Fluorescence Microscopy Research Articles

Comparative Analysis of Canonical Inflammasome Activation by Flow Cytometry, Imaging Flow Cytometry and High-Content Imaging.

Inflammasome activation occurs in various diseases, including rare diseases that require multicenter studies for investigation. Flow cytometric analysis of ASC speck+ cells in patient samples can be used to detect cell type-specific inflammasome activation. However, this requires standardized sample processing and the ability to compare data from different flow cytometers. To address this issue, we analyzed stimulated and unstimulated PBMCs from healthy donors using seven different flow cytometers. Additionally, human PBMCs were analyzed by fluorescence microscopy, imaging flow cytometry and high-content imaging (HCI). Flow cytometers differed significantly in their ability to detect ASC speck+ cells. AriaIII, Astrios EQ, and Canto II performed best in separating ASC speck+ from diffuse ASC cells. Imaging flow cytometry and HCI provided additional insight into ASC speck formation based on image-based parameters. For optimal results, the ability to separate cells with diffuse ASC from ASC speck+ cells is decisive. Image-based parameters can also differentiate cells with diffuse ASC from ASC speck+ cells. For the first time, we analyzed ASC speck detection by HCI in PBMCs and demonstrated advantages of this technique, such as high-throughput, algorithm-driven image quantification and 3D-rendering. Thus, inflammasome activation by ASC speck formation can be detected by various technical methods. However, the results may vary depending on the device used.

Smooth muscle cells clonally expand in a murine carotid allograft model complicated by immune reactions to reporter transgenes

Background and aimsMost experimental studies of allograft vasculopathy (AV) have relied on transplantation between major histocompatibility complex-mismatched inbred mouse strains, but this leads to the complete eradication of donor smooth muscle cells (SMCs) and lesions formed by recipient cells. This is unlike human AV which is thought to form mainly by donor SMCs. Here, we studied sources of neointimal cells in a minor histocompatibility antigen-mismatched AV model by combining male-to-female orthotopic carotid transplantations and lineage tracing by SMC-specific expression of fluorescent proteins. MethodsTo track SMC-derived cells in allograft vasculopathy, we used male donor mice with SMC-restricted Cre recombination of the mT/mG reporter transgene, which switches expression of membrane-bound red fluorescent protein (RFP) to green fluorescent protein (GFP), or the stochastically recombining Confetti reporter transgene, which yields a mosaic expression of four fluorescent proteins. Donor carotid segments were harvested and orthotopically allografted to female recipients that were wildtype or had non-recombined reporter transgenes. Inhibition of T cell responses by CTLA4Ig was used in some experiments. Sections of lesions harvested after 4 weeks were analyzed by fluorescence microscopy. ResultsDonor-derived SMCs survived and gave rise to part of the neointimal cells in experiments where carotid segments from recombined mT/mG male mice were transplanted into wild-type or non-recombined mT/mG female mice. Sex-mismatched transplants developed significant lesions, increasing the intimal and medial area 4.6-fold (p = 0.038) and 2.0-fold (p = 0.024) compared to sex- and fluorescence-matched controls, respectively. Interestingly, sex-matched fluorescence-positive transplants developed intimal lesions in 50% of fluorescence-naïve recipient controls. To study the clonal structure of the neointimal donor-derived SMC lineage cells, we then transplanted male carotids with heterozygous or homozygous recombined Confetti transgenes into female recipients. These transplants developed lesions with few surviving donor SMCs, indicating that expression of the Confetti reporter increased rejection and donor-specific SMC death. Some of the few remaining donor SMCs underwent clonal expansion. CTLA4Ig administration at the time of surgery did not improve SMC survival in mT/mG or Confetti transplants. ConclusionMale-to-female transplant models feature donor-derived SMCs, some of which undergo clonal expansion, but immune rejection to fluorescence reporters appears to bias results in lineage tracing models. Overcoming these challenges with alternative reporter transgenes or tolerant recipients is necessary to study the mechanisms by which donor SMCs contribute to allograft vasculopathy.

