Inverted Microscope Research Articles

Long-term live-cell imaging of GFAP+ astroglia and laminin+ vessels in organotypic mouse brain slices using microcontact printing

Organotypic brain slices are three-dimensional, 150-μm-thick sections derived from postnatal day 10 mice that can be cultured for several weeks in vitro. However, these slices pose challenges for live-cell imaging due to their thickness, particularly without access to expensive two-photon microscopy. In this study, we present an innovative method to label and visualize specific brain cell populations in living slices. Using microcontact printing, antibodies are applied directly onto the slices in a controlled 400-μm-diameter pattern. Astrocytes are labeled with glial fibrillary acidic protein (GFAP), and vessels are labeled with laminin. Subsequently, slices are incubated with secondary fluorescent antibodies (green fluorescent Alexa-488 or red fluorescent Alexa-546) and visualized using an inverted fluorescence microscope. This approach offers a cost-effective and detailed visualization technique for astroglia and vessels in living brain slices, enabling investigation to be conducted over several weeks.

Mesenchymal Stem Cell-conditioned Medium Attenuated CoCl2-induced Injury of Renal Tubular Epithelial Cells by Inhibiting NCOA1, HIF-1α, and Sox9.

Renal interstitial fibrosis (RIF) constitutes the ultimate pathological alteration in nearly all chronic kidney diseases (CKD). Mesenchymal stem cell conditioned medium (MSC-CM) exhibits an alleviating impact on renal fibrosis; however, the underlying mechanism remains unclear. The objective of this study was to explore whether MSC-CM regulates the expression of α-smooth muscle actin (α-SMA), Transforming growth factor-β1 (TGF-β1), Hypoxia-inducible factor-1α (HIF-1α), Nuclear receptor coactivators (NCOA1), and SRY-related high mobility (Sox9). Rat renal tubular epithelial cells (RTECs), NRK-52E, were treated with diverse concentrations of Cobalt chloride (CoCl2) for 24 hours. The survival rate and protein expression of NRK-52E cells exposed to different concentrations of CoCl2 were determined to identify the final concentration. Three groups of NRK-52E cells were employed in the experiment: the normal control group, the 400 μM CoCl2 group, and the MSC-CM + 400 μM CoCl2 group. The cell morphology was observed by an inverted phase contrast microscope and scanning electron microscope, and the protein expressions of α-SMA, TGF-β1, HIF-1α, NCOA1, and Sox9 were detected. The microscopic findings demonstrated that MSC-CM was able to decrease the degree of cytochemical hypoxia damage in NRK-52E cells induced by CoCl2. Immunofluorescence and Western blot analyses also affirmed a similar tendency. The upregulation of α-SMA, TGF-β1, HIF-1α, NCOA1, and Sox9 triggered by CoCl2 could be inhibited following MSC-CM intervention. Our findings indicate that MSC-CM exerts a protective effect on RTECs by down-regulating α-SMA, TGF-β1, HIF-1α, NCOA1, and Sox9.

The total extract of Abelmoschus manihot (L.) medic flowers (TEA) mediated Nrf2-TFAM signalling to regulate mitochondrial antioxidant mechanism

Skin, as the first line of defence of the human body, is exposed to dangers such as overheating substances, ultraviolet rays, and environmental pollutants, and the incidence of skin diseases is increasing annually. Oxidative stress plays a dominant role in most skin diseases. Abelmoschus manihot (L.) medic flower (TEA) is a traditional Chinese medicine widely used to treat injuries to the skin such as water and fire scalds. It has been reported that TEA has excellent antioxidant effects. In this study, we aimed to explore the antioxidant and mitochondrial protection effects of TEA in H2O2-mediated HaCaT cell damage. HaCaT cells were incubated with H2O2 to simulate oxidative stress in the skin. The effect of TEA on HaCaT cells was also evaluated. Cell morphology was observed via inverted microscopy, and cell viability was measured via the MTT reagent. The cells were stained with Hoechst 33,324 solution. Reactive oxygen species (ROS), superoxide dismutase (SOD), malondialdehyde (MDA) and ATP detection kits were used to detect the corresponding indicators. The mitochondrial membrane potential was detected by JC-1. RT-PCR was used to detect mRNA and mtDNA expression. The expression of the target protein was detected by Western blotting and immunofluorescence. H2O2 triggered oxidative damage in HaCaT cells, which manifested as apoptosis, increased ROS and MDA contents, and decreased SOD activity. H2O2 activates the KEAP1/Nrf2/NQO1 signalling pathway, which decreases the expression of the intracellular KEAP1 protein and slightly increases the expression of the Nrf2 and NQO1 proteins, further causing mitochondrial oxidative stress, resulting in changes in the mitochondrial membrane potential, a reduction in the mtDNA copy number, and decreased expression of the PGC-1α and TFAM proteins. In addition the expression of mitochondrial respiratory chain genes and proteins decreased. TEA promoted the expression of Nrf2 in HaCaT cells, activated the downstream antioxidant response, and alleviated the oxidative stress and mitochondrial damage caused by H2O2. ML385 is an Nrf2 inhibitor, under which the antioxidant and mitochondrial protective effects of TEA are inhibited. When TFAM was knocked down, the protective effect of TEA on mitochondria was also inhibited. TEA protects HaCaT cells from H2O2-induced oxidative damage and mitochondrial oxidative damage through the KEAP1/Nrf2/NQO1/PGC-1α/TFAM pathway.

