Cells Stained Research Articles

Functional characterization of OR51B5 and OR1G1 in human lung epithelial cells as potential drug targets for non-type 2 lung diseases

Abstract Background Hypersensitivity to odorants like perfumes can induce or promote asthma with non-type 2 inflammation for which therapeutic options are limited. Cell death of primary bronchial epithelial cells (PBECs) and the release of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8 are key in the pathogenesis. Extra-nasal olfactory receptors (ORs) can influence cellular processes involved in asthma. This study investigated the utility of ORs in epithelial cells as potential drug targets in this context. Methods We used the A549 cell line and primary bronchial epithelial cells using air–liquid interface culture system (ALI-PBECs). OR expression was investigated by RT-PCR, Western blot, and Immunofluorescence. Effects of OR activation by specific ligands on intracellular calcium concentration, cAMP, Phospholipase C (PLC), cell viability, and IL-6 and IL-8 secretion were analyzed by calcium imaging, enzyme immunoassays, Annexin V/ propidium iodide -based fluorescence-activated cell staining or by ELISA, respectively. Results By screening A549 cells, the OR51B5 agonists Farnesol and Isononyl Alcohol and the OR1G1 agonist Nonanal increased intracellular Ca2 + . OR51B5 and OR1G1 mRNAs and proteins were detected. Both receptors showed a preferential intracellular localization. OR51B5- but not OR1G1-induced Ca2 + dependent on both cAMP and PLC signaling. Farnesol, Isononyl Alcohol, and Nonanal, all reduced cell viability and induced IL-8 and IL-6 release. The data were verified in ALI-PBECs. Conclusion ORs in the lung epithelium might be involved in airway-sensitivity to odorants. Their antagonism could represent a promising strategy in treatment of odorant-induced asthma with non-type 2 inflammation. Graphical Abstract

TMIC-57. EFFECT OF HYPOXIA ON SPHINGOSINE-1-PHOSPHATE (S1P) METABOLIZING ENZYMES IN DIFFUSE INTRINSIC PONTINE GLIOMA CELL LINES

Abstract Diffuse Midline Glioma H3K27 altered (DMG) is a devastating and incurable paediatric brain tumour. Previously a bioinformatic approach was taken to evaluate genes involved in the sphingosine-1-phosphate (S1P) biosynthesis pathway in diffuse intrinsic pontine gliomas (DIPG). S1P is known to interact with epigenetic partners (HDAC1) as well as play a role in proliferation, immune cell trafficking, angiogenesis and apoptosis. As a part of a broad bioinformatic approach evaluating the expression of genes associated with the sphingomyelin (SM) biosynthesis pathway, S1P metabolism, S1P receptors and downstream targets, 14 of 25 genes were found to be dysregulated in DIPG relative to normal brain (p<0.0002). Herein we focus on the imbalance of the S1P metabolic enzymes sphingosine kinase 2 (SPHK2) and sphingosine-1-phosphate lyase (SGPL1). Bioinformatic data revealed higher levels of SGPL1 and lower levels of SPHK2 compared with normal brain. DIPG cell lines (SU-DIPG-IV, VUMC-DIPG-08, VUMC-DIPG-A) were used to validate gene and protein expression and to evaluate changes following exposure to hypoxic conditions. Confocal images reveal an increase in nuclear SPHK2 staining in cells cultured for 24- and 48-hours under hypoxic conditions. HDAC1 immunostaining was localised in the nucleus. SGPL1 immunostaining, while mostly cytoplasmic, nuclear staining was observed. qRT-PCR assays were used to evaluate changes in mRNA expression in SPHK2, SGPL1 and HDAC1 in DIPG cell lines incubated under hypoxic conditions. The Pfaffl method was used to analyse gene expression relative to the housekeeping gene TBP. Under hypoxic conditions, the levels of SPHK2 and HDAC1 were higher in DIPG cells cultured in hypoxic conditions for 48 hours compared to DIPG cells cultured in normoxic conditions, whereas SGPL1 expression was lower in hypoxic treated cells. Current experiments targeting the modulation of these S1P-metabolizing enzymes are underway to determine the role of the S1P signalling pathway and determine the potential advantage of targeting this pathway in DIPG.