Impact of Synthesis Method on the Structure and Function of High Entropy Oxides.

The term sample dependence describes the troublesome tendency of nominally equivalent samples to exhibit different physical properties. High entropy oxides (HEOs) are a class of materials where sample dependence has the potential to be particularly profound due to their inherent chemical complexity. In this work, we prepare a spinel HEO of identical nominal composition by five distinct methods, spanning a range of thermodynamic and kinetic conditions: solid state, high pressure, hydrothermal, molten salt, and combustion syntheses. By structurally characterizing these five samples across all length scales with a variety of X-ray methods, we find that while the average structure is unaltered, the samples vary significantly in their local structures and their microstructures. The most profound differences are observed at intermediate length scales, both in terms of crystallite morphology and cation homogeneity. As revealed by X-ray fluorescence microscopy ideal cation homogeneity is achieved only in the case of combustion synthesis. These structural differences in turn significantly alter the observed functional properties, which we demonstrate via characterization of their magnetic response. While ferrimagnetic order is retained across all five samples, the sharpness of the transition, the size of the saturated moment, and the coercivity all show marked variations with synthesis method. We conclude that the chemical flexibility inherent to HEOs is complemented by strong synthesis method dependence, providing another axis along which to optimize these materials for a wide range of applications.

Million Microfiber Releases: Comparing Washable and Disposable Face Masks.

The extensive use of single-use or disposable face masks has raised environmental concerns related to microfiber contamination. In contrast, research on the potential release and ecological impact of microfibers from washable masks (WMs), suggested as an eco-friendly alternative, is currently lacking. Here, we comprehensively investigated the release of microfibers from disposable and WMs of different types in simulated aquatic environments and real-life scenarios, including shaking, disinfection, hand washing, and machine washing. Using a combination of wide-field fluorescence microscopy, He-ion microscopy, and confocal μ-Raman spectroscopy, we revealed that disposable masks (DMs) released microfibers ranging from 18 to 3042 microfiber/piece, whereas WMs released 6.1 × 104-6.7 × 106 microfibers/piece depending on the simulated conditions above. Another noteworthy finding was the observed negative correlation between microfiber release and the proportion of reinforcement (embossing) on the DM surfaces. Microfibers from tested DMs primarily comprised polypropylene (PP), while WMs predominantly released poly(ethylene terephthalate) (PET) and cellulose microfibers. Furthermore, acute toxicological analyses unveiled that PP microfibers (0.01-50 mg/L) from DMs impacted zebrafish larval swimming behavior, while PET microfibers from WMs delayed early-stage zebrafish hatching. This study offers new insights into the source of microfiber contamination and raises concerns about the environmental implications linked to the use of washable face masks.

Degradation of Amyloid-β Species by Multi-Copper Oxidases.

Reduction of the production of amyloid-β (Aβ) species has been intensively investigated as potential therapeutic approaches for Alzheimer's disease (AD). However, the degradation of Aβ species, another potential beneficial approach, has been far less explored. To investigate the potential of multi-copper oxidases (MCOs) in degrading Aβ peptides and their potential benefits for AD treatment. We investigated the degradation efficiency of MCOs by using electrophoresis and validated the ceruloplasmin (CP)-Aβ interaction using total internal reflection fluorescence microscopy, fluorescence photometer, and fluorescence polarization measurement. We also investigated the therapeutic effect of ascorbate oxidase (AO) by using induced pluripotent stem (iPS) neuron cells and electrophysiological analysis with brain slices. We discovered that CP, an important MCO in human blood, could degrade Aβ peptides. We also found that other MCOs could induce Aβ degradation as well. Remarkably, we revealed that AO had the strongest degrading effect among the tested MCOs. Using iPS neuron cells, we observed that AO could rescue neuron toxicity which induced by Aβ oligomers. In addition, our electrophysiological analysis with brain slices suggested that AO could prevent an Aβ-induced deficit in synaptic transmission in the hippocampus. To the best of our knowledge, our report is the first to demonstrate that MCOs have a degrading function for peptides/proteins. Further investigations are warranted to explore the possible benefits of MCOs for future AD treatment.