Oral Cancer's New Enemy: Goniothalamus umbrosus Targets Oral Squamous Cell Carcinoma and Spare Human Gingival Fibroblast Cells.

Oral squamous cell carcinoma (OSCC) is the prevailing type of oral cancer, representing poor prognosis and elevated mortality rates. Major risk factors for OSCC include the use of tobacco products, alcohol consumption, betel quid chewing, and genetic mutation. Goniothalamus umbrosus is traditionally consumed by cancer patients to fight against tumor growth. To date, research on the anticancer potential of G. umbrosus in oral cancer remains deficient. This study aimed to evaluate the anticancer potential of G. umbrosus in OSCC cell lines (SCC-15 and HSC-3) and compare its cytotoxic activity on human gingival fibroblast (HGF) cell lines. Leaves of G. umbrosus were cleaned, air dried, ground, and soaked for 24 hours with methanol and hexane repeatedly three times, respectively. Pooled extracts of each solvent were then dried with a rotary evaporator. Anticancer potential of G. umbrosus extracts was evaluated on two OSCC cell lines (SCC-15 and HSC-3) and a normal HGF cell line incubated for 24, 48, and 72 hours by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cytotoxicity of cisplatin was assessed as a positive control. Morphological changes of cells were observed under an inverted microscope. MTT assay revealed that G. umbrosus methanol extract (GUME) displayed moderate anticancer activity on SCC-15, HSC-3, and HGF cell lines with IC50 values of 126.67, 90.5, and 87.33 µg/mL following 72 hours' incubation times, respectively. G. umbrosus hexane extract (GUHE) exerted moderate anticancer activity against SCC-15 and HSC-3 cell lines with IC50 values of 171 and 174 µg/mL, respectively, but weak cytotoxicity against the HGF cell line with IC50 value of 343.5 µg/mL. Cisplatin exerted a strong cytotoxic impact on both OSCC and HGF cell lines. Morphological observation revealed the characteristics of cells undergoing apoptosis. The findings show that GUHE was more selective in inhibiting the proliferation of oral cancer cells than GUME by exerting moderate cytotoxicity on OSCC cell lines and weak cytotoxicity in HGF cells, while GUME exerted moderate cytotoxicity on both. These findings suggest a more targeted anticancer effect by GUHE as compared with cisplatin, which exerted nonselective cytotoxic activity. These findings provide a groundwork for the development of more targeted plant-based treatment for oral cancer.

Origanum syriacum Induces Apoptosis in Lung Cancer Cells by Altering the Ratio of Bax/Bcl2.

The lung cancer is the leading cause of death worldwide. Although methods such as surgery, chemotherapy, radiotherapy, and immunotherapy are used for treatment, these treatments are sometimes inadequate. In addition, the number of chemotherapeutic agents used is very limited, and it is very important to use new natural agents that can increase the effect of these methods used in treatment. The present study was designed to determine the suppression of proliferation and induction of apoptosis activities and phenolic content of Origanum syriacum methanol extract (OsME) on lung cancer cells (A549). For this purpose, the cell viability of A549 cells exposed to OsME was first determined. The morphological changes of the cell were observed by an inverted phase contrast microscope. Moreover, the percentage of apoptotic and necrotic cells was determined by FACS with AnnexinV/Propodium iodide staining. Additionally, proapoptotic Bax and antiapoptotic Bcl-2 mRNA levels were determined by Real-time PCR. Phenolic compounds of OsME were detected by LC-MS-MS. It was observed that the viability and proliferation of lung cancer cells decreased after the treatment of different concentrations of OsME. At a concentration of 200 mg/ml of OsME, most of the cell membrane structures were observed to disintegrate. Meanwhile, a 25 μg/ml concentration of OsME increased the Bax expression and percentage of late apoptotic cells. Vanillic acid and luteolin were identified as the main phenolic compounds of OsME. OsME exhibited antiproliferation activity on A549 cells and induced apoptosis at low doses.

