Decellularized matrix-hyaluronic acid-alginate hybrid hydrogels to enable a multi-layered full-thickness oral mucosa-on-a-chip.

Abstract High-grade gliomas (HGGs), including glioblastoma and anaplastic astrocytoma, represent some of the most aggressive and treatment-resistant forms of brain tumours in adults. Despite advances, prognosis remains poor largely due to the infiltrative nature of these tumours and the limitations imposed by the BBB on drug delivery. OATPs, particularly SLCO1A2, have emerged as promising targets due to their ability to transport a wide variety of compounds and their upregulation in hypoxic tumour microenvironments. we evaluated the expression of SLCO1A2 in high-grade gliomas using immunohistochemistry (IHC) and immunocytochemistry (ICC). IHC was performed on, paraffin-embedded tumour tissues obtained from over 150 patients diagnosed with HGGs. A panel of antibodies targeting key cellular markers was used to delineate cell types, including CD44 (tumour cells), PDGFRB (stromal cells), IBA1 (myeloid cells), and UEA1 (endothelial cells). These were co-labelled with anti-SLCO1A2 to assess its single-cell expression across these cellular populations. For in vitro validation, ICC was employed on both commercially available and primary human glioma cell lines to determine the expression levels of SLCO1A2 and its potential function. Our IHC results demonstrated strong SLCO1A2 immunoreactivity predominantly in tumour cells marked by CD44, with additional but lower levels of expression observed in the stromal (PDGFRB+) and endothelial (UEA1+) compartments. Notably, the transporter showed minimal colocalization with IBA1+ myeloid cells, suggesting cell-type specificity in its expression. In vitro, ICC analysis revealed high levels of SLCO1A2 expression in all investigated cell lines, with clear localisation to the plasma membrane, indicating its functional readiness to mediate transport of substrates across the cell membrane. We also demonstrated that a reported drug carrier molecule, Heptamethine cyanine dyes (HMCDs), utilised SLCO1A2 to deliver anticancer agents into tumour cells. These findings suggest SLCO1A2 may aid in drug uptake, highlighting its potential as a therapeutic target to improve drug delivery and treatment specificity in malignant gliomas.

Background: We previously demonstrated that GPR39 is present in vascular smooth muscle cells (VSMCs) and pericytes in cardiac tissue. We also showed that genetic deletion of GPR39 reduces infarct size in a rodent acute myocardial infarction model. Here we investigated whether selective pharmacological blockade of GPR39 by VC108 reduces myocardial infarct size by increasing blood flow. Methods: Rats were administered 0.5 mg/kg of VC108 intravenously, a dose that occupies >90% of receptors in the body. Tissue pO 2 was measured, followed by 1 h of coronary occlusion and 1 h of reperfusion. At 30 min each into occlusion and reperfusion tissue pO 2 was again measured. After euthanasia, infarct volume from multiple heart slices was measured using triphenyl tetrazolium chloride. From separate rats, whole heart tissue, cardiomyocytes, pericytes, and VSMCs were isolated for qPCR. Isolated cardiomyocytes also underwent immunocytochemistry (ICC) for presence of GPR39 and cardiac tissue was subjected to immunohistochemistry (IHC) for the same. Results: Figure 1 illustrates markedly smaller infarct volume in VC108 (A) versus vehicle treated (B) animals. Aggregate data from 27 animals demonstrate the same (C). However, tissue pO 2 levels were not associated with infarct volume reduction implying a direct tissue effect of GPR39 inhibition not attributable to vasodilation. ICC revealed presence of GPR39 in a cardiomyocyte (Figure 2A). IHC showed co-localization of GPR39 in pericytes, VSMCs, and cardiomyocytes (Figure 2C). qPCR revealed GPR39 presence in cardiomyocytes equivalent to that of VSMCs, which has not been described before. Conclusions: The marked reduction in infarct volume noted in VC108 treated animals is not associated with increases in tissue pO 2 during coronary occlusion, indicating a flow-independent mechanism of cardioprotection. Here we show that cardiomyocytes possess GPR39, which is a new finding. Inhibition of downstream signaling from GPR39 may reduce infarction by preventing ferroptosis, apoptosis and/or necrosis.

