Tissue Slices Research Articles

TMIC-40. TARGETING GLIOMA TUMOR MICROENVIRONMENT BY SPECIFIC NEUTRALIZATION OF EXTRACELLULAR NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE (ENAMPT) USING ALT-100, A NOVEL HUMANIZED MONOCLONAL ANTIBODY

Abstract BACKGROUND Intracellular nicotinamide phosphoribosyltransferase (iNAMPT is the rate-limiting enzyme of NAD salvage pathway and is preferentially used by glioma cells for regulating energy metabolism and homeostasis. When secreted, extracellular NAMPT (eNAMPT) exerts cytokine-like functions and is associated with poor prognosis in cancer patients. We have previously reported an essential role of iNAMPT for glioma cell proliferation and survival. Here, we examined whether eNAMPT influences glioma biology and tumor microenvironment by neutralizing eNAMPT using ALT-100, a novel humanized monoclonal antibody. METHODS Native levels of eNAMPT in blood samples of patients with primary/recurrent gliomas were determined using ELISA under an IRB approved protocol. Next, the effects of ALT-100 on glioma microenvironment were determined by treating viable human glioma tissue slices with ALT-100 and assessing downstream effects on RNA and protein using RNASeq, reverse phase protein array and spatial transcriptomics. Lastly, the in vivo effect of ALT-100-mediated eNAMPT neutralization on survival was determined in a mouse intracranial glioma model. RESULTS Measurable levels of eNAMPT were noted in all patient samples with a range of (3.7-71.2 ng/ml). Preliminary results showed a correlation of eNAMPT levels with grade of tumor. Human organotypic glioma slice cultures treated with ALT-100, showed a profound alteration of gene and protein expression particularly related to key energy metabolism and proliferation pathways and the PD-1/PD-L1 axis. Additionally, results of spatial profiling of ALT-100 treated human organotypic glioma slices will be presented. Survival and response data from ALT-100 treatment in ongoing in vivo experiment will also be reported. CONCLUSIONS This is the first report of ALT-100-mediated eNAMPT neutralization in gliomas and demonstrates profound effects on the tumor and its microenvironment. Given that ALT-100 is in phase 2 trials (NCT05938036) against ARDS, data from our studies can potentially accelerate translating ALT-100-mediated eNAMPT neutralization to treat gliomas.

EXTH-83. CHARACTERIZATION OF SPLICING ABERRATIONS INDUCED BY PRMT5 INHIBITION IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) are lethal tumors with limited treatment options, with a high unmet need for novel therapies. PRMT5, an arginine methyltransferase that regulates several histone and non-histone targets, is overexpressed in cancers including GBM and is a promising therapeutic target given its role in promoting oncogenic processes. Here, we characterized the effects of pharmacological inhibition of PRMT5 by the brain-penetrant, orally bioavailable inhibitors, PRT811 and PRT808 on treatment-induced splicing aberrations using a panel of patient-derived glioma stem cells (GSC) and organotypic human glioma slice cultures. Changes in protein expression upon PRMT5 inhibition were assessed using reverse phase protein array (RPPA) analysis. Additionally, splicing aberrations due to PRMT5 inhibition in were tested in several GSCs using RNA-seq. We observed a potent reduction of symmetrical dimethylation of arginine (SDMA protein marks by both PRMT5 inhibitors across various GSC lines and ex-vivo brain tumor tissue slices. Notably, GSCs with both mutant and wild-type p53 displayed similar sensitivity to PRMT5 inhibition, independent of MTAP status. Moreover, PRMT5 inhibition elicited reduced cell proliferation, altered cell survival pathways, and apoptosis. RNA-seq analysis revealed that splicing alterations affecting numerous oncogenes and regulatory factors, encompassing 42 cancer driver genes, 80 splicing factors, and 42 transcriptional factors across these GSC lines, with specific splicing events observed in nuclear and cytoplasmic subcellular compartments. Additional analysis is ongoing to determine which of the aberrant transcripts are translated to yield tumor specific neoantigens that can be potential targets for tumor specific immune strategies. These results underscore the efficacy of pharmacological PRMT5 inhibition in genetically and epigenetically diverse GSCs, and highlight its potential for developing novel immune and non-immune therapeutic strategies against GBM.