IMMU-35. TARGETING THE AHR IMMUNOMETABOLIC HUB DURING VIROIMMUNOTHERAPY OF GLIOMAS

Abstract Glioblastoma (GBM) poses a significant therapeutic challenge in oncology. Recent clinical trials have shown the feasibility of virotherapy to treat patients with GBM. Our laboratory developed an oncolytic adenovirus termed Delta-24-RGD that has been tested in clinical trials for recurrent GBM patients with encouraging results (NCT00805376, NCT02798406). These clinical studies, together with preclinical data, showed that the efficacy of Delta-24-RGD was due to direct tumor cell oncolysis and indirect activation of anti-tumor immune response. To further leverage this immune component, our group generated Delta-24-RGDOX, an armed version of Delta-24-RGD expressing the T-cell activator OX40-L, which exhibits a superior anti-cancer effect. In this project, we aim to further boost the effectiveness of Delta-24-RGDOX by targeting factors that maintain the immunosuppressive characteristic of gliomas. Specifically, we identified the environmental sensor and transcription factor Aryl Hydrocarbon Receptor (AhR) as one of the immunosuppressive factors. We show that AhR is overexpressed in GBM surgical specimens compared to healthy brain tissue, and high AhR expression correlates with poorer survival in glioma patients. Immunofluorescence staining of glioma cells treated with Delta-24-RGDOX demonstrated that AhR translocates to the nucleus, and qPCR showed that translocation resulted in the enhanced transcription of AhR-responsive elements. Additionally, bulk RNA-seq reveals that in vivo glioma models treated with Delta-24-RGDOX exhibit activation of the AhR pathway network. This unexpected and paradoxical activation may be responsible for immune suppression mechanisms, which might hinder the effect of Delta-24-RGDOX. Based on these data, we combined two innovative strategies, the oncolytic virus Delta-24-RGDOX and AhR inhibitors. Survival analysis in immunocompetent models of gliomas treated with this combined therapy showed the greatest therapeutic benefit compared to monotherapy controls. These findings highlight the critical role of AhR activation triggered by Delta-24-RGDOX and position AhR as a potential therapeutic target for enhancing the efficacy of oncolytic adenovirus therapy in GBM.

Lactiplantibacillus plantarum inhibited the growth of primary liver cancer by inducing early apoptosis and senescence, in vitro

Primary liver cancer (PLC), comprising hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), is a severe form of cancer associated with a high mortality and morbidity rate and increasing incidence worldwide. Current treatment options are limited and chemotherapeutics demonstrate strong side effects. New therapies are highly required. Lactobacilli represent the most diverse lactic acid-producing bacteria group and a prominent example of probiotics. Several studies have highlighted the anticancer efficacy of probiotics, especially of Lactiplantibacillus plantarum. However, there are limited studies on its activity on two PLC types, hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). This study evaluated the inhibitory mechanism and properties of L. plantarum ONU 12 (Lp 12) and L. plantarum ONU 355 (Lp 355), isolated from grapes in Ukraine and France, in murine PLC cell lines, in vitro. Strain Lacticaseibacillus casei ATCC 393 (Lc 393) has been taken for a direct comparison, as the most studied probiotic strain. The three Lactobacillus species were used in three forms: as live and heat-killed suspensions, and as sonicated extracts, and tested either as a monotherapy or in combination with standard chemotherapeutics (sorafenib for HCC and gemcitabine for CCA). Cell proliferation and viability were assessed via crystal violet staining assay and cell counting kit-8 assay. The induction of senescence was investigated by senescence-associated β-galactosidase assay. Fluorescence-activated cell sorting analysis was used to determine the apoptotic mechanism behind the inhibitory property of lactobacilli. The results showed that the live suspensions and sonicated extracts of Lp 12, Lp 355, and Lc 393 demonstrated inhibitory properties in CCA and HCC cells after 48 h of incubation. In combinations with standard chemotherapeutics, lactobacilli treatments have shown strong synergistic effects. The combination therapy allowed to reduce the chemotherapeutic doses of gemcitabine from 50 μM to 0.1 and 0.05 μM and sorafenib from 13.8 μM to 6.9 and 3.45 μM. Successful treatment regimes induced early apoptosis and cellular senescence in PLC, as the mechanism of inhibition. Heat-killed suspensions showed no inhibitory effect in none of the cell lines. Both strains, Lp 12 and Lp 355, showed successful results and need further testing in vivo, using autochthonous HCC and CCA models.