Introduction: The LMNA gene encodes the nuclear envelope proteins Lamins A and C, which are essential for nuclear structure and function. Mutations in LMNA are commonly associated with dilated cardiomyopathy (DCM), the second leading cause of heart failure in the U.S. and one of the most lethal inherited cardiomyopathies, with a five-year mortality rate of 55.9%. To investigate the morphological and functional consequences of a patient-specific intronic LMNA mutation (c.937-1G>A), we employed an iPSC-derived 3D cardiac organoid model. Hypothesis: The (c.937-1G>A) intronic LMNA mutation reduces the differentiation efficiency into cardiomyocytes and leads to impaired cardiomyocyte function in 3D-cardiac organoids. Methods: Peripheral blood mononuclear cells of a patient with dilated cardiomyopathy carrying a uncharacterized novel LMNA (c.937-1G>A) mutation were reprogrammed into iPSCs. We then utilized CRISPR-Cas9 to generate an isogenic corrected control. Both mutant and corrected iPSCs were differentiated into 3D-cardiac organoids that recapitulate key features of human heart tissue. Morphology, protein expression and calcium handling differences were evaluated using immunocytochemistry (ICC) and calcium imaging in an IonOptix system. Results: Western blot analysis showed reduced Lamin A protein expression in mutant organoids, with restored expression in CRISPR-corrected controls. Immunocytochemistry revealed marked reduction in cTnT-positive (cardiomyocytes) and disrupted nuclear morphology in mutant organoids compared to corrected controls. Calcium imaging showed altered calcium handling in LMNA mutants, including depressed systolic Ca 2+ levels, prolonged time to peak (n = 3, p < 0.05) and slow departure velocity (d[Ca 2+ ]/dt max ), which were improved with the correction of the mutation. Conclusions: These findings suggest that the early effects of the novel intronic LMNA mutation (c.937-1G>A) that occur during development can be modeled in 3D-cardiac organoids. The mutation impairs cardiomyocyte differentiation and disrupts calcium dynamics, underscoring its pathogenic potential during cardiac development.

Melanin-rich cytologic specimens, particularly those from melanocytic lesions, present diagnostic challenges due to pigment-induced obscuration of cellular details and interference with immunocytochemistry (ICC) interpretation. These limitations are especially pronounced in cell transfer preparations, which differ significantly from tissue sections in cellular distribution and density. Existing bleaching protocols are inconsistent, often incomplete in pigment removal and can compromise cellular morphology. This study aimed to develop and evaluate an automated platform incorporating optimised melanin bleaching, ICC and cytomorphologic staining to enhance diagnostic accuracy in heavily pigmented cytologic samples. Ten melanoma cell transfer smears were processed using an optimised protocol. Slides underwent melanin bleaching with 10% hydrogen peroxide at 60°C for 25 min, followed by automated ICC for Melan-A and SOX-10. Chromogenic detection was performed using either 3,3'-diaminobenzidine (DAB) or alkaline phosphatase (AP). In a parallel workflow, Papanicolaou (Pap) staining was performed after bleaching to assess cytomorphologic preservation. The integrated protocol completed bleaching and staining within 2 h, with bleaching effectively removing melanin pigment while enhancing nuclear and cytoplasmic visibility without compromising morphological detail. Post-bleaching Pap staining preserved cytologic features, enabling accurate morphological interpretation. Both markers exhibited strong, specific immunoreactivity with either chromogen; however, AP yielded superior contrast and clearer antigen localisation. In contrast, residual melanin occasionally masked DAB signals, limiting interpretability. This automated protocol, combining melanin bleaching, Pap staining and ICC, improves visualisation and diagnostic interpretation of melanin-rich cytologic specimens. The bleaching step preserves cellular and antigenic integrity, while AP chromogen provides enhanced clarity in the presence of residual pigment. This reproducible and practical workflow facilitates more accurate cytopathologic evaluation of melanocytic lesions.