Cryo-electron tomographic investigation of native hippocampal glutamatergic synapses

Chemical synapses are the major sites of communication between neurons in the nervous system and mediate either excitatory or inhibitory signaling. At excitatory synapses, glutamate is the primary neurotransmitter and upon release from presynaptic vesicles, is detected by postsynaptic glutamate receptors, which include ionotropic AMPA and NMDA receptors. Here, we have developed methods to identify glutamatergic synapses in brain tissue slices, label AMPA receptors with small gold nanoparticles (AuNPs), and prepare lamella for cryo-electron tomography studies. The targeted imaging of glutamatergic synapses in the lamella is facilitated by fluorescent pre- and postsynaptic signatures, and the subsequent tomograms allow for the identification of key features of chemical synapses, including synaptic vesicles, the synaptic cleft, and AuNP-labeled AMPA receptors. These methods pave the way for imaging brain regions at high resolution, using unstained, unfixed samples preserved under near-native conditions.

Cryo-electron tomographic investigation of native hippocampal glutamatergic synapses.

Chemical synapses are the major sites of communication between neurons in the nervous system and mediate either excitatory or inhibitory signaling. At excitatory synapses, glutamate is the primary neurotransmitter and upon release from presynaptic vesicles, is detected by postsynaptic glutamate receptors, which include ionotropic AMPA and NMDA receptors. Here, we have developed methods to identify glutamatergic synapses in brain tissue slices, label AMPA receptors with small gold nanoparticles (AuNPs), and prepare lamella for cryo-electron tomography studies. The targeted imaging of glutamatergic synapses in the lamella is facilitated by fluorescent pre- and postsynaptic signatures, and the subsequent tomograms allow for the identification of key features of chemical synapses, including synaptic vesicles, the synaptic cleft, and AuNP-labeled AMPA receptors. These methods pave the way for imaging brain regions at high resolution, using unstained, unfixed samples preserved under near-native conditions.

Subcutaneously transplanted xenogeneic protein recruits treg cells and M2 macrophages to induce browning of inguinal white adipose tissue.

To study whether subcutaneously embedding xenogeneic protein threads or synthetic polymer absorbable threads can improve obesity phenotypes and metabolic conditions, and to further explore its underlying mechanism. Thirty-six 8-week-old ob/ob mice were randomly allocated to three groups, respectively, receiving catgut embedding, PGA thread embedding or sham treatment bilaterally to the groin. Individual parameters including weight, food intake, and core temperature are recorded and metabolism assessment, energy expenditure analysis, and PET/CT scanning are also performed at fixed timepoints. After surgical incision, the inguinal white adipose tissue was histologically examined and its expression profile was tested and compared among groups 4 weeks and 12 weeks after operation. Catgut embedding reduced weight gain and improved metabolic status in ob/ob mice. Browning of bilateral inguinal WAT (white adipose tissue) was induced after catgut embedding, with massive infiltration of Treg cells and M2 macrophages in the tissue slices of fat pads. IL-10 and TGF-β released by Treg cells targeted macrophages and the induced M2 macrophages increased the expression of thermogenic and anti-inflammatory genes in fat. The secretion of catecholamines by polarized M2 macrophages led to the activation of β3-AR-related pathways in adipocytes and the browning of adipose tissue. Abdominal subcutaneous catgut embedding has the potential to combat obesity through the induction of WAT browning mediated by infiltrated Treg cells and macrophages.