Protective Effects of Heat-Killed Lactobacilli against Plasma-Induced Neurotoxicity in Multiple Sclerosis.

Heat-killed lactobacilli seem to have protective effects against oxidative stress and neurotoxicity. This study aimed to evaluate the antioxidant properties of specific heat-killed lactobacilli extracts and determine their neuroprotective effects against the neurotoxicity induced by blood plasma from people with multiple sclerosis (MS). The antioxidant activity of the three heat-killed lactobacilli was measured using the DPPH assay. For neuroprotective evaluations of lactobacilli, human neuroblastoma cells (SK-N-SH) were exposed to plasma from individuals with relapsing-remitting MS (RRMS) and healthy controls, with or without pre-treatment of heat-killed lactobacilli including Lactiplantibacillus plantarum (L. plantarum), Levilactobacillus brevis (L. brevis), and Lacticaseibacillus rhamnosus (L. rhamnosus). The morphological changes of SK-N-SH cells associated with plasma-induced apoptosis were observed using an inverted microscope. The neurotoxic effects of plasma samples were assessed using flow cytometry as the percentage of apoptosis in neuronal cells treated with plasma from RRMS patients and healthy controls. The neuroprotective effects of the lactobacilli were also evaluated using flow cytometry, which showed an increased viability percentage in cells pretreated with heat-killed lactobacilli extracts compared to those without pre-treatment. Compared to plasma from healthy controls, plasma from RRMS patients caused morphological changes characteristic of apoptosis such as rounding, detachment, and shrinkage in SK-N-SH cells on microscopy observations. Significant apoptosis in MS plasma-treated neuronal cells was identified by flow cytometry analysis compared to cells treated with plasma from healthy controls (p < 0.01). Heat-killed lactobacilli extracts showed antioxidant activity above 50% in the DPPH radical scavenging assay. Pre-treatment of cells with heat-killed lactobacilli significantly reduced the morphological changes and apoptosis percentage in neuronal cells induced by MS plasma samples. L. plantarum and L. rhamnosus had considerable neuroprotective effects (p < 0.001), followed by L. brevis (p < 0.05). These findings demonstrate that heat-killed lactobacilli extracts as bacterial fractions free of live microorganisms, are suitable safe candidates for adjunctive therapy with potential antioxidant and neuroprotective properties in MS.

Investigating the Anticancer Properties of Bacterial Toxoid in Combination Vaccines.

The use of bacterial vaccines as a potential Bacterial-Based Cancer Therapy (BBCT) presents an innovative approach, transforming these vaccines into multifunctional tools capable of serving dual roles in medicine. This study aimed to conduct in vitro, immunity-independent experiments to investigate the anticancer properties of vaccine-derived bacterial toxoids on various cancer cell lines. Six concentrations of the DTP vaccine (5 x 10-4, 25 x 10-5, 125 x 10-6, 625 x 10-7, 312 x 10-7, and 15 x 10-6 µg/ml) were tested on two cancer cell lines (SKG and HCAM) and a normal Rat Embryonic Fibroblast (REF) cell line. The cytotoxic effects were evaluated using the Crystal Violet assay to determine the percentage of cell death for each toxoid concentration, leading to the calculation of IC50 values. Apoptotic effects and other cytopathological changes were observed under an inverted microscope. The findings revealed significant toxic effects of the bacterial toxoid on the SKG and HCAM cancer cell lines (p < 0.001). In contrast, the toxic effects on the normal REF cell line were evident only at the highest toxoid concentrations. Microscopic analysis showed marked cytological changes in cancer cells treated with the toxoid, with minimal impact on normal cells.

Effects of Tongluo Zhitong formula on synovial fibroblast proliferation in human knee osteoarthritis.