Biomimetic modification of macrophage membrane-coated prussian blue nanoparticles loaded with SN-38 to treat colorectal cancer by photothermal-chemotherapy.

SN-38 is the active metabolite of irinotecan and acts as an effective topoisomerase I inhibitor with therapeutic effects on many malignant tumors, including some drug-resistant cancers. However, the poor solubility, low bioavailability, and severe dose-dependent toxicity limits the clinical application of SN-38. Currently, emerging macrophage membrane-coated nanoparticles provide an efficient biomimetic approach to develop novel SN-38 formulations for the reduction of its side effects. Photothermal therapy (PTT) is a promising methods in tumor treatment to thermally ablate tumors using various materials such Prussian blue nanoparticles (NPs) and can combined with chemotherapy to synergistically work. There is no report that combined SN38 and photothermal therapy for the treatment of colorectal cancer (CRC). SN38-PB@CM NPs were constructed by loading SN-38 into macrophage cell membrane-coated hollow mesoporous Prussian blue (PB) NPs. The morphology, size and zeta potential were evaluated by transmission microscopy and dynamic light scatter (DLS). Coomassie bright blue staining was performed to assess total protein profile. The photothermal properties of it were also investigated via near-infrared imaging. CCK8 and calcein-AM/PI staining were used to evaluate cell viability. Flow cytometry was performed to assess cell apoptosis. The fluorescent microscopy was used to observe cellular uptake of SN38-PB@CM NPs to assess its internalization in vitro. The biodistribution, tumor-targeting efficacy, antitumor efficacy and safety of SN38-PB@CM NPs in vivo were assessed in CT26 tumor-bearing mice via In Vivo Imaging System. SN38-PB@CM NPs were successfully constructed and exhibited a uniform size distribution (140.5 ± 4.3nm) and an excellent drug-loading capacity (5.61 ± 0.64%). SN38-PB@CM NPs showed stable release properties within 72h. It can also enhance the selective intracellular delivery of SN38 in vitro and showed good near-infrared (NIR) photothermal properties. And the NPs showed excellent tumor targeting, effective photothermal therapy, improved biosafety and antitumor efficacy on CT26-bearing mice. Multifunctional SN38-PB@CM NPs could achieve improved biosafety, great tumor-targeting, high-efficiency PTT and excellent antitumor efficacy, which provided a promising and attractive combination therapy for the treatment of CRC.

Digital quantitation of bridging fibrosis and septa reveals changes in natural history and treatment not seen with conventional histology.

Metabolic dysfunction-associated steatohepatitis (MASH) with bridging fibrosis is a critical stage in the evolution of fatty liver disease. Second harmonic generation/two-photon excitation fluorescence (SHG/TPEF) microscopy with artificial intelligence (AI) provides sensitive and reproducible quantitation of liver fibrosis. This methodology was applied to gain an in-depth understanding of intra-stage fibrosis changes and septa analyses in a homogenous, well-characterised group with MASH F3 fibrosis. Paired liver biopsies (baseline [BL] and end of treatment [EOT]) of 57 patients (placebo, n = 17 and tropifexor n = 40), with F3 fibrosis stage at BL according to the clinical research network (CRN) scoring, were included. Unstained sections were examined using SHG/TPEF microscopy with AI. Changes in liver fibrosis overall and in five areas of liver lobules were quantitatively assessed by qFibrosis. Progressive, regressive septa, and 12 septa parameters were quantitatively analysed. qFibrosis demonstrated fibrosis progression or regression in 14/17 (82%) patients receiving placebo, while the CRN scoring categorised 11/17 (65%) as 'no change'. Radar maps with qFibrosis readouts visualised quantitative fibrosis dynamics in different areas of liver lobules even in cases categorised as 'No Change'. Measurement of septa parameters objectively differentiated regressive and progressive septa (p < .001). Quantitative changes in individual septa parameters (BL to EOT) were observed both in the 'no change' and the 'regression' subgroups, as defined by the CRN scoring. SHG/TPEF microscopy with AI provides greater granularity and precision in assessing fibrosis dynamics in patients with bridging fibrosis, thus advancing knowledge development of fibrosis evolution in natural history and in clinical trials.