SpaInGNN: Enhanced clustering and integration of spatial transcriptomics based on refined graph neural networks

Recent developments in spatial transcriptomics (ST) technology have markedly enhanced the proposed capacity to comprehensively characterize gene expression patterns within tissue microenvironments while crucially preserving spatial context. However, the identification of spatial domains at the single-cell level remains a significant challenge in elucidating biological processes. To address this, SpaInGNN was developed, a sophisticated graph neural network (GNN) framework that accurately delineates spatial domains by integrating spatial location data, histological information, and gene expression profiles into low-dimensional latent embeddings. Additionally, to fully leverage spatial coordinate data, spatial integration using graph neural network (SpaInGNN) refines the graph constructed for spatial locations by incorporating both tissue image distance and Euclidean distance, following a pre-clustering of gene expression profiles. This refined graph is then embedded using a self-supervised GNN, which minimizes self-reconfiguration loss. By applying SpaInGNN to refined graphs across multiple consecutive tissue slices, this study mitigates the impact of batch effects in data analysis. The proposed method demonstrates substantial improvements in the accuracy of spatial domain recognition, providing a more faithful representation of the tissue organization in both mouse olfactory bulb and human lateral prefrontal cortex samples

Mechanisms of atrial-fibrillation-induced ventricular dysfunction and recovery in the human left ventricular myocardium

Abstract Background Atrial fibrillation (AF) frequently occurs alongside heart failure (HF). Clinical studies indicate improved left ventricular (LV) function following rhythm restoration but the underlying myocardial processes remain unclear. Purpose This study investigated effects of AF and its termination on human LV myocardium as well as potential molecular mechanisms involved in the AF-induced LV dysfunction. Methods Human LV tissue slices obtained from HF patients were long-term cultivated in-vitro. AF was simulated by tachy-arrhythmic culture pacing at 100 bpm with 30% irregularity for 7 days and contractile LV function was compared to slices undergoing sinus rhythm (SR)-simulation (60 bpm/0%). After 7 days, rhythm restoration was simulated by switching to SR-simulation. To elucidate cellular mechanisms involved in AF-induced LV dysfunction, human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were subjected to AF-simulation and examined via epifluorescence microscopy (FURA-2) and confocal microscopy (Fluo-4). Additionally, to investigate the impact of reactive oxygen species (ROS) on cardiomyocytes, iPSC-CM were subjected to treatment with the ROS-scavenger N-acetylcysteine (NAC). Results AF-simulation caused a progressive decline in systolic force of human LV myocardium (n=14 slices) with significantly reduced twitch amplitudes compared to SR-simulation (n=11). Within just 5 days of AF-termination, a significant recovery in LV function was observed, indicated by improved systolic contraction amplitudes. IPSC-CM measurements revealed a significant reduction in systolic Ca2+-transient amplitudes as well as an increased diastolic Ca2+ leak after AF-simulation (n=41) compared to SR-simulation (n=44). However, 5 days after SR-restoration, iPSC-CM exhibited no significant differences anymore, indicating a fast recovery of systolic Ca2+-cycling. We further investigated the impact of ROS on LV function and could show that Ca2+-transient amplitude was preserved after AF-simulation in iPSC-CM when treated with NAC (n=35), suggesting an interplay of ROS with the Ca2+-homeostasis of cardiomyocytes. Conclusions This study demonstrates that AF impairs LV function in human HF myocardium. However, the involved changes in contractility and cardiomyocyte Ca2+ handling are reversible upon AF termination leading to improved LV function already within few days after AF cessation. Moreover, we provided an indication for an interplay of ROS with the Ca2+ homeostasis, endorsing an AF-induced LV-dysfunction, which merits further investigation.

Mechanisms of inducing cancer extracellular vesicles secretion and transfer for cancer malignancy