EXTH-39. A BENCH-TOP MODEL FOR THE OPTIMIZATION OF ULTRASOUND PARAMETERS FOR SONODYNAMIC THERAPY OF GLIOBLASTOMA

Abstract BACKGROUND In-vitro studies have demonstrated that low frequency sound waves can interact with hemo-porphyrin molecule to induce the production of reactive oxygen species. This biophysical phenomenon can induce tumour cell death in glioblastoma cells in-vitro. The purpose of this study was to evaluate the production of reactive oxygen species and expression of apoptotic markers in glioblastoma cells in response to varying total treatment time and ultrasound power using a bench-top focused ultrasound (FUS) device. METHODS U251 cells were exposed to 5-Aminolevulenic Acid (5-ALA) at varying concentrations for up to 24 hours. The time point with peak proto-porphyrin IX (PPIX) fluorescence was established using fluorescence activated cell sorting (FACS). A transducer utilized at 0.91MHz was placed on an inverted stage with the focus targeted to the bottom of a standard 96-well cell culture plate. Pressure wave delivery was confirmed using a hydrophone. GBM cells were treated with 5-ALA alone, FUS alone, or 5-ALA + FUS and the cells were incubated for 24 hours prior to measuring ROS induction and cell death. ROS was measured using FACS with the CellROX Green Kit for Oxidative Stress Detection and Annexin-V was measured using the Dead Cell Stain Kit. RESULTS Peak fluorescence intensity for PPIX in the U251 cell line was found to be induced through incubation with 5-ALA for 24 hours with a concentration 200mg/mL. The fluorescence intensity of PPIX was found to be 313% greater in the treated group compared to the control group. Peak ROS was observed using a 0.91MHz transducer frequency for a total time of 120s with 6W average power. ROS was 61% and 66% greater in the SDT group compared to the FUS and 5-ALA group respectively. CONCLUSION Using this bench-top set up we successfully induced ROS and apoptosis in U251 cells. Establishing a reliable in-vitro model will allow us to further investigate the effects of other sonication parameters such as burst length.

Expression of ALKBH5 in Odontogenic Lesions.

N6-methyladenosine (m6A) is the most abundant epigenetic RNA modification in eukaryotes and plays a role in various cancers in humans. This m6A modification is regulated by m6A writers, erasers, and readers. One of the m6A erasers is α-ketoglutarate-dependent dioxygenase homolog 5 (ALKBH5). Previous studies have suggested that ALKBH5 is involved in the pathogenesis of head and neck squamous cell carcinoma. However, the role of ALKBH5 in odontogenic lesions has never been investigated. This study aimed to examine ALKBH5 expression in dental follicles (DFs), dentigerous cysts (DCs), odontogenic keratocyst (OKC), and ameloblastoma (AM) using immunohistochemistry. Six cases of DF, 20 cases of DC and OKC, respectively, and 30 cases of AM were included. The expression patterns, percentage of ALKBH5-positive cells, staining intensities, and immunoreactive scores were examined. ALKBH5 was mainly expressed in the nuclei of the epithelial cells in odontogenic lesions. The percentage of ALKBH5-positive cells was significantly higher in OKC and AM samples compared with DF samples (P < 0.01). The percentage of ALKBH5-positive cells was also higher in OKC and AM samples than in DC samples; however, these results did not show statistical significance (P > 0.05). ALKBH5 cell staining intensities and immunoreactive scores were significantly greater in OKC and AM samples than in DF and DC samples (P < 0.01). Our results suggested that ALKBH5 might play a role in the pathogenesis of odontogenic lesions. Further investigation is needed to elucidate the precise molecular mechanism of the role of ALKBH5 in these diseases.