A-scan fluorescence microscopy for rapid cross-sectional imaging

This paper presents a microscopy technique that can perform snapshot depth resolved optical imaging in the same manner as A-scan in ultrasound imaging and optical coherence tomography. In this technique, a laser line along the axial dimension is used to illuminate a sample to create a fluorescent line object. By transforming the line object along the axial dimension (Z) to a ring image on the lateral dimensions (X-Y) using a full cone mirror, common optics can be used to relay and acquire the ring image precisely. Then, by converting half of the ring image back to a line image using a half cone mirror, the opening side of the half cone mirror allows the line image, which contains the full depth resolved information of the line object, to be taken in one snapshot. This eliminates the requirement of axial scanning in traditional depth resolved imaging techniques such as confocal microscopy to obtain the same information. The technique is demonstrated by imaging fluorescent microspheres of different diameters. This technique offers a simple alternative to traditional depth resolved imaging techniques such as confocal microscopy and light sheet microscopy. It is particularly useful in imaging samples with multiple layers in which multiple A-scans or a few B-scans are sufficient to represent the entire sample.

Evaluation of the cytotoxic effects and apoptosis inducing of Cuscuta epithymum extract on esophageal squamous cell carcinoma

Background. Esophageal cancer is the eighth most common cancer in the world. The antitumor effects of medicinal plants have been shown as a therapeutic strategy to treat esophageal cancer. This study aimed to evaluate in vitro effects of Cuscuta epithymum extract on the survival and apoptosis of esophageal cancer cell line. Material and Methods. Here, the hydroalcoholic Soxhlet extract of C. epithymum plant was prepared. The cell viability of esophageal cancer cell line KYSE-30 was evaluated by MTT assay after 24 h treatment with concentrations of 50, 100, 200, 400, 600, 800 and 1000 μg/ml of the extract. Then, the apoptotic effect of extract was evaluated by flow cytometry using Propidium Iodide (PI) staining and sub-G1 peak analysis, and Annexin V-FITC/PI staining in cells treated with concentrations of 125, 250, 500 and 750 μg/ml as well as morphological change of healthy cells to apoptotic and necrotic form. Results. The hydroalcoholic extract of C. epithymum decreases the viability of KYSE-30 cells in a dose-dependent manner with an IC50 of 646 µg/ ml at 24 h. A significant increase was observed in the percentage of sub-G1 phase in cells treated with 500, 750, and 1000 μg/ml of C. epithymum extract for 24 h compared to the control group (p&lt;0.01 and p&lt;0.001). The results also showed a significantly enhanced the percentage of primary and secondary apoptotic cells compared to untreated cells. At concentrations of 250, 500, and 750 µg/ml, approximately 17, 33 and 45 % of cells was apoptotic. The apoptotic and necrotic cells morphology after treatment with 250 and 500 µg/ml of the extract was also confirmed by fluorescence microscopy. Conclusion. The findings showed the apoptotic effect of the hydroalcoholic extract of C. epithymum on KYSE-30 cells in vitro. The effect of this extract on the genes involved in apoptosis as well as the mechanism of action of this extract are recommended.