Assistant Professor Nao Nishida-Aoki, Waseda Institute for Advanced Study (WIAS), is investigating the role of extracellular vesicles (EVs) in physiological tumour tissue environments in order to improve cancer treatments. Communication tools like EVs are used by cancer cells to educate the surrounding cells and make them supportive to cancer cell growth and metastasis. Nishida-Aoki’s research focuses on understanding EV-mediated communication within the complex tissue environment of patient tumours, where they are secreted in densely packed cells and non-cellular components forming a 3D structure. She has developed ex vivo brain and lung metastatic models using organotypic tissue slice culture (TSC). This enables her to observe EV transfer within tissue architecture in real time, identify target cells affected by cancer EVs and determine how they are distributed. Nishida-Aoki is also exploring the mechanisms of inducible secretion of cancer extracellular vesicles upon cell recognition and contribution to cancer malignancy. Her research is investigating the molecular mechanics by which cancer cells regulate and the extent of interactions with neighbouring cells using EVs at metastatic initiation. She has hypothesised that cancer cells should increase communication frequency using EVs when they meet other types of cells in a new environment to benefit themselves. By elucidating the molecular basis of cancer-normal cell interactions mediated by EVs, Nishida-Aoki aims to develop novel cancer therapeutic strategies.

Proteomic profiling of extracellular vesicles in takotsubo syndrome

Abstract Background/Purpose Takotsubo syndrome (TS) is an acute form of heart failure characterized by transient regional wall motion abnormalities triggered by a stressful event. The mechanisms underlying its development and recovery are poorly understood, resulting in a lack of disease-specific treatments and diagnostic markers. Extracellular vesicles (EVs) play a significant role in cellular communication and have been shown to be important in various diseases. Their involvement in cardiac conditions like TS is an emerging area of research. The primary objective of this study is to isolate and characterize EVs, as well as profile their proteomic profile, from the heart tissue of rats induced with TS. Methods Rats underwent TS induction through the infusion of 1 mg/kg isoprenaline over 15 minutes (TS24h), or were given saline (control). High-resolution echocardiography verified apical ballooning at 6 hours. After 24 hours, heart tissues from apical and basal segments were collected and processed. EV isolation involved tissue slicing, enzymatic digestion, and differential centrifugation steps, including a final iodixanol cushion separation for large and small EVs (Figure 1). Tandem mass tags and mass spectrometry were utilized for global proteomics and any differentially expressed proteins (DEPs) were identified. Analytical techniques such as volcano plots, heatmaps, enrichment analysis, and protein clustering/networks were employed. The strongest clusters (lowest False Discovery Rate and highest intensity) were selected, and their Gene Ontology (GO) terms/pathways were identified. Results Pure, cup-shaped, vesicles ranging from 50-800nm were successfully isolated (figure 1). EVs from apex of control and TS24h hearts had lower protein concentrations compared to their corresponding basal segments. Global proteomic analysis revealed a distinct protein profile in EVs from the apical segment of TS hearts at 24 hours. A total of 256 (TS24h-apex vs control-apex) and 561 proteins (TS24h-apex vs TS24h-base) were differentially expressed. No DEPs were observed when comparing the basal segments of TS24h and control hearts. Functional enrichment analysis of the DEPs showed an abundant change of biological processes related to metabolic lipid processes, inflammatory response, complement activation, reorganization of extracellular matrix. A downregulation was seen for biological processes related to mitochondrial ATP synthesis coupled electron transport and muscle contraction. Conclusion This study demonstrates a distinct EV protein profile in the apical segments of TS hearts, with marked changes in proteins related to metabolic lipid processes, inflammation, and mitochondrial activity. This extensive catalogue of novel proteins sets the stage for future research aimed at identifying potential therapeutic targets and diagnostic biomarkers, and enabling proof-of-concept studies in TS.

Deep-Learning-Based Segmentation of Cells and Analysis (DL-SCAN)

With the recent surge in the development of highly selective probes, fluorescence microscopy has become one of the most widely used approaches to studying cellular properties and signaling in living cells and tissues. Traditionally, microscopy image analysis heavily relies on manufacturer-supplied software, which often demands extensive training and lacks automation capabilities for handling diverse datasets. A critical challenge arises if the fluorophores employed exhibit low brightness and a low signal-to-noise ratio (SNR). Consequently, manual intervention may become a necessity, introducing variability in the analysis outcomes even for identical samples when analyzed by different users. This leads to the incorporation of blinded analysis, which ensures that the outcome is free from user bias to a certain extent but is extremely time-consuming. To overcome these issues, we developed a tool called DL-SCAN that automatically segments and analyzes fluorophore-stained regions of interest such as cell bodies in fluorescence microscopy images using deep learning. We demonstrate the program’s ability to automate cell identification and study cellular ion dynamics using synthetic image stacks with varying SNR. This is followed by its application to experimental Na+ and Ca2+ imaging data from neurons and astrocytes in mouse brain tissue slices exposed to transient chemical ischemia. The results from DL-SCAN are consistent, reproducible, and free from user bias, allowing efficient and rapid analysis of experimental data in an objective manner. The open-source nature of the tool also provides room for modification and extension to analyze other forms of microscopy images specific to the dynamics of different ions in other cell types.