ANGI-11. SEX DIFFERENCES IN SOX2 GLIOBLASTOMA EXPRESSION OUTSIDE MRI-DEFINED CONTRAST ENHANCEMENT AT AUTOPSY

Abstract INTRODUCTION Sex-determining region Y-box 2 (SOX2) has recently been used as a marker for pluripotency to identify cellular glioblastoma invasion pathologically. This can be used to identify areas of tumor invasion consisting of sparse individual cells, likely highlighting the furthest margin of tumor invasion. This study aligned large format SOX2 stained tissue samples to conventional MRI to determine demographic factors that impact the presence of SOX2 positive invasion outside the T1 contrast-enhanced tumor margin. METHODS The cohort for this study consisted of 36 IDH-wildtype glioblastoma patients (23 male, 13 female, mean age=61.9). Tissue samples from areas of suspected tumor invasion and normal tissue were collected at autopsy, stained for SOX2, digitized at 40X resolution, and automatically segmented for SOX2 positive staining cell percent (SOX2+%) at MR resolution using a color deconvolution algorithm. Contrast enhanced T1 scans from acquisitions close to death were annotated manually for tumor presence and aligned to the T2-weighted FLAIR image. Tissue data was then aligned to MR images using a custom in-house software that relies on manually defined control points to generate a nonlinear transform that warps tissue into MR space while correcting for tissue shrinkage. Mean SOX2+% was computed for each patient outside of contrast enhancement and compared across age and sex as demographic factors. RESULTS Males were found to have a higher SOX2+% than females outside contrast enhancement (t=2.09, p=0.045), while no association with age was observed (r=0.146, p=0.397). CONCLUSIONS Sex differences were observed for non-enhancing SOX2 positivity, which may relate to the more aggressive tumors identified in male patients in some studies. Further research into the mechanisms behind sex differences in tumor growth patterns and molecular characteristics impacting the infiltration of tumor beyond the MR defined region is needed.

Antibacterial Activity and Mechanism of Rose Essential Oil against Aeromonas veronii isolated from Northern snakehead (Channa argus).

To investigate and identify the antibacterial action and mechanism of rose essential oil (REO) against Aeromonas veronii isolated from northern snakehead for the first time by the phenotypic and metabolic analysis. The two-fold broth microdilution and spread-plate method identified that the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of REO against A. veronii were 1.25 μL mL-1 and REO impaired the growth in a concentration-dependent manner, indicating that REO possessed a significant bacteriostatic activity. Electron microscopy and live-dead cell staining found that REO caused a severe disruption of cellular morphology and increased the membrane permeability. Additionally, REO treatment induced the leakage of intracellular biomolecules such as proteins and nucleic acids from the bacteria. Metabolomics analysis showed that compared with the control, the REO treatment group exhibited a total of 190 differential metabolites (118 down-regulated and 72 up-regulated), which involved in the main metabolic pathways such as biotin metabolism, arginine biosynthesis, glutathione metabolism, lysine degradation, and histidine metabolism and the TCA cycle. These results verified that REO disturbed the metabolic processes of A. veronii to achieve the bacteriostatic effect. The REO exhibited the effective antibacterial activity against A. veronii via breaking the cellular structure, increasing the membrane permeation and disrupting the metabolic processes.

Analysis of antinuclear antibody in 9 528 pregnant women during early pregnancy in a hospital in Qingdao City

To analyze the positivity rate and titer of antinuclear antibody (ANA), as well as nuclear pattern and target antigen of ANA in healthy pregnant women during early pregnancy in Qingdao area. A prospective cohort study design was used to include a total of 9 528 healthy pregnant women registered at the Women and Children's Hospital Affiliated to Qingdao University from March 2023 to June 2024.Indirect immunofluorescence assay (IIF) was used to detect ANA, its titer and cell staining pattern. Fifteen specific antibodies were tested using the magnetic bar code immunofluorescent luminescence method. Logistic regression model was used to analyze the risk factors of pregnancy with autoimmune disease(AID). The results showed that among 9 528 pregnant women in early pregnancy, 1 346 cases (14.1%) were positive of ANA, including 1 011 cases with a titer of 1∶100 (10.6%), 236 cases (2.5%) with a titer of 1∶320, and 99 cases (1.0%) were detected at a titer >1∶320. Among the 1 346 ANA-positive pregnant women, nuclear granular type accounted for the highest proportion (483 cases, 35.9%), followed by speckled type (347 cases, 25.8%) and cytoplasmic type (176 cases, 13.1%).Then, pregnant women with ANA titers ≥1∶100 were detected 15 specific antibodies.Anti-SSA was tested in 121 cases accounted for the majority, followed by 110 cases with anti-Ro-52, 56 cases with anti-SSB, 51 cases with anti-mitochondrial M2 subtype antibodies and 37 cases with anti-centromere B. In conclusion,in healthy pregnant women in Qingdao area, ANA positivity rate was 14.1%, and the titer of ANA was mainly at 1∶100.The predominant nuclear patterns were nuclear granular and speckled types.The specific autoantibodies were mainly anti-SSA antibodies and anti-Ro-52 antibodies.The detection of ANA and specific autoantibodies is of great significance for early prediction, diagnosis, and intervention of autoimmune diseases during pregnancy.