Abstract Background and Aims Lupus nephritis (LN) is one of the most frequent and severe manifestations of systemic lupus erythematosus (SLE), affecting 40-65% of SLE patients and resulting in end-stage renal disease (ESRD) in 10%–20% of them. Clinical and histological prognostic factors such as race, chronicity index or tubulointerstitial damage have already been described, but little is still known about the prognostic value of immunocytochemical (ICC) data from renal biopsies. Objectives To identify demographic, clinical, histological and immunocytochemical factors which can help to predict the prognosis of LN patients. To build a predictive model based on these factors for estimating disease outcome in a cohort of LN patients. Method Retrospective study including patients diagnosed of LN in a tertiary clinic between January 2014 and May 2024, confirmed by renal biopsy. Data was recorded regarding clinical and laboratory variables as well as information from renal biopsies that included histology, indirect immunofluorescence (IIF) and immunohistochemistry (IHC). A univariate logistic regression analysis was first carried out to preselect possible predictive variables, selecting those which were considered clinically relevant or had a determination of p-value <0.30. These were included in a multivariate logistic regression analysis to build different prognostic models, a first model with clinical variables and a second one with data from renal biopsies. The best predictive model was chosen based on criteria such as goodness-of-fit tests and its discriminatory power. A local Ethics Committee approved the execution of this project. Results From an initial sample of 55 patients, 38 fulfilled criteria to be included in our study. 84.2% were female with a mean age of 39.5 ± 14.4 years at onset of LN and a mean of 4.57 ± 2.81 years of follow-up during the study. 94.7% of patients had extrarenal SLE manifestations, being articular involvement the most frequent (79.0%), while the most common types of renal disease were proliferative forms (81.6%) with mean baseline values of proteinuria of 2.6 ± 3.3 g/24 h. Complete renal response was achieved by 47.4% of patients. The multivariate analysis allowed us to select the best predictive models for poor outcome in our cohort. The clinical model included as statistically significant variables age at onset, baseline eGFR and quantification of hematuria, while the model based on information from renal biopsies related the presence of interstitial inflammation and CD68+ macrophages with worse outcomes (Table 1). Both of these models presented adequate discriminatory power with values of AUC >0.8 as well as correct classification rates over 80%. Conclusion This study has aimed to develop a prognostic index based on clinical variables and data obtained from renal biopsies of LN patients, which could be useful in clinical practice to help predict which patients will have a worse disease outcome and to adjust therapeutic regimens and management accordingly. Other than clinical and histological variables, the inclusion of ICC characteristics from renal biopsies, such as CD68+ macrophage infiltration, could play a role in predicting renal prognosis in LN.

Thyroid nodules are frequently encountered, mostly detected during ultrasound evaluation of the head and neck, frequently performed for other reasons. Albeit most nodules arise in the setting of multinodular goiter, hypoechoic subcentimeter nodules can be recognized that show suspicious features, requiring them to undergo fine needle aspiration cytology (FNAC) evaluation. ATA guidelines suggest performing FNAC only on nodules greater than 1.5cm. Nevertheless, the issue emerges if these subcentimeter lesions are aspirated and diagnosed as follicular neoplasms (FN). Herein, we describe an algorithm used to approach such subcentimeter thyroid nodules in our tertiary medical center. All subcentimeter thyroid nodules sampled by FNAC were retrieved, performed between 2014 and 2023 with a diagnosis of indeterminate lesion of high-risk malignancy (Italian Classification system) that corresponds to follicular neoplasm (FN) in the Bethesda Reporting System. These cases were processed only with liquid-based cytology (LBC) with immunocytochemistry (ICC) and molecular testing performed when necessary. The series included 174 indeterminate subcentimeter nodules analyzed with FNAC, based on suspicious ultrasound criteria. The cytological diagnosis included 101 cases with atypia of undetermined significance (AUS) and 74 FN. All FN cases underwent surgery, and the subsequent histological diagnoses revealed 24 (32%) benign and 49 malignant lesions including 38 papillary thyroid carcinoma (PTC) and its variants as well as 11 cases of invasive follicular variant of PTC (I-FVPTC). Furthermore, 30.6% (15/49) of these malignant lesions had lymph node involvement, and 34.6% were multifocal. Among the histological malignant cases, only three (6.1%) cases had a moderate positivity for VE1-BRAF, with eight (16.3%) cases showing a concordant positive HMBE1 and Galectin-3 panel. Although some guidelines do not recommend sampling subcentimeter thyroid nodules, in clinical practice, these may undergo FNAC to help elucidate concerning ultrasound findings. In our series, in the presence of suspicious ultrasound criteria, 66% of the nodules turned out to be malignant. Although ICC is unable to help make a definitive diagnosis, it serves as a useful pathology ancillary tool in the algorithmic work-up of subcentimeter thyroid lesions.