Calcium Imaging and Analysis in Beta Cells in Acute Mouse Pancreas Tissue Slices.

Ca2+ ions play a central role in the stimulus-secretion coupling cascade of pancreatic beta cells. The use of confocal microscopy in conjunction with the acute pancreas tissue slice technique offers valuable insights into changes in theintracellular calcium concentration following stimulation by secretagogues. This allows the study of beta cells on a single cell level, as well as their behavior on a multicellular scale within an intact environment. With the use of advanced analytical tools, this approach offers insight into how single cells contribute to the functional unit of islets of Langerhans and processes underlying insulin secretion. Here we describe a comprehensive protocol for the preparation and utilization of acute pancreas tissue slices in mice, the use of high-resolution confocal microscopy for observation of glucose-stimulated calcium dynamics in beta cells, and the computational analysis for objective evaluation of calcium signals.

Calcium Imaging in Brain Tissue Slices.

Calcium imaging is a method that was first developed in the mid-1970s yet kept developing until current days to allow accurate measurement of free calcium ions in tissues. This widely used method has provided significant advances to our understanding of cellular signal transduction, including the discovery of subcellular compartmentalization of neurons and astrocytes, the identification of multiple signaling pathways, and mapping the functional connectivity between astrocytes and neuronal networks. Here we describe a method for the loading and imaging of cell-permeable AM ester calcium-sensitive dyes for the in vitro measurement of free intracellular Ca2+ ions in acute brain slices.

Ex vivo fluorescence-guided resection margin assessment in breast cancer surgery using a topically applied, cathepsin-activatable imaging agent

Up to 40 % of breast cancer patients have a tumor-positive resection margin (TPRM) – defined as cancer cells at the surface of the resected specimen – after breast-conserving surgery (BCS), necessitating re-resection or boost radiation. To prevent these additional treatments, intraoperative near-infrared (NIR) fluorescence imaging with the topically applied, cathepsin-activatable imaging agent AKRO-6qcICG might be used to detect TPRMs and guide additional resection. Here, to validate its performance, the agent is topically applied to all surfaces of freshly resected breast cancer specimens (n = 11 patients) and to 3–5 mm thick tissue slices of the specimens (n = 26 patients). NIR fluorescence images of the resection surfaces and tissue slices are acquired and correlated to final histopathology. AKRO-6qcICG detects TPRMs with a sensitivity, specificity, PVV, and NPV of 100 %, 67 %, 10 %, and 100 %, respectively. On the tissue slices, the fluorescence signal has a median tumor-to-background ratio of 1.8. These findings indicate that topically applied AKRO-6qcICG can visualize TPRMs ex vivo with a high sensitivity and NPV, with sufficient contrast to adjacent healthy breast tissue.

Vitamin U alleviates AFB1-induced hepatotoxicity in pregnant and lactating mice by regulating the Nrf2/Hmox1 pathway