Microfluidic device-assisted 3D cell spheroids isolation, staining and embedding

3D cell spheroids have emerged as indispensable tools in cell biology research. In this study, the efficacy of an open-close switchable microfluidic device for on-chip staining and agar-embedding of 3D spheroids was thoroughly validated, especially in preparation for immunohistochemistry (IHC) analysis. The on-chip staining procedure, employing Trypan blue and Calcein-AM/PI staining, was demonstrated to evaluate the cell viability of spheroids. During overextended culture periods, a discernible decline in cell viability was observed at the central region of the spheroids, offering valuable information on the temporal aspects of spheroid development. On-chip agar embedding stands out for its precise control over the location and orientation of three-dimensional spheroids, a key factor in exploring cellular architecture, especially in fused spheroids using IHC analysis. In comparison to tube-based staining, this method significantly cuts down on reagent use and analysis time. This not only boosts efficiency but also lowers the risk of spheroid damage or loss during staining, highlighting its potential for handling miniaturized tissue samples and biopsies in cell assays. The on-chip staining and agar-embedding techniques presented here not only provide valuable insights into spheroids’ behavior but also pave the way for further developments in miniaturized tissue analysis and cell-based assays.

Hypoxia-Induced Mitochondrial ROS and Function in Pulmonary Arterial Endothelial Cells.

Pulmonary artery endothelial cells (PAECs) are a major contributor to hypoxic pulmonary hypertension (PH) due to the possible roles of reactive oxygen species (ROS). However, the molecular mechanisms and functional roles of ROS in PAECs are not well established. In this study, we first used Amplex UltraRed reagent to assess hydrogen peroxide (H2O2) generation. The result indicated that hypoxic exposure resulted in a significant increase in Amplex UltraRed-derived fluorescence (i.e., H2O2 production) in human PAECs. To complement this result, we employed lucigenin as a probe to detect superoxide (O2-) production. Our assays showed that hypoxia largely increased O2- production. Hypoxia also enhanced H2O2 production in the mitochondria from PAECs. Using the genetically encoded H2O2 sensor HyPer, we further revealed the hypoxic ROS production in PAECs, which was fully blocked by the mitochondrial inhibitor rotenone or myxothiazol. Interestingly, hypoxia caused an increase in the migration of PAECs, determined by scratch wound assay. In contrast, nicotine, a major cigarette or e-cigarette component, had no effect. Moreover, hypoxia and nicotine co-exposure further increased migration. Transfection of lentiviral shRNAs specific for the mitochondrial Rieske iron-sulfur protein (RISP), which knocked down its expression and associated ROS generation, inhibited the hypoxic migration of PAECs. Hypoxia largely increased the proliferation of PAECs, determined using Ki67 staining and direct cell number accounting. Similarly, nicotine caused a large increase in proliferation. Moreover, hypoxia/nicotine co-exposure elicited a further increase in cell proliferation. RISP knockdown inhibited the proliferation of PAECs following hypoxia, nicotine exposure, and hypoxia/nicotine co-exposure. Taken together, our data demonstrate that hypoxia increases RISP-mediated mitochondrial ROS production, migration, and proliferation in human PAECs; nicotine has no effect on migration, increases proliferation, and promotes hypoxic proliferation; the effects of nicotine are largely mediated by RISP-dependent mitochondrial ROS signaling. Conceivably, PAECs may contribute to PH via the RISP-mediated mitochondrial ROS.