We aimed to investigate the effects of Tongluo Zhitong formula on synovial fibroblast proliferation in human knee osteoarthritis (KOA). Discarded synovial tissue collected from patients undergoing total knee arthroplasty at our hospital was digested with type I collagenase. Primary culture was performed on three to four generations of fibroblasts, which were treated with high, medium, and low concentrations of Tongluo Zhitong formula. The KOA synoviocyte proliferation level was detected through applying the methyl-thiazol-tetrazolium (MTT) method, and cell morphology at each concentration was observed under inverted microscopy. Significant differences were observed between three concentration categories and a blank control (p<0.05). MTT assay test results indicated that as the Tongluo Zhitong formula concentration decreased, the absorbance/optical density value of KOA synovial cells at 490nm increased. As the concentration increased, a positive inhibition rate was observed in the high concentration category. As the concentration decreased, a negative inhibition rate was observed in the low concentration category. In phase S, the Tongluo Zhitong formula inhibited deoxyribonucleic acid replication, which became more pronounced at increasing concentrations. In the G2 phase, the proportion of cells in the mitotic preparation phase did not change in the low concentration category (p>0.05). The proportion of cells in the mitotic preparation phase significantly increased in the high and medium concentration categories (p<0.05). High and medium concentrations of Tongluo Zhitong formula inhibited cell proliferation; therefore, the formula had an inhibitory effect on the cell cycle of synovial fibroblasts.

Molecular Characteristics of High-Grade Glioma in Relation to 5-Aminolevulinic Acid (5-ALA) Fluorescence Intensity.

5-aminolevulinic acid (5-ALA) fluorescence used in glioma surgery has different intensities within tumors and amongdifferent patients, some molecular and external factors have been implicated, but there is no clear evidence analyzing the difference of fluorescence according to glioma molecular characteristics. This study aimed to compare molecular factors of glioma samples with fluorescence intensity to identify potential cofounders and associations with clinically relevant tumor features. Tumor samples of high-grade glioma patients operated using 5-ALA for guided resection were included for comparative analysis of fluorescence intensity and molecular features. All the samples were processed under the same conditions. The power for fluorescent stimulation and acquisition time was the same between samples. An inverted fluorescence microscope compared the mean fluorescence for each molecular variation. p53, ATRX and Ki67 expression and IDH1 mutation were assessed by immunohistochemistry. Follow-up of the patients for progression-free survival and overall survival was made. We found that the fluorescence intensity for each specific tumor was independent of the methylation of the methylguanine-DNA-methyltransferase (MGMT) promoter region assessed by pyrosequencing, there was no association of fluorescence with p53, ATRX, IDH1 mutation as assessed by immunochemistry. Also, fluorescence intensity has no relation with time of tumor recurrence or overall survival. With the results, we argue that many factors are involved in fluorescence intensity that may be related to the specific metabolic status of the glioma cells analyzed, which is more likely to be responsible for the variation of fluorescence.

Matairesinol repolarizes M2 macrophages to M1 phenotype to induce apoptosis in triple-negative breast cancer cells

Objective Triple-Negative Breast Cancer (TNBC), the most challenging subtype of Breast Cancer (BC), currently lacks targeted therapy, presenting a significant therapeutic gap in its management. Tumor Associated Macrophages (TAMs) play a significant role in TNBC progression and could be targeted by repolarizing them from M2 to M1 phenotype. Matairesinol (MAT), a plant lignan, has been shown to exhibit anticancer, anti-inflammatory and immunomodulatory activities. In this study, we explored how MAT-induced repolarization of THP-1-derived M2 macrophages towards the M1 phenotype, which could effectively target the TNBC cell line, MDA-MB-231. Methods The differential expression of genes in THP-1-derived macrophages at mRNA levels was evaluated by RNAseq assay. An inverted microscope equipped with a CMOS camera was utilized to capture the morphological variations in THP-1 cells and THP-1-derived macrophages. Relative mRNA expression of M1 and M2 specific marker genes was quantified by qRT-PCR. Cell viability and induction of apoptosis were evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1 dye) assays, respectively. Results MAT reduced the viability of M2a and M2d macrophages and repolarized them to M1 phenotype. Conditioned medium (CM) from MAT-treated M2a and M2d macrophages significantly reduced the viability of TNBC cells by apoptosis. Conclusion Targeting M2 macrophages is an important strategy to regulate cancer progression. Our study provides evidence that MAT may be a promising drug candidate for developing novel anti-TNBC therapy. However, further studies are warranted to thoroughly elucidate the molecular mechanism of action of MAT and evaluate its therapeutic potential in TNBC in vitro and in vivo models.