Composite fluorescence probe towards a smart coating for early corrosion detection of carbon steel

Herein, a composite fluorescent probe was developed as a smart indicator in acrylic resin-based coatings for the early detection of steel corrosion. The Fe3+-sensitive Rhodamine B derivative probe (Rxbxja) was synthesized, and the SiO2 nanoparticle-supported probe S-Rxbxja was prepared through the hydrolysis of tetraethyl orthosilicate (TEOS). The successful synthesis of S-Rxbxja was confirmed using a variety of techniques i.e. SEM and FTIR. A fluorescence microscope was utilized to assess the self-reporting performance of coatings. EIS and seawater immersion tests were employed to evaluate the corrosion resistance of the coating. The fluorescence test results revealed the absence of premature fluorescence in the composite coating, owing to the exceptional compatibility between S-Rxbxja and the coating. The scratched area in the 0.5 wt% sensor-doped coatings exhibited fluorescent and colorimetric dual-channel signals after soaking in 3.5 wt% NaCl solution for 2 h before any visible corrosion phenomenon. The EIS results demonstrated that the |Z|0.01Hz value of composite coating maintained 107 Ω cm2 even after being immersed in a 3.5 wt% NaCl solution for 7 days, indicating the excellent anti-corrosion properties of the S-Rxbxja coating. Moreover, mechanical property tests showed that the S-Rxbxja coating had excellent mechanical properties due to the introduction of SiO2 nanoparticles.

Parecoxib Enhances Resveratrol against Human Colorectal Cancer Cells through Akt and TXNDC5 Inhibition and MAPK Regulation.

In this study, we discovered the mechanisms underlying parecoxib and resveratrol combination's anti-cancer characteristics against human colorectal cancer DLD-1 cells. We studied its anti-proliferation and apoptosis-provoking effect by utilizing cell viability 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, fluorescence microscope, gene overexpression, Western blot, and flow cytometry analyses. Parecoxib enhanced the ability of resveratrol to inhibit cell viability and increase apoptosis. Parecoxib in combination with resveratrol strongly enhanced apoptosis by inhibiting the expression of thioredoxin domain containing 5 (TXNDC5) and Akt phosphorylation. Parecoxib enhanced resveratrol-provoked c-Jun N-terminal kinase (JNK) and p38 phosphorylation. Overexpression of TXNDC5 and repression of JNK and p38 pathways significantly reversed the inhibition of cell viability and stimulation of apoptosis by the parecoxib/resveratrol combination. This study presents evidence that parecoxib enhances the anti-cancer power of resveratrol in DLD-1 colorectal cancer cells via the inhibition of TXNDC5 and Akt signaling and enhancement of JNK/p38 MAPK pathways. Parecoxib may be provided as an efficient drug to sensitize colorectal cancer by resveratrol.

Preparation and Performance Study of Ultraviolet-Responsive Self-Healing Epoxy Asphalt.

In this study, a self-healing epoxy asphalt material was developed by incorporating coumarin groups. This material achieved microcrack self-repair under UV irradiation at 50 °C. Fluorescence microscopy observations and mechanical performance tests demonstrated significant advantages in crack filling and mechanical property recovery after repair, with the fracture toughness of the repaired epoxy asphalt reaching 69% of that in its original state. Furthermore, the synergistic effect of temperature and UV irradiation in the self-healing process enhanced the material's durability and service life. This research offers new insights and methods for developing more durable and long-lasting self-healing asphalt materials, showcasing the great potential of smart materials in infrastructure applications.

A nanoscale visual exploration of the pathogenic effects of bacterial extracellular vesicles on host cells

BackgroundBacterial extracellular vesicles (EVs) are pivotal mediators of intercellular communication and influence host cell biology, thereby contributing to the pathogenesis of infections. Despite their significance, the precise effects of bacterial EVs on the host cells remain poorly understood. This study aimed to elucidate ultrastructural changes in host cells upon infection with EVs derived from a pathogenic bacterium, Staphylococcus aureus (S. aureus).ResultsUsing super-resolution fluorescence microscopy and high-voltage electron microscopy, we investigated the nanoscale alterations in mitochondria, endoplasmic reticulum (ER), Golgi apparatus, lysosomes, and microtubules of skin cells infected with bacterial EVs. Our results revealed significant mitochondrial fission, loss of cristae, transformation of the ER from tubular to sheet-like structures, and fragmentation of the Golgi apparatus in cells infected with S. aureus EVs, in contrast to the negligible effects observed following S. epidermidis EV infection, probably due to the pathogenic factors in S. aureus EV, including protein A and enterotoxin. These findings indicate that bacterial EVs, particularly those from pathogenic strains, induce profound ultrastructural changes of host cells that can disrupt cellular homeostasis and contribute to infection pathogenesis.ConclusionsThis study advances the understanding of bacterial EV-host cell interactions and contributes to the development of new diagnostic and therapeutic strategies for bacterial infections.