Enhancing osteogenesis and mandibular defect repair with magnesium-modified acellular bovine bone matrix

An acellular bovine bone matrix modified to release Magnesium ions (Mg2+) (ABBM-Mg) was prepared and evaluated for its potential in osteogenesis and mandibular defect repair. Mg2+ was incorporated into ABBM using an ion exchange method. The microstructure and mechanical properties of both ABBM and ABBM-Mg were analyzed using SEM and a biomechanical testing machine. Cytocompatibility, cell adhesion, and osteogenic differentiation were assessed using various methods including CCK-8, Live/Dead staining, SEM, ALP staining, and qPCR analysis in MC3T3-E1 cells. Additionally, a mandibular defect model in rats was established. The bone defect repair outcomes were evaluated using Micro-CT, histological HE staining, and Masson staining. The study showed that mineralization containing magnesium was redeposited on the surface of the three-dimensional porous ABBM, and the ABBM-Mg scaffold promoted cell proliferation and osteogenic differentiation compared to the ABBM scaffold. In the rat mandibular defect model, the ABBM-Mg scaffold demonstrated superior bone repair ability. This study successfully incorporated Mg2+ into ABBM without significantly affecting its microstructure and compressive strength. Furthermore, ABBM-Mg showed sustained release of Mg2+ which enhanced cell proliferation, adhesion, and osteogenic differentiation in vitro, and promoted mandibular defect healing in rats. This research opens up new possibilities for the clinical application of functionalized acellular bone matrix.Graphical

Reduced Sox7 contributes to abnormal valve development resulting in congenital aortic stenosis

Abstract Background Abnormal valve development is the most common congenital heart malformation. During valve remodeling, abnormal changes in valve cell proliferation, apoptosis, and extracellular matrix synthesis affect valve development, resulting in malformations. The molecular mechanisms underlying pathophysiology of aortic stenos is remain unclear. Purpose We would like to explore how Sox7 is involved in aortic valve development. Methods and Results We proved Sox7 was mainly expressed in endocardial and mesenchymal cells of aortic valves by immunofluorescence staining determined by at different stages of mice (E10.5～8 weeks) and human embryos and RNA-seq of E9.5 mouse embryonic hearts. We constructed a conditional Sox7 floxed allele in endothelial cells using an endocardial specific Cre. Echocardiography data showed that Nfat-KO mice had increased blood velocity, pressure gradient across aortic valves, left ventricular mass and dilated aortic roots. Then, HE and Sirius red staining to examine histological changes in aortic valves of Nfat-KO mice were performed, and we observed a remarkable increase in the size and increased staining of fibrotic cells in aortic valves of KO mice. Next, we collected embryonic hearts of Sox7 CNC-specific knockout mice (WNT1-KO) and controls at E16.5. According to HE staining results and echocardiography analysis, there was no difference in aortic valves between WNT1-KO mice and controls. Meanwhile, we collected Nfat-KO mice and control embryos at E11.5 days. HE staining results showed that EndMT process was not impaired. Interestingly, leaflets and cusps of Nfat-KO mice showed significantly augmented proliferation levels, decreased apoptosis levels, and calcium deposition in fibrin layer. A significant decrease in MMP9 levels were observed in both mRNA and protein levels. Co-transfection of Sox7-expressing vector and MMP9 luciferase in MEEC suggested that Sox7 significantly activated MMP9. We used adenovirus to overexpress Sox7 in valvular interstitial cells. Compared with that in control group, the Sox7-overexpression group increased protein level of MMP9 and cleaved caspase-3, and increased Tunel-positive rate and lower Ki-67 positive rate. Finally, we detected expression of Sox7 protein in human fetal aortic valves derived from patients with aortic stenosis using immunofluorescence. The results demonstrated that the number of Sox7 positive mesenchymal cells in human fetal aortic valves of patients was significantly lower than that in control group. Conclusion We demonstrated that targeted inactivation of Sox7 in the endocardium of mice affects valve development and leads to aortic stenosis. Mechanistically, no influence of Sox7 on the EndMT process of the OFT cushion. Moreover, CNC-derived Sox7 was not involved in aortic valve development. Interestingly, we found that Sox7 regulated ECM remodeling through matrix MMP9 and Sox7 had an impact on apoptosis, proliferation, and calcification.