This study investigated the protective effect of Vitamin U on liver injury induced by aflatoxin B1 (AFB1) in maternal mice. 25 pregnant ICR mice were randomly divided into five groups: the AFB1 group (AF, 0.3 mg AFB1/kg b.w.), the Vitamin U group (U, 50 mg Vitamin U/kg b.w.), the AFB1 + Vitamin U group (AU, 50 mg Vitamin U /kg b.w. + 0.3 mg AFB1/kg b.w.), the control group (DMSO), and the MOCK group (distilled water). They were administered substances by gavage every day for 28 days. Results indicated that exposure to AFB1 increased the liver index and caused histological disruptions. Elevated serum levels of ALT and ALP were observed, along with a significant increase in liver MDA content and a decrease in GSH-Px and T-SOD levels. Moreover, the Keap1 and Hmox1 gene was downregulated with statistical significance, while the IL1β and TNFα gene were significantly upregulated. Vitamin U was demonstrated by the organized structure of liver cells in tissue slices, effectively reducing liver cell necrosis. This intervention was associated with a significant decrease in serum ALT and ALP activities, as well as a significant decrease in liver MDA content. Additionally, there were significant increases in liver T-SOD and GSH-Px levels, along with upregulation of mRNA and protein expression of Nfr2, Hmox1 and Keap1, and downregulation of mRNA expression of the IL1β gene. In summary, Vitamin U mitigated oxidative stress-induced liver injury by modulating the Nrf2/Hmox1 signaling pathway and inflammatory factors affected by AFB1.

Neuroprotective Role of AQP4 Knockdown in Astrocytes After Oxygen-Glucose Deprivation.

Aquaporin-4 (AQP4), predominantly expressed in astrocytes, has been implicated in the development of brain edema following ischemic events. However, its role in post-stroke neuroinflammation is not fully understood. Using a middle cerebral artery occlusion (MCAO) mouse model, we assessed AQP4's role in post-stroke inflammation. Brain tissue slices from male C57BL/6 mice were subjected to immunohistochemistry and western blot post-MCAO. Additionally, primary astrocytes were isolated for quantitative real-time PCR and immunofluorescence assays to evaluate the expression of inflammatory markers glial fibrillary acidic protein (GFAP) and AQP4. AQP4 modulation was achieved using viral knockdown and overexpression methods. Neuronal damage was assessed using flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) tests in co-culture studies. MCAO mice exhibited a significant upregulation in GFAP. This reactive astrogliosis corresponded with an elevation in inflammatory markers. AQP4 expression responded to this inflammatory trend, peaking at 6 h after OGD and returning to baseline levels at 24 and 48 h. Co-culture experiments revealed that AQP4(+) astrocytes exacerbated injury in OGD-treated neurons, as evidenced by increased TUNEL positivity and apoptotic events. Conversely, AQP4(-) astrocytes appeared to have a protective effect. Knockdown of AQP4 resulted in reduced post-OGD inflammatory response, whereas AQP4 overexpression intensified the injury to neurons post-OGD. In vivo experiments also confirmed that AQP4 inhibitor TGN-020 reduced and overexpression of AQP4 increased behavioral abnormalities and brain infarcts. Our findings underscore AQP4's pivotal role in modulating post-stroke neuroinflammation. Targeting AQP4 may present a novel therapeutic avenue for mitigating ischemia-induced neuronal damage.

Multiple tail ionizable lipids improve in vivo mRNA delivery efficiency with biosafety

Ionizable lipid-based lipid nanoparticles (LNP) play a crucial role in the delivery of mRNA. The hydrophobic tail of ionizable lipid affects the formation of LNP and the release of mRNA. In this report, we focus on the effect of the number, chain length, and double bond number of the hydrophobic tail on the delivery efficiency. First, a series of ionizable lipids with two, three and four tails were synthesized and characterized featured with imidazole group as the head. The ionizable lipids derived LNP were prepared using a microfluidic co-mixing device, yielding particles primarily in the size range of 100 to 150 nm, with a polydispersity index (PDI) below 0.2. Screening identified ionizable lipids with four tails exhibiting superior delivery efficiency, of which U-15, U-17, U-18 and U-19 demonstrated the highest performance. Additionally, the U-19 significantly prolongs mRNA expression duration, and along with specific extrahepatic delivery effect compared to ALC-0315. Tissue slice tests on representatives (U-06: two tails, U-19: four tails, U-29: three tails) revealed no notable abnormalities. Analysis of immunogenicity, liver and kidney function tests indicated that all samples exhibited no evident immunogenicity or in vivo toxicity. Findings from tests on lysosome escape, cell transfection, and cytotoxicity revealed excellent in vitro delivery effectiveness. In summary, among the 35 imidazole-based ionizable lipids screened, optimal effects were exhibited by four tails, which providing a new strategy for the development of ionizable lipids.