Uptake of substances into living mammalian cells by microwave induced perturbation of the plasma membrane

Delivering foreign molecules and genetic material into cells is a crucial process in life sciences and biotechnology, resulting in great interest in effective cell transfection methods. Importantly, physical transfection methods allow delivery of molecules of different chemical composition and are, thus, very flexible. Here, we investigated the influence of microwave radiation on the transfection and survival of mammalian cells. We made use of an optimized microwave-poration device and analyzed its performance (frequency and electric field strength) in comparison with simulations. We, then, tested the effect of microwave irradiation on cells and found that 18 GHz had the least impact on cell survival, viability, cell division and genotoxicity while 10 GHz drastically impacted cell physiology. Using live-cell fluorescence microscopy and image analysis, we tested the uptake of small chemical substances, which was most efficient at 18 GHz and correlated with electric field strength and frequency. Finally, we were able to obtain cellular uptake of molecules of very different chemical composition and sizes up to whole immunoglobulin antibodies. In conclusion, microwave-induced poration enables the uptake of widely different substances directly into mammalian cells growing as adherent cultures and with low physiological impact.

Applications of Lightsheet Fluorescence Microscopy by High Numerical Aperture Detection Lens.

This Review explores the evolution, improvements, and recent applications of Light Sheet Fluorescence Microscopy (LSFM) in biological research using a high numerical aperture detection objective (lens) for imaging subcellular structures. The Review begins with an overview of the development of LSFM, tracing its evolution from its inception to its current state and emphasizing key milestones and technological advancements over the years. Subsequently, we will discuss various improvements of LSFM techniques, covering advancements in hardware such as illumination strategies, optical designs, and sample preparation methods that have enhanced imaging capabilities and resolution. The advancements in data acquisition and processing are also included, which provides a brief overview of the recent development of artificial intelligence. Fluorescence probes that were commonly used in LSFM will be highlighted, together with some insights regarding the selection of potential probe candidates for future LSFM development. Furthermore, we also discuss recent advances in the application of LSFM with a focus on high numerical aperture detection objectives for various biological studies. For sample preparation techniques, there are discussions regarding fluorescence probe selection, tissue clearing protocols, and some insights into expansion microscopy. Integrated setups such as adaptive optics, single objective modification, and microfluidics will also be some of the key discussion points in this Review. We hope that this comprehensive Review will provide a holistic perspective on the historical development, technical enhancements, and cutting-edge applications of LSFM, showcasing its pivotal role and future potential in advancing biological research.

Glycyrrhetinic acid-modified redox-sensitive polymeric mixed micelles for tumor-specific intracellular delivery of cantharidin.