Topiramate Inhibits Capsaicin-Induced Mast Cell Degranulation and CGRP Release in Rat Dura Mater

Background/Objectives: Migraine is a disease that stands out for its high prevalence and socioeconomic costs. It involves the entire trigeminovascular system, the signaling substances, and their targets. However, the role of meningeal mast cells in migraine is still unclear. To better understand one of the components of neurogenic inflammation underlying migraine pathophysiology, we developed an in vivo rat model in which the dura mater was exposed bilaterally to investigate the influence of topiramate on capsaicin-induced mast cell degranulation and CGRP release from dura mater. Methods: On the day of the experiment, rats were anesthetized, and a craniectomy was performed on each parietal bone. Test substances were applied in situ over the dura mater using the right and left sides of the dura mater for the test and control, respectively. After exposure, the dura mater was processed for mast cell staining and counting. Using this setup, the effect of capsaicin (10−3 M) was evaluated in rats of both sexes, and subsequently the effect of in situ (10−3 M, 20 µL) and (20 mg/kg/day for 10 days) topiramate treatment on mast cell degranulation and CGRP release were evaluated. Results: In both female and male rats, there was a greater amount of degranulated mast cells in the side stimulated by capsaicin compared to the control side in both females (18 ± 3% vs. 74 ± 3%; p = 0.016) and males (28 ± 2% vs. 74 ± 3%, p = 0.016). In the group treated with topiramate for 10 days prior to the experiments, capsaicin did not induce mast cell degranulation (control 20 ± 1% vs. capsaicin 22 ± 1%, p = 0.375) in contrast to animals treated for 10 days with gavage control (control 25 ± 1% vs. capsaicin 76 ± 1%, p = 0.016). Topiramate applied in situ concomitant with capsaicin did not protect the mast cells from degranulation in response to capsaicin (38 ± 2% vs. 44 ± 1%, p = 0.016). There was a significant reduction in CGRP release from the dura mater in the group treated with topiramate for 10 days compared to the control. Conclusions: This study demonstrates a novel experimental model wherein systemic administration of topiramate is observed to modulate the impact of capsaicin on meningeal mast cell degranulation.

Characterization of E-Cadherin, SSEA-1, MSI-1, and SOX-2 Expression and Their Association with Pale Cells in Adenomyosis

Adenomyosis (AM) is a gynecological disease characterized by the invasion of endometrial glands and stroma within the myometrium. The etiology and pathogenesis of AM remain inadequately understood. Pale cells were identified as a novel cell type characterized by the absence of desmosomal contacts and light-colored cytoplasm. These cells were observed to migrate individually through ultra-micro ruptures in the basal membrane of the endometrial glands, translocating into the stroma and then further into the myometrium. Our study aimed to explore the possible stem cell properties of these pale cells. Forty hysterectomy specimens were analyzed using immunohistochemistry and immunofluorescence to assess negative E-cadherin expression and the positive expression of stem cell markers SSEA-1, MSI-1, and SOX-2. Immunohistochemical analysis revealed the presence of pale cells and occasionally rounded, enlarged E-cadherin-negative cells predominantly in the basal endometrial epithelium. The stem cell marker SSEA-1 was significantly elevated in the basalis epithelium, as well as in the ectopic epithelium. SSEA-1 positive cells were also identified in the stroma and myometrium. Sporadic colocalization of SSEA-1+/E-cadherin– cells was confirmed through immunofluorescence. The positive staining of pale cells for SSEA-1 and MSI-1 was also confirmed at the ultrastructural level by immunoelectron microscopy. These findings indicate that pale cells may possess stem cell characteristics, particularly a positive SSEA-1 profile, warranting further in vitro investigation into their role in the pathogenesis of adenomyosis.

Role of hypothetical protein PA1-LRP in antibacterial activity of endolysin from a new Pantoea phage PA1.