Quantification of elemental composition of the thin tissue slices with the commercial benchtop micro-XRF spectrometer

In this paper we present the scheme for quantification of local elemental composition by micro-XRF in thin tissue slices. The proposed procedure involves simultaneous determination of the geometry factor and the primary spectrum distribution from the measurement of set of the thin film calibrations standards. The procedure has been applied for the commercial M4 Tornado Plus spectrometer by Bruker. The quantification of the thin tissue slice has been performed as well.

First Report of Botryosphaeria dothidea Causing Leaf Blight on Parrotia subaequalis in China.

Parrotia subaequalis is of great ornamental value due to its unique bark, featuring interesting textures and colors, and its large, striking galls. These characteristics make it a popular choice for bonsai cultivation. (Yan et al. 2022) . In July 2023, an outbreak of leaf blight was observed on 40, six-month-old P. subaequalis seedlings in Anqing, Anhui, China, with an incidence rate of 80%. In the early stages of infection, small brown spots appear on the leaf surface, which gradually become round or irregular and darken to a deep brown color. As the disease progresses, the affected areas expand from the leaf margins towards the center, causing the leaf surface to become concave, wilt, and necrotize. This resulted in restricted plant growth, and in severe cases, partial or complete plant death. For isolation, 30 tissue slices (5 × 5 mm) were taken from the leaves of 10 symptomatic seedlings and surface sterilized with 75% ethanol for 5 seconds, followed by five rinses with sterilized distilled water. After two days of dark incubation at 28°C, hyphal tips of fungi were transferred onto new potato dextrose agar (PDA) plates and incubated until conidia production. Six unidentified isolates with similar morphological characteristics were obtained. The colonies, initially white, darken to black after 7 to 10 days of incubation. They produced colorless, aseptate conidia that were oblong or fusiform, measuring 18-26 μm in length and 5-8 μm in width (n=50). The morphological characteristics of the isolates resembled those of Botryosphaeria (Udayanga et al. 2015) . Isolate IS2116-1 was further confirmed through molecular methods. The rDNA internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF1-α), and beta-tubulin (TUB2) genes were amplified and sequenced using the primers ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R, and Bt2a/Bt2b (Ferreira et al., 2021; Carbone et al., 1999), respectively. BLAST analysis revealed that the ITS (OR958722) sequence was 100% similar to the B. dothidea isolate HZ5(MH329650.1), TEF1-a (PP214058) sequence was 100% similar to the B. dothidea strain JZB310220(ON890458.1), and strain TUB2 (PP214057) sequence was 99.78% similar to the B. dothidea strain L14 (KR260833.1). Maximum likelihood analyses were performed for the combined ITS、TUB2、TEF datasets using PhyloSuite v1.2.2, the resulting phylogenetic tree indicated that isolate IS2116-1 clustered together with Botryosphaeria dothidea in a clade with 97% bootstrap support(Zheng et al. 2020) . Pathogenicity tests were conducted on 3-6 month-old P. subaequalis seedlings (n = 5) grown in a greenhouse. A conidial suspension (106 spores/ml) collected from the isolates was sprayed onto P. subaequalis seedlings, while the control was treated with distilled water. All plants were maintained in a growth chamber at 28°C with a 12-h photoperiod. The experiment was conducted twice independently . After 20 days of inoculation, brownish lesions similar to those observed in the field appeared on the treated plants, while the noninoculated control plants remained symptomless. The pathogen was reisolated from the leaves of the obviously diseased seedlings and confirmed as B. dothidea through morphological and sequence analysis. No isolates were obtained from uninoculated control plants, thus fulfilling Koch's hypothesis. This report marks the first record of B. dothidea causing leaf blight in P. subaequalis. In light of the rarity of natural P. subaequalis populations, it is imperative to assess both the extent of disease spread and its economic impact. These insights are crucial for devising strategies to protect this endangered species from disease threats and to preserve its ecological significance.