Cantharidin (CTD) has been widely used to treat hepatocellular carcinoma (HCC) in clinical practice. However, the current CTD preparations may induce hepatic and renal damage due to their non-specific distribution. Therefore, redox-sensitive polymer Pluronic F127-disulfide bond-poly(d,l-lactide) (F127-SS-PDLA) and active targeting polymer F127-glycyrrhetinic acid (F127-GA) were synthesized to prepare mixed micelles (GA/F127-SS-PDLA/CTD) for effective delivery of CTD. Fourier transform infrared (FTIR) spectroscopy and 1H nuclear magnetic resonance (1H-NMR) spectroscopy were used to verify the successful synthesis of F127-SS-PDLA and F127-GA. During the preparation, this study was the first to screen the density of GA by cellular uptake assay. The results indicated that mixed micelles with 10% and 15% F127-GA (weight fraction) exhibited superior cellular uptake in comparison to micelles with 5% and 20% F127-GA. GA/F127-SS-PDLA/CTD micelles prepared by thin film hydration method demonstrated excellent drug loading capacity for CTD (16.12 ± 0.11%). The particle size and zeta potential of GA/F127-SS-PDLA/CTD micelles were 85.17 ± 1.24 nm and -11.71 ± 0.86 mV, respectively. Hemolysis and stability assay showed that the mixed micelles had good blood compatibility and could remain stable for 30 days at 4 °C. The redox-sensitivity of GA/F127-SS-PDLA/CTD micelles in vitro was verified under reducing conditions through dynamic light scattering (DLS) and an in vitro drug release experiment, which showed obvious particle size variation and rapid drug release ability. In cellular experiments, GA/F127-SS-PDLA/CTD micelles could induce superior cytotoxicity, apoptosis and intracellular reactive oxygen species (ROS) levels compared with free CTD, non-sensitive F127-PDLA/CTD micelles and redox-sensitive F127-SS-PDLA/CTD micelles. The cellular uptake ability of nile red-labeled GA/F127-SS-PDLA micelles, which was evaluated via fluorescent microscope and flow cytometry, indicated that the modification of GA significantly increased micelle uptake in HepG-2 cells. Consequently, GA/F127-SS-PDLA/CTD micelles could be considered as a satisfactory drug administration strategy in the treatment of HCC.

Auto-Combustion of Corn Straw: Production and Characterization of Corn Straw Ash (CSA) for Its Use in Portland Cement Mortars.

Agricultural waste availability implies the possibility of recovering energy as biomass. The collateral effect is the production of ashes that, in some cases, have the potential to be reused in the manufacture of cement, mortar, and concrete. This article presents the study of the auto-combustion (unlike all previous studies) of corn (maize) straw (stems and leaves). The auto-combustion temperature was monitored, and the obtained corn straw ash (CSA) was characterized by means of X-ray fluorescence, X-ray diffraction, thermogravimetry, and scanning electron microscopy. Finally, the behavior of ground CSA was analyzed in both the fresh state by measurement of workability on the spreading table and the hardened state by compressive strength measurement on mortars in which 10% of ordinary Portland cement (OPC) was replaced with CSA. These values were compared to both a control mortar (OPC) and a mortar in which OPC was partially replaced with 10% limestone filler. Ashes showed adequate pozzolanic reactivity because, at 90 curing days, the compressive strength of the mortars with 10% replacement of OPC with CSA was practically equal (98% of the strength) to the control mortar without pozzolan replacement. The auto-combustion of biomass is a process that can be easily available, and the results on pozzolanic reactivity of CSA are satisfactory. The auto-combustion could be used by low-income communities to reduce Portland cement clinker use and to recover waste.

ColocZStats: a z-stack signal colocalization extension tool for 3D slicer.

Confocal microscopy has evolved to be a widely adopted imaging technique in molecular biology and is frequently utilized to achieve accurate subcellular localization of proteins. Applying colocalization analysis on image z-stacks obtained from confocal fluorescence microscopes is a dependable method of revealing the relationship between different molecules. In addition, despite the established advantages and growing adoption of 3D visualization software in various microscopy research domains, there have been few systems that can support colocalization analysis within a user-specified region of interest (ROI). In this context, several broadly employed biological image visualization platforms are meticulously explored in this study to understand the current landscape. It has been observed that while these applications can generate three-dimensional (3D) reconstructions for z-stacks, and in some cases transfer them into an immersive virtual reality (VR) scene, there is still little support for performing quantitative colocalization analysis on such images based on a user-defined ROI and thresholding levels. To address these issues, an extension called ColocZStats (pronounced Coloc-Zee-Stats) has been developed for 3D Slicer, a widely used free and open-source software package for image analysis and scientific visualization. With a custom-designed user-friendly interface, ColocZStats allows investigators to conduct intensity thresholding and ROI selection on imported 3D image stacks. It can deliver several essential colocalization metrics for structures of interest and produce reports in the form of diagrams and spreadsheets.