Pantoea ananatis has emerged as a significant plant pathogen affecting various crops worldwide, causing substantial economic losses. Bacteriophages and their endolysins offer promising alternatives for controlling bacterial infections, addressing the growing concerns of antibiotic resistance. This study isolated and characterized the Pantoea phage PA1 and investigated the role of PA1-LRP in directly damaging bacteria and assisting endolysin PA1-Lys in cell lysis, comparing its effect to exogenous transmembrane domains following the identification and analysis of the PA1-Lys and the PA1-LRP based on whole genome analysis of phage PA1. Additionally, this study also explored how hydrophobic region of PA1-LRP (HPP) contributes to bacterial killing when combined with PA1-Lys and examined the stability and lytic spectrum of PA1-Lys under various conditions. Phage PA1 belonging to the Chaseviridae family exhibited a broad host range against P. ananatis strains, with a latent period of 40 minutes and a burst size of 17.17 phages per infected cell. PA1-Lys remained stable at pH 6-10 and temperatures of 20-50°C and showed lytic activity against various Gram-negative bacteria, while PA1-Lys alone could not directly lyse bacteria, its lytic activity was enhanced in the presence of EDTA. Surprisingly, PA1-LRP inhibited bacterial growth when expressed alone. After 24 h of incubation, the OD600 value of pET28a-LRP decreased by 0.164 compared to pET28a. Furthermore, the lytic effect of co-expressed PA1-LRP and PA1-Lys was significantly stronger than each separately. After 24 h of incubation, compared to pET28a-LRP, the OD600 value of pET28a-Lys-LRP decreased by 0.444, while the OD420 value increased by 3.121. Live/dead cell staining, and flow cytometry experiments showed that the fusion expression of PA1-LRP and PA1-Lys resulted in 41.29% cell death, with bacterial morphology changing from rod-shaped to filamentous. Notably, PA1-LRP provided stronger support for endolysin-mediated cell lysis than exogenous transmembrane domains. Additionally, our results demonstrated that the HPP fused with PA1-Lys, led to 40.60% cell death, with bacteria changing from rod-shaped to spherical and exhibiting vacuolation. Taken together, this study provides insights into the lysis mechanisms of Pantoea phages and identifies a novel lysis-related protein, PA1-LRP, which could have potential applications in phage therapy and bacterial disease control.

Seasonal changes in the viability and abundance of bacterial cells in the snowpack ecosystem of a High Arctic ice cap

ABSTRACT Microbes play an essential role in nutrient turnover within Arctic environments, and their contribution to biogeochemical cycles can depend on several factors, including but not limited to cell viability. In this study, we employed the SYBR-PI dual cell stain to epifluorescence microscopy to enumerate proportions of potentially viable and non-viable bacterial cell populations within a melting snowpack on an ice cap, Foxfonna in Svalbard. Non-viable cells dominated on Foxfonna (2.5 ± 0.36 × 107 cells m−2) during the June to early July period, when biological production was usually at its peak. Furthermore, non-viable cells also dominated the total cell abundance within superimposed ice (223 ± 242 cells mL−1) and glacial ice (695 ± 717 cells mL−1) beneath the snow. We propose that the rapid, early loss of cell viability was caused by a number of abiotic and biotic factors. Hence, necromass (dead cell residue) contributed to the export of organic matter to downstream ecosystems.

Reactive oxygen species-responsive nano gel as a carrier, combined with photothermal therapy and photodynamic therapy for the treatment of brucellosis.

Brucellosis is an intracellular infectious disease that is primarily treated with antibacterial therapy. However, most antibacterial drugs struggle to penetrate the cell membrane and may be excluded or inactivated within the cell. In a recent study, researchers developed a nanogel coated with polydopamine (PDA) that responds to reactive oxygen species (ROS) and has enhanced adhesion properties. This nanogel encapsulates photosensitized zinc phthalocyanine (ZnPc) and an antibacterial drug, and is further modified with folic acid (FA) for active targeting. The resulting ROS-responsive nanogel, termed PDA@PMAA@ZnPc@DH-FA, can reach temperatures up to 50°C under near-infrared light, leading to a 72.1% improvement in drug release through increased ROS production. Cell staining confirmed a cell survival rate above 75%, with a low hemolysis rate of only 4.633%, indicating excellent biocompatibility. Furthermore, the study's results showed that the nanogel exhibited stronger killing effects against Brucella compared to administering the drug alone. Under near-infrared irradiation, the nanogel achieved a bacteriostatic rate of 99.8%. The combined approach of photothermal therapy and photodynamic therapy offers valuable insights for treating Brucella.

Nuclear staining for pan-Trk by immunohistochemistry is highly specific for secretory carcinoma of breast: pan-Trk in various subtypes of breast carcinoma

AimsSecretory carcinoma of breast (SCB) typically harbours ETV6-NTRK3 gene fusion. Pan-Trk immunohistochemistry analysis (IHC) has been shown to be sensitive for SCB diagnosis. However, weak focal pan-Trk nuclear staining was...

PD-L1 protein expression in breast cancer

AimsThe immune checkpoint marker, Programmed cell death-ligand 1 (PD-L1), is expressed by both cancer epithelial cells and tumour-infiltrating immune cells (TICs) thus constituting a potential target for immunotherapy. This is...