A time course analysis of the electrophysiological and gene expression properties during differentiation of hair cell-like cells in culture.

Autophagy, a conserved intracellular degradation process, plays dual roles in cancer, promoting survival under stress or mediating cell death through deregulated autophagy. Atypical cadherin FAT1 functions as an oncogene or tumor suppressor in a context-dependent manner. Our previous work identifies the oncogenic role of FAT1 in glioblastoma. Deregulated autophagy has been documented in glioma. Here, we investigated the role of FAT1 in regulating autophagy and its implications for glioblastoma growth and progression. CRISPR-Cas9 mediated FAT1 knockout was generated in glioblastoma (U87MG and LN229) and other cancers such as hepatocellular carcinoma (HepG2 and HUH7) and pancreatic adenocarcinoma (MIAPaca-2 and Panc-1) cells. The cell viability and growth under hypoxia ± serum deprivation were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, and Annexin V-FITC assays. Autophagy markers were assessed by quantitative polymerase chain reaction (qPCR), Western blot, immunocytochemistry (ICC), and immunohistochemistry (IHC). Autophagosomes were visualized by transmission electron microscopy (TEM), and puncta formation was analyzed by transfecting the cells with pEGFP-LC3. Autophagy flux was evaluated by analyzing p62/SQSTM1 levels, and the GFP/RFP ratio using pMRX-IP-GFP-LC3-RFP-LC3ΔG. In vivo, FAT1-knockout U87MG xenografts in nude mice were analyzed for tumor growth and autophagy marker expression. Surgically resected glioblastoma tumors from our hospital and The Cancer Genome Atlas (TCGA) dataset were analyzed for autophagy marker expression and patient survival correlations. FAT1-knockout glioblastoma (U87MG and LN229) cells demonstrated reduced survival and colony numbers under normoxia and hypoxia with serum deprivation, facilitated by autophagy-dependent cell death. These cells exhibited upregulated autophagy markers, increased LC3 puncta, autophagosomes, and autophagy flux. FAT1-knockout glioblastoma cells showed decreased total and phospho-mTOR levels. FAT1-knockout xenografts showed reduced tumor progression with increased LC3II, Beclin1, and autophagosomes. Human glioblastoma tumors and TCGA glioblastoma data revealed an inverse expression correlation of FAT1 with LC3B/Beclin1, tumors with high-FAT1/low-LC3B expression were associated with poor patient survival. FAT1 also regulated autophagy in hepatocellular and pancreatic cancers. Our findings indicate that FAT1 mediates pro-tumorigenic function by suppressing autophagic cell death in glioblastoma and other cancers. FAT1 may serve as a potential therapeutic adjuvant along with standard therapeutic regimens for treating cancers with high FAT1 expression having an oncogenic role.

