to form a detached spherical droplet. So there is a transition from a spherical cap-shaped embryo before crossing the equatorial line of the particle to a detached spherical droplet after crossing the equatorial line.

Development of phenyllactic acid ionic liquids and evaluation of cytotoxicity to human cervical epithelial cells.

Phenyllactic acid (PLA), is a naturally produced, broad-spectrum antimicrobial compound with activity against bacteria and fungi. PLA can be produced by a variety of lactic acid bacteria, including vaginal Lactobacillus species, which are healthy constituents of the vaginal microbiome with a protective role against invading pathogenic bacteria and/or fungi. Additionally, PLA has been shown to exhibit anti-inflammatory and immunomodulatory properties, overall indicating its therapeutic potential as an intravaginally delivered compound for modulation of the vaginal microbiome. However, PLA has low kinetic solubility in water. Hence, strategies to improve the solubility of PLA are necessary to facilitate its intravaginal delivery. Using biocompatible cations, choline and carnitine, we successfully transformed both d- and l-enantiomers of crystalline PLA into amorphous low-melting ionic liquids (ILs) with high water solubility. We further evaluated the in vitro cytotoxicity of PLA ILs to human cervical epithelial cells. Microscopic visualisation of cellular morphology using crystal violet staining and MTT cell proliferation assay revealed that PLA ILs result in minimal morphological changes and low cytotoxicity to human cervical epithelial cells. Overall, we successfully demonstrated that transforming PLA into ILs efficiently enhances its solubility in water and these formulations are not toxic to human epithelial cells. This investigation lays the groundwork for future testing of PLA ILs for their antimicrobial properties and metabolic activity within the cervicovaginal microenvironment.

An Overexpression of SLC30A6 Gene Contributes to Cardiomyocyte Dysfunction via Affecting Mitochondria and Inducing Activations in K-Acetylation and Epigenetic Proteins.

Intracellular free Zn2+ ([Zn2+]i) is less than 1-nM in cardiomyocytes and its regulation is performed with Zn2+-transporters. However, the roles of Zn2+-transporters in cardiomyocytes are not defined exactly yet. Here, we aimed to examine the role of an overexpression and subcellular localization of a ZnT6 in insulin-resistance mimic H9c2 cardiomyoblasts (IR-cells; 50-μM palmitic acid for 24-h incubation). We used both IR-cells and ZnT6-overexpressed (ZnT6OE) cells in comparison to those of H9c2 cells (CON-cells). The IR-cells have higher ZnT6-protein levels than CON-cells while this level was similar to those of ZnT6OE-cells. The [Zn2+]i in IR-cells was increased significantly and mitochondrial localization of ZnT6 was demonstrated in these cells by using confocal microscopy visualization. Furthermore, electron microscopy analysis demonstrated abnormal morphological appearance in both IR-cells and ZnT6OE-cells characterized by irregular mitochondrion cristae and condensed and dilated cisterna in the sarcoplasmic reticulum. Mitochondria were similarly depolarized in both IR-cells and ZnT6OE-cells. The protein expression level of a mitofusin protein MFN2 in the IR-cells was decreased, significantly, whereas, it was found significantly upregulated in both ZnT6-OE-cells and IR-incubated ZnT6OE-cells, which demonstrates the role of ZnT6-overexpression but not IR. Additionally, the total protein level of a mitochondrial fission protein, dynamin-related protein 1, DRP1 was found to be increased over 1.5-fold in IR-cells while this increase was found to be higher in the ZnT6OE-cells than those of IR-cells, demonstrating an additional effect on IR-increase. ZnT6-overexpression induced also significant increases in K-acetylation, trimethylation of histone H3 lysine27, and mono-methylation of histone H3 lysine36, in a similar manner to those of IR-cells. Overall, our data point out an important contribution of ZnT6-overexpression to IR-induced cellular changes, such as alteration in mitochondria function and activation of epigenetic modifications.