Light-sheet microscopy enabled by a miniaturized plane illuminator.

We present a implementation method of light-sheet microscopy utilizing a highly miniaturized device that produces light-sheet illumination while immersed in the sample container. Our miniaturized plane illuminator (MPI) internally equips a two-axis beam-scanning mechanism based on a magnetostatically driven optical fiber cantilever. A light sheet is produced by fast scanning of the focused beam in an axis while the illumination plane can move in the other axis for positioning and 3D imaging. Our MPI device is so compact in a 1.5 mm-thick needle form that it can be conveniently placed in the right vicinity of the imaging sample. Because the illumination is directly given in the sample-surrounding medium, a great deal of operational flexibility is obtained with an uncompromised beam quality. We could build a light-sheet microscopy system with a conventional inverted microscope frame by attaching our MPI upgrade kit as an add-on module. In this study, the optical and electromechanical characteristics of our MPI device were carefully investigated. As well, light-sheet microscopy imaging of various samples was performed to validate the practical power of our technique. We found our MPI can provide a low-cost and easy-to-use imaging mode, and make the light-sheet microscopy more available in various applications.

Effect of Sintering Temperature on Microstructure and Properties of Zirconia Ceramics for the Needs of Nuclear Energy

The paper provides experimental results of obtaining high-density ZrO2+3%Y2O3 ceramics, which is promising for use as a matrix for immobilization of HLW. The effect of sintering temperature in the range of 1100...1650 °C on the microstructure of the sintered tablets was studied. Phase composition and microstructure of experimental samples were characterized by XRD and SEM. Grain size distribution analysis was carried out using the "Thixomet" image analyzer. Microhardness was determined using a metallographic complex LECO (USA), an inverted microscope IM-3MET and a hardness tester UIT HVB-30. It was established that increase in the sintering temperature leads to a significant increase in the average grain size (from 85 nm to 1000 nm) and increase in the density of the sintered tablets. Sintering temperature should be at least 1550...1650 ºС to produce high-dense ceramics (97...98 % of theoretical value). Obtained ceramics is characterized by high values of microhardness HV &gt; 12 GPa and crack resistance of 5.5...6.3 MPa·m1/2.

Effect of Aconitum diphtheria on the Proliferation, Apoptosis, and Inflammatory Response of Rheumatoid Arthritis Fibroblast-Like Synoviocytes.

Rheumatoid arthritis (RA), a highly disabling autoimmune disease, is characterized by joint damage and synovial hyperplasia. This paper was designed to explore the effect and mechanisms of Aconitum diphtheria on the proliferation and apoptosis of RA fibroblast-like synoviocytes (RA-FLS). First, RA-FLS was treated with different doses of A. diphtheria extracts. Then, an inverted biological microscope was adopted to observe the RA-FLS morphology. Subsequently, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining, Cell Counting Kit-8 and western blot were utilized to analyze the apoptosis, proliferation, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway-related proteins in RA-FLS, respectively. The experimental results disclosed that, after treatment of RA-FLS with A. diphtheria extracts, the cells became round and the intercellular space was increased. Besides, the RA-FLS proliferation was effectively inhibited by A. diphtheria extracts, while the apoptosis was promoted. Additionally, A. diphtheria extracts could effectively downregulate the expression levels of p-NF-κB (p50), NF-κB (p50), p-NF-κB (p65), and NF-κB (p65). Collectively, A. diphtheria can effectively inhibit RA-FLS proliferation and promote apoptosis in a dose-dependent manner. Similarly, A. diphtheria is able to downregulate p-NF-κB and NF-κB protein expression, but there is no dose-response relationship.

Fast autofluorescence imaging to evaluate dynamic changes in cell metabolism.