12/15-Lipooxygenase Inhibition Reduces Microvessel Constriction and Microthrombi After Subarachnoid Hemorrhage in Mice.

Impaired cerebral circulation, induced by blood vessel constrictions and microthrombi, leads to delayed cerebral ischemia after subarachnoid hemorrhage (SAH). 12/15-Lipooxygenase (12/15-LOX) overexpression has been implicated in worsening early brain injury outcomes following SAH. However, it is unknown if 12/15-LOX is important in delayed pathophysiological events after SAH. Since 12/15-LOX produces metabolites that induce inflammation and vasoconstriction, we hypothesized that 12/15-LOX leads to microvessel constriction and microthrombi formation after SAH, and thus, 12/15-LOX is an important target to prevent delayed cerebral ischemia. SAH was induced in C57BL/6 and 12/15-LOX-/- mice of both sexes by endovascular perforation. Expression of 12/15-LOX was assessed in brain tissue slices and in vitro. C57BL/6 mice were administered either ML351 (12/15-LOX inhibitor) or vehicle. Mice were evaluated for daily neuroscore and euthanized on day 5 to assess cerebral 12/15-LOX expression, vessel constrictions, platelet activation, microthrombi, neurodegeneration, infarction, cortical perfusion, and development of delayed deficits. Finally, the effect of 12/15-LOX inhibition on platelet activation was assessed in SAH patient samples using a platelet spreading assay. In SAH mice, 12/15-LOX was upregulated in brain vascular cells, and there was an increase in12-S-HETE.Inhibition of 12/15-LOX improved brain perfusion on days 4-5 and attenuated delayed pathophysiological events, including microvessel constrictions, microthrombi, neuronal degeneration, and infarction. Additionally, 12/15-LOX inhibition reduced platelet activation in human and mouse blood samples. Cerebrovascular 12/15-LOX overexpression plays a major role in brain dysfunction after SAH by triggering microvessel constrictions and microthrombi formation, which reduces brain perfusion. Inhibiting 12/15-LOX may be a therapeutic target to improve outcomes after SAH.

Use of Melinex film for flat embedding tissue sections in LR White.

Tissue slices can undergo distortions during processing into resin for light and electron microscopy as a result of differential shrinkage of the various tissue components, and this may necessitate removal of a considerable amount of material from the final resin-embedded tissue block to ensure production of complete sections of the sample. To mitigate this problem, a number of techniques have been devised that ensure the sample is held flat during the final curing/polymerisation of the resin. For embedding in acrylic resins, oxygen must be excluded as it inhibits polymerisation, and methods devised for epoxy resin embedding are generally unsuitable. The method describes the preparation and use of air-tight flat-embedding chambers prepared from Melinex film and provides an inexpensive, technically simpler, and versatile alternative to chambers formed from either Thermanox coverslips or Aclar films that have previously been advocated for such purposes. Lay description: Tissue slices can undergo distortions during processing into resin for light and electron microscopy as a result of differential shrinkage of the various tissue components. Such distortions may necessitate removal of a considerable amount of material to ensure production of complete sections of the sample. For embedding in acrylic resins, oxygen must be excluded as it inhibits polymerisation, and methods devised for epoxy resin flat-embedding are generally unsuitable. Air-tight flat-embedding chambers prepared from either Thermanox coverslips, or a combination of PTFE-coated glass slides, polycarbonate film gaskets, and Aclar film have been advocated for such purposes. Thermanox coverslips are expensive and limited in size to 22mm × 60mm, and the alternative method is technically complicated. Melinex film is commercially available as 210mm × 297mm sheets and is approximately 1/20th the price of Thermanox and less than half the price of Aclar film. The method describes the preparation and use of embedding chambers made from Melinex film, glass slides and double-sided adhesive tape as a technically simpler, inexpensive and versatile alternative to both Thermanox coverslips and the Aclar film method.