Introduction: Mesenchymal stem cells (MSCs), especially those derived from the umbilical cord (UC-MSCs), have emerged as promising candidates for cancer therapy due to their immunomodulatory and anti-tumour properties. However, the mechanisms through which UC-MSCs affect cervical cancer cells remain unclear. This study aimed to investigate the effects of UC-MSCs on HeLa cervical cancer cells, focusing on cytoskeletal alterations and apoptosis-related pathways. Methods: HeLa cells were co-cultured with UC-MSCs at varying concentrations (5%, 10%, 20%, and 30%). Doxorubicin (3 ppm) was used as a positive control, and untreated HeLa cells served as the negative control. Apoptosis markers (caspase-3 and caspase-8) were analysed using immunocytochemistry (ICC), while DAPI/phalloidin staining was used to observe cytoskeletal changes. Levels of cytokines (CCL2 and TGF-α) were measured using ELISA. Results: Activation of caspase-3 was observed at 5% UC-MSC concentration, indicating the induction of apoptosis. However, higher UC-MSC concentrations (10–30%) led to a decrease in HeLa cell viability without caspase-3 or caspase-8 activation, suggesting alternative cell death pathways such as necrosis or autophagy. No caspase-8 activation was observed across all treatments, indicating resistance to extrinsic apoptosis, possibly due to the expression of anti-apoptotic proteins like c-FLIP. ELISA analysis showed no significant changes in CCL2 and TGF-α levels. Conclusion: UC-MSCs can induce HeLa cell death via caspase-independent mechanisms, particularly at higher concentrations. The selective activation of caspase-3 at low concentrations highlights the complex interaction between UC-MSCs and cervical cancer cells. These findings contribute to a deeper understanding of MSC-based cancer therapy and offer insights for the development of novel therapeutic strategies.

PURPOSE:This study aimed to develop a high-resolution full-field optical coherence tomography (HR-FFOCT) system for automated three-dimensional (3D) imaging of cultivated epithelial cell sheets and biopsied tissue used in corneal regenerative therapy.MATERIALS AND METHODS:A commercial HR-FFOCT system (ApolloVue® S100, AMO, Taiwan), originally for dermatological imaging, was re-engineered for <2 μm resolution imaging of rabbit limbal and oral mucosal tissues for limbal stem cell deficiency treatment. Modifications included a piezoelectric transducer for precise Z-stack acquisition, customized LED illumination for registration, and a specialized platform for culture dishes. The system enabled en face and cross-sectional imaging with 3D reconstruction. Rabbit-derived products for cultivated limbal epithelial transplantation (CLET) and cultivated oral mucosal epithelial transplantation (COMET) were imaged before cultivation. Morphology, stratification, and protein expression were analyzed and validated with immunocytochemistry (ICC).RESULTS:The optimized system produced high-resolution en face and Z-stack images with accurate alignment, capturing stratified epithelial layers in CLET and oral mucosal tissue. Reconstructed 3D images revealed structural detail consistent with ICC-verified expression of junctional proteins, including occludin and actin. Both two-dimensional and 3D visualization of biopsied oral mucosal tissue was achieved. The system enabled noninvasive monitoring of epithelial sheet architecture and thickness without sectioning.CONCLUSION:The reconstructed HR-FFOCT system provides a noninvasive, real-time imaging platform for assessing epithelial cell sheets and biopsied tissue in corneal regenerative therapy. It offers potential for standardizing quality evaluation of cell-based products before transplantation and advancing translational applications in regenerative medicine.