Cellular metabolic dynamics can occur within milliseconds, yet there are no optimal tools to spatially and temporally capture these events. Autofluorescence imaging can provide metabolic information on the cellular level due to the intrinsic fluorescence of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD). Our goal is to build and evaluate a widefield microscope optimized for rapid autofluorescence imaging of metabolic changes in cells. A widefield, fluorescence microscope was assembled from an inverted microscope base, an light-emitting diode (LED) for excitation, and an image splitter for simultaneous but separate imaging of two bandwidths of emission (451/106 and 560/94nm) on a single scientific complementary metal-oxide-semiconductor (sCMOS) camera. MCF-7 cells and primary murine hippocampal neurons were metabolically perturbed using cyanide and imaged to optimize illumination and camera exposure. To capture a rapid change in metabolism, MCF-7 cells were starved for 1h and imaged while reintroduced to glucose. Significant differences in the optical redox ratio (ORR) and intensity of NAD(P)H divided by the summed intensities of NAD(P)H and FAD were quantified for cyanide-treated neurons and MCF-7 cells at illumination powers above 0.30mW and camera exposures as low as 5ms; however, low illumination and camera exposures hindered the ability to identify subcellular features. Minimal photobleaching was quantified for 30s of continuous imaging for illuminations at 4.14mW and below. Using the optimized illumination power of 4.14mW and an exposure of 10ms, continuous autofluorescence imaging of starved MCF-7 cells demonstrated a rapid, yet heterogeneous, increase in the ORR of cells upon exposure to glucose. Ultimately, this widefield autofluorescence imaging system allowed for dynamic imaging and quantification of cellular metabolism at 99.6Hz.

Mesoporous silica nanoparticles based on a dual environmental response corresponding to temperature and α-amylase for the control of Spodoptera litura

The design and development of stimuli-responsive nanocarriers hold the promise of the smart delivery of pesticides to target organisms. Herein, we successfully prepared mesoporous silica nanoparticles using the self-templating method, which were grafted with both N-isopropylacrylamide (NIPAM) and cyclodextrin (CD) and loaded with indoxacarb (IN) to afford a pesticide release system with a dual-stimulated response of temperature and α-amylase, referred to as IN@MSNs@NIPAM@CD. Physicochemical characterisation revealed the successful preparation of IN@MSNs@NIPAM@CD with 46.30 % indoxacarb loading. The results showed that IN@MSNs@NIPAM@CD was able to release IN rapidly in the environment of high temperature and α-Amylase, with excellent dual-responsive property. The UV resistance evaluation showed that IN@MSNs@NIPAM@CD could effectively improve the photodegradation resistance of indoxacarb. Fluorescent inverted microscope showed that FITC@MSNs@NIPAM@CD has excellent uptake and translocation properties in corn plants. Bioactivity tests showed that at 14 d, IN@MSNs@NIPAM@CD (LC50 = 9.83 mg L−1) exhibited higher insecticidal activity and longer effective duration than IN@EC (LC50 = 14.64 mg L−1). Acute toxicity test revealed that the IN@MSNs@NIPAM@CD nanoparticles were less acutely toxic to earthworms, zebrafish, BEAS-2B cells and MSNs@NIPAM@CD nanocarriers were safe for the growth of corn. Thus, the dual-responsive nano-controlled-release formulation of IN@MSNs@NIPAM@CD can provide an effective way of achieving the precise delivery and reduced use of pesticides.

Pyropheophorbide-α methyl ester-mediated photodynamic therapy triggers pyroptosis in osteosarcoma cells via the ROS/caspase-3/GSDME pathway

BackgroundPyropheophorbide-α methyl ester-mediated photodynamic therapy(MPPa-PDT) is a candidate treatment for solid tumors, including osteosarcoma. Pyroptosis has garnered significant attention in cancer research due to its pro-inflammatory and immunomodulatory nature. This study investigated the mechanism and role of MPPa-PDT-induced pyroptosis in osteosarcoma cells. MethodsWe treated human osteosarcoma 143b and HOS cells with MPPa at concentrations of 0.5 μM and 0.25 μM, respectively, then irradiated the cells with LED light at 630 nm wavelength with an energy density of 4.8 J/cm2. Cell viability and apoptosis ratio were detected using CCK-8 and Annexin V–Propidium Iodide staining, respectively. Intracellular reactive oxygen species (ROS) levels and mitochondrial membrane potential (MtΔψ) were assessed using 2ʹ,7ʹ-Dichlorofluorescin diacetate, and JC-1 staining kits, respectively. Scanning Electron Microscopy (SEM) was utilized to examine cell ultrastructure. The morphological changes of the cells were observed by an inverted microscope. Western blotting analysis was conducted to measure protein levels. To elucidate the mechanism and role, we re-evaluated relevant parameters after pretreating with NAC,Si caspase-3, and Si GSDME. ResultsMPPa-PDT inhibited the activity of osteosarcoma 143b and HOS cells and induced pyroptosis with mitochondrial damage, ROS aggregation, and activation of Caspase-3 and GSDME. The effects of MPPa-PDT on the activity and apoptosis of osteosarcoma cells were partially reversed after pretreating with Si GSDME. After NAC pretreatment, the activation of pyroptosis and Caspase-3 induced by MPPa-PDT was partially reversed. After Si Caspase-3 pretreatment, the pyroptosis induced by MPPa-PDT was partially reversed. ConclusionMPPa-PDT can induce pyroptosis in osteosarcoma cells, which has the effect of enhancing apoptotic processes. Mitochondrial damage and ROS/caspase-3/GSDME pathway are the possible mechanisms of pyroptosis induced by MPPa-PDT.