Mechanism of bacteriophage-induced vaterite formation

This study shows how bacterial viruses (bacteriophages, phages) interact with calcium carbonate during precipitation from aqueous solution. Using electron microscopy, epifluorescence microscopy, X-ray diffraction, and image analysis, we demonstrate that bacteriophages can strongly influence the formation of the vaterite phase. Importantly, bacteriophages may selectively bind both amorphous calcium carbonate (ACC) and vaterite, and indirectly affect the formation of structural defects in calcite crystallites. Consequently, the surface properties of calcium carbonate phases precipitating in the presence of viruses may exhibit different characteristics. These findings may have significant implications in determining the role of bacterial viruses in modern microbially-rich carbonate sedimentary environments, as well as in biomedical technologies. Finally, the phage-vaterite system, as a biocompatible material, may serve as a basis for the development of promising drug delivery carriers.

Killing of xenogenous and virally infected homogenous target cells by shrimp lymphocyte-like haemocytes

Haemocytes play a crucial role in the invertebrate's immune system. In our lab, five subpopulations of shrimp haemocytes were identified in the past: hyalinocytes, granulocytes, semi-granulocytes and two subpopulations of non-phagocytic cells. In the latter two subpopulations, their characteristics such as having small cytoplasmic rims and not adhering to plastic cell-culture plates are very similar to those of mammalian lymphocytes. Therefore, they were designated lymphocyte-like haemocytes. Although little is known about their function, we hypothesize, based on their morphology, that they may have a cytotoxic activity like natural killer cells, with the ability to recognize and kill target cells. In our study, K562 cells and Sf9 cells were used as xenogenous target cells to detect the cytotoxic activity of the shrimp non-adherent lymphocyte-like haemocytes. Non-adherent haemocytes were collected and mixed with K562 cells and Sf9 cells at a 5:1 ratio and the binding activity was examined under a microscope. The binding rate of non-adherent haemocytes to K562 cells and Sf9 cells reached 6.6 % and 2.4 % after 240 min of culture, respectively. Then, the killing activity of non-adherent haemocytes was detected by an EMA staining (fluorescence microscopy), which showed 3.75 % dead K562 cells and 1.025 % dead Sf9 cells, and by Sytox® blue staining (flow cytometry), which showed 4.97 % of dead K562 cells. Next, a killing assay was developed to visualize the killing activity of shrimp non-adherent haemocytes. Non-adherent haemocytes were pre-labeled in blue (CellTracker blue) and K562/Sf9 cells in green (CFSE); dead cells were differentially stained red with ethidium bromide. The cytotoxic activity increased and reached a level of 2.59 % in K562 cells and 0.925 % in Sf9 cells at 120 min after co-culture. Furthermore, in the co-cultures of non-adherent haemocytes with K562 cells and Sf9 cells, upregulation of the gene and protein expression of the cytotoxic molecules torso-like protein and granzyme B was observed by RT-qPCR at 240 min and western blotting at 180 min. Additionally, non-adherent haemocytes were co-cultured with WSSV-inoculated shrimp ovary and lymphoid organ cells to detect the cytotoxicity to homogenous target cells. The binding activity started at 60 min in both the ovary and lymphoid organ cultures and reached at 240 min 50.62 % and 40.7 %, respectively. The killing activity was detected by EMA staining and the percentage of dead ovary and lymphoid organ cells increased respectively from 10.84 % to 6.89 % at 0 min to 13.09 % and 8.37 % at 240 min. In conclusion, we demonstrated the existence of cytotoxic activity of shrimp lymphocyte-like haemocytes against xenogenous cells from mammals and insects and against WSSV-infected homogenous shrimp cells.