INTRODUCTIONPatients with LMD have poor prognosis and limited treatment options. Oncogene amplification of primary, metastatic, and CNS metastatic tumors can be heterogeneous. Therefore, patients with LMD may benefit from assessment of clinically relevant biomarkers in CSF, which may guide the choice of a targeted therapy specifically for the LMD tumor. CNSide is a CLIA-validated laboratory-developed test ordered commercially at the discretion of physicians for CSF-TC enumeration, immunocytochemical (ICC) and fluorescence in situ hybridization (FISH) analysis of oncogene amplification. We longitudinally analyze oncogenes in CSF-TCs in patients with LMD of various primary cancers. METHODSCSF was collected from patients with suspected or confirmed LMD; 613 tests were ordered on 218 individual patients with breast (N=105 patients), lung (N=65), gastrointestinal (N=10), and other cancers. Using CNSide, CSF-TCs were isolated and tested via ICC (ER, PD-L1, and PR), and FISH (ALK, cMET, cMyc, EGFR, FGFR1, HER2, NTRK1, NTRK3, PTEN, RET, and ROS1). RESULTSIn patients with lung cancer, ALK was detected in 14% (17/118) of samples, CMET in 61% (78/128), HER2 in 73% (16/22), and RET in 4% (4/90). In patients with breast cancer, HER2 was detected in 39% (65/168) of samples, FGFR1 in 32% (19/60), ER in 26% (44/168) and PR in 4% (5/120). 66 patients underwent 2+ CSF draws; 24 underwent 5+. Among these, there were 13 ICC flips (7 acquired mutations) and 58 FISH probe detection flips (26 acquired mutations). 20/66 patients (30.3%) had at least one flip in their ordered biomarkers. CONCLUSIONCNSide can be used to detect oncogene amplification on CSF-TCs of patients with LMD, and mutational status of the LMD tumor may differ from the original tumor biopsy. CSF-TC analysis may provide therapeutic insights that vary from the original tumor and could open the door to additional treatment targets for patients struck with LMD.

Heparan sulfate proteoglycans (HSPGs) within the neuronal niche are expressed during brain development, contributing to multiple aspects of neurogenesis, yet their roles in glial lineage commitment remain elusive. This study utilised three human cell models expanded under basal culture conditions followed by media-induced lineage induction to identify a reproducible and robust model of gliogenesis. SH-SY5Y human neuroblastoma cells (neuronal control), ReNcell CX human neural progenitor cells (astrocyte inductive) and ReNcell VM human neural progenitor (mixed neural induction) models were examined. The cultures were characterised during basal and inductive states via Q-PCR, Western Blotting, immunocytochemistry (ICC) and calcium signalling activity analyses. While the ReNcell lines did not produce fully mature or homogeneous astrocyte cultures, the ReNcell CX cultures most closely resembled an astrocytic phenotype with ReNcell VM cells treated with platelet-derived growth factor (PDGF) biased toward an oligodendrocyte lineage. The glycated variant of surface-bound glypican-2 (GPC2) was found to be associated with lineage commitment, with GPC6 and 6-O HS sulfation upregulated in astrocyte lineage cultures. Syndecan-3 (SDC3) emerged as a lineage-sensitive proteoglycan, with its cytoplasmic domain enriched in progenitor-like states and lost upon differentiation, supporting a role in maintaining neural plasticity. Conversely, the persistence of transmembrane-bound SDC3 in astrocyte cultures suggest continued involvement in extracellular signalling and proteoglycan secretion, demonstrated by increased membrane-bound HS aggregates. This data supports HSPGs and HS GAGs as human neural lineage differentiation and specification markers that may enable better isolation of human neural lineage-specific cell populations and improve our understanding of human neurogenesis.

Background: Neural progenitor cells (NPCs) in specific regions of the adult brain hold promise for treating central nervous system (CNS) disorders and degenerative diseases, such as Parkinson's and Alzheimer's. Rosa damascena, enriched with flavonoids, offers restorative, inhibitory, and protective advantages against nerve damage. Objectives: This study investigates the neuroprotective effects of R. damascena essential oil (ESO) on NPCs exposed to ethanol (ETH)-induced toxicity. Methods: The study involved two phases: The NPCs were isolated from the brains of young Wistar rats and cultured following standard protocols. Differentiation into mature neural cells (NCs) was induced by withdrawing supportive factors. MTT assays were used to determine effective ETH and ESO doses, followed by assessments of their impact on NPCs and NCs. Gene expression analysis of neuroepithelial stem cell protein (Nestin), Sex determining region Y-box 2 (Sox2), and Paired Box 6 (Pax6) was conducted using real-time PCR during the NPC phase, while class III β-tubulin (Tuj1) and microtubule associated protein 2 (Map2) were evaluated during the NC stage. TAU protein expression was analyzed via immunocytochemistry (ICC). Results: The ETH exposure significantly reduced cell viability in a dose- and time-dependent manner (24, 48, 72 hours), while treatment with 200 µM ESO significantly improved survival in NPCs and NCs (P &lt; 0.001). Additionally, ESO reduced TAU protein expression in NCs exposed to ETH. The NPCs treated with ETH + ESO showed increased expression of Nestin, Sox2, and Pax6 compared to the ETH-only group (P &lt; 0.01). Similarly, Tuj1 and Map2 expression levels were significantly higher in the ETH + ESO group compared to ETH-treated NCs (P &lt; 0.01). Conclusions: The ESO demonstrates significant neuroprotective properties, mitigating ETH-induced toxicity in NPCs and NCs. These findings highlight its potential as a therapeutic agent for reducing neurodegenerative damage and supporting nervous system function.