MORPHOLOGICAL CHANGES IN LEYDIG TM3 CELLS INDUCED BY NEONICOTINOID ACETAMIPRID

Numerous studies demonstrate the vulnerability of the male reproductive system to various exogenous environmental factors, including agrochemicals such as pesticides, herbicides, and insecticides. Acetamiprid, a widely used chlornicotinyl insecticide of a new generation, was developed as a safer alternative to harmful organophosphates and carbamates for controlling insect pests. Our study aimed to investigate the impact of acetamiprid on Leydig TM3 cells cultivated as three-dimensional (3D) cultures after prolonged repeated exposure. The 3D model was treated with acetamiprid in a concentration range of 4 to 500 µM. Morphological parameters, specifically the size and shape of the cell spheroids, were evaluated for up to 7 days. Using a Zeiss Z1 Observer automated inverted microscope and TissueFAXS software, the development of spheroids was monitored. Three separate iterations yielded the experimental findings. Microsoft Office 365 and GraphPad Prism 8.01 were used to process the results. The obtained results showed no statistically significant changes (P&gt;0.05) in morphological parameters at any of the tested acetamiprid concentrations.

Electroacupuncture Serum Protects Blood-brain Barrier Damage after Ischemic Stroke BY Regulating the Pericytes in vitro.

Electroacupuncture (EA) exerts a protective role in Blood-Brain Barrier (BBB) damage after ischemic stroke, but whether this effect involves the regulation of the pericytes in vitro is unclear. The in vitro BBB models were established with brain microvascular endothelial cells (BMECs) and pericytes, and the co-cultured cells were randomly divided into three groups: the control group, oxygen-glucose deprivation/reoxygenation (OGD/R) group and EA group. OGD/R was performed to simulate cerebral ischemia-reperfusion in vitro. EA serum was prepared by EA treatment at the "Renzhong" (GV26) and "Baihui" (GV20) acupoints in middle cerebral artery occlusion/ reperfusion rats. Furthermore, the characteristics of BMECs and pericytes were identified with immunohistochemistry staining. The cell morphology of the BBB model was observed using an inverted microscope. The function of BBB was measured with transendothelial electrical resistance (TEER) and sodium fluorescein, and the viability, apoptosis, and migration of pericytes were detected by cell counting kit-8, flow cytometry, and Transwell migration assay. BMECs were positive staining for Factor-VIII, and pericytes were positive staining for the α-SMA and NG2. EA serum improved cell morphology of the BBB model increased TEER, and decreased sodium fluorescein in OGD/R condition. Besides, EA serum alleviated pericytes" apoptosis rate and migration number, and enhanced pericytes' viability rate in OGD/R condition. EA serum protects against BBB damage induced by OGD/R in vitro, and this protection might be achieved by attenuating pericytes apoptosis and migration, as well as enhancing pericytes viability. The findings provided new evidence for EA as a medical therapy for ischemic stroke.

(Invited) Development of Bipolar Electrochemical Microscopy for High-Speed and High-Resolution Monitoring of Biochemical Phenomena

Closed bipolar electrochemistry (cBPE) involves simultaneous oxidation and reduction at opposite poles of a conductor in two separated solutions. (Figure left). The unique property of cBPE to be operated without direct connection to an external power source is expected to lead to higher resolution in electrochemical imaging than the conventional microelectrode array type imaging platforms. We have applied cBPE to an electrochemical microscopy (BEM) (Figure right) for high-resolution biochemical imaging.1- 7 The operation principle of BEM has been successfully verified,1 then the current focus is on fabrication method of cBPE array. Previously, we filled Au into pores of a polyethylene terephthalate track-etched membrane having D=8 µm pores by electroless plating using 50 mM HAuCl4 aqueous solution and 50 mM NaBH4 EtOH solution.2 Using this cBPE array, an image of oxygen concentration distribution around cell spheroids was successfully obtained, but this method has a problem of the inability to completely fill the pores with gold, resulting in large ionic currents. In this study, we use conductive pastes for fabrication of vertical closed bipolar electrode array to completely fill the pores with conductive materials.Epoxy-based carbon paste (ECP) was prepared by mixing acetylene carbon black (ACB; particle size=42 nm; Strem Chemicals) and epoxy resin (mixture of Epok 812 (62 mL), DDSA (100 mL), and DMP-30 (1.5%); Okenshoji). Oil-based carbon paste (OCP) was prepared by mixing the ACB and the liquid paraffin. For Au paste (AuP), commercially available AURoFUSE (particle size (D50) 0.2–0.4 µm; Tanaka Kikinzoku Kogyo) was used as is. Vertical cBPE arrays using the carbon paste filled in pores of a glass capillary plate was fabricated by filling ECP (ACB: epoxy resin=1:4 in weight ratio) cured at 60°C for 15 h. After cooling, the surface was coated by OCP (ACB: liquid paraffin=1:1 in weight ratio) to fill the dented parts shrank by heat curing. For [Fe(CN)6]3 − imaging, a sample side of the array was modified with reduced graphene oxide (RGO) by coating OCP containing 5 wt.% RGO (RGO-OCP). For H2O2 imaging, a sample side of the array was modified with Prussian blue (PB) by coating commercially available PB containing carbon paste (Carbon Mediator Paste, C2070424P2; SunChemical Gwent). The BEM system was constructed using two solution chambers (detection/sample chambers), BPE array, and driving electrodes. The detection chamber located at the bottom side of the BPE array was filled with 10 mM [Ru(bpy)3] Cl2/20 mM tripropylamine (TPA) solution containing phosphate buffered saline (PBS). The sample chamber located at the bottom side was filled with the sample solution for each measurement. A platinum driving electrode inserted into the detection chamber, another platinum driving electrode and a Ag/AgCl reference electrode inserted into the sample chamber were connected WE, CE and RE connector of a potentiostat, respectively. The entire system was mounted on an inverted microscope with a high sensitivity CCD camera to observe the electrochemiluminescence (ECL) generated at the underside of the BPEs.By using RGO and PB modified carbon paste (PC) cBPE array for BEM element, we successfully obtained an image of 5 mM [Fe(CN)6]3− and H2O2 inflow, respectively.5 These results show that cBPE arrays for BEM can be fabricated by filling micropores with carbon paste taking advantage of the flexibility and ease of modification of the paste electrode. We are now investigating the use of AuP as an electrode material of cBPE array. We have obtained the result that ECL can be enhanced by using AuP as an electrode material.7 This may be due to the surface plasmon enhancement effect by the Au particles contained in the AuP. The establishment of a simple method for fabricating vertical cBPE arrays without ion leakage is expected to further accelerate the research and development of BEM.

The culture and identification of human placenta-derived mesenchymal stem cells

Objective: This study aimed to establish a reliable protocol for the cultivation and characterization of human placenta-derived mesenchymal stem cells (hPMSCs) to evaluate their growth dynamics and immunophenotype. Methods: hPMSCs were thawed and cultured under controlled conditions using specialized serum-free medium. Cell viability and morphology were assessed using an inverted microscope, and medium changes were performed bi-daily. For cell identification, immunofluorescence staining was conducted with antibodies CD44, CD90, and CD45, and cells were characterized based on surface marker expression. Results: Cultured hPMSCs exhibited a fibroblast-like morphology with rapid proliferation, particularly after reaching a seeding density of 50%. Growth curves indicated peak proliferation between days 3 and 4. Immunofluorescence analysis confirmed that hPMSCs were positive for CD90 and CD44, but negative for CD45, aligning with typical mesenchymal stem cell profiles. Conclusion: The established protocol successfully cultivated and characterized hPMSCs, demonstrating their viability and specific surface markers. These findings support their potential application in regenerative medicine and therapeutic research.