Abstract Background: Metastatic involvement of the brain is a common and serious event in the progression of various cancers. Determining the primary source of brain metastases (BM) presents a significant diagnostic challenge, especially when histopathological features are inconclusive. Immunocytochemistry (ICC) has become a crucial diagnostic tool in this context, enabling pathologists to distinguish tumor types and identify the primary neoplasm with greater accuracy. This study aims to assess the effectiveness of immunocytochemical markers in diagnosing and classifying metastatic brain tumors. Materials and Methods: This prospective cross-sectional study was conducted in the Cytopathology Section of the Department of Pathology at S.C.B. Medical College and Hospital, Cuttack, Odisha, India, from October 2023 to October 2024. All cytopathologically suspicious cases of metastatic carcinoma involving the brain were examined. Relevant clinical data, including patient age and sex, along with radiological findings, were collected to assist in the final diagnosis. A panel of immunocytochemical markers, including cytokeratins, CD markers, and hormone receptors, was used to help determine the tumor’s primary origin. Results: A total of 11 cases were examined. The most common metastatic deposits found were adenocarcinomas (5 cases, 45.45%), followed by papillary thyroid carcinoma deposits (3 cases, 27.27%), squamous cell carcinoma (2 cases, 18.18%), and metastatic breast carcinoma (1 case, 9.09%). The average age of patients was 54.3 years, with an age range from 35 to 69 years. Males were more frequently affected, with a male-to-female ratio of 1.75:1. Conclusion: Immunocytochemical markers, such as CK7, CK20, TTF-1, and hormone receptors, are essential for differentiating tumor subtypes, including lung adenocarcinoma, breast carcinoma, and gastrointestinal cancers. ICC is a vital and effective tool in the diagnostic process for metastatic brain tumors. When combined with clinical history and pathological data, ICC provides valuable insights that inform both prognosis and treatment decisions.

The increasing number of contact lens users correlates with a rise in the incidence of fungal keratitis. Fungal keratitis can lead to blindness if not treated promptly, and its early symptoms are similar to those of bacterial and amoebic keratitis, making rapid diagnosis challenging. This study aimed to assess the potential of using a peptide antibody against the fungal-specific protein ERG24, which encodes sterol C-14 reductase, to differentiate fungal keratitis from other forms of keratitis. The specificity of the ERG24 antibody was assessed through Western blot and enzyme-linked immunosorbent assay (ELISA). Immunocytochemistry (ICC) was performed by co-culturing two types of fungi, Acanthamoeba, and two bacterial strains with human corneal epithelial (HCE) cells. Additionally, to evaluate the diagnostic potential of the ERG24 antibody, animal models of fungal, amoebic, and bacterial keratitis were developed, and ELISA was conducted on tear and ocular lysates from these models. The results demonstrated that the antibody specifically reacted with Fusarium solani in Western blot, and both ELISA and ICC confirmed that the ERG24 antibody did not react with HCE cells, Acanthamoeba, or bacteria, but was specific to the two fungal species. In vivo experiments further showed that the ERG24 antibody significantly detected F. solani in tear-wash samples and eyeball lysates from the fungal keratitis model, without reacting with samples from amoebic and bacterial keratitis models. This study suggests that the ERG24 peptide antibody could provide valuable information for developing differential diagnostic methods for fungal keratitis compared to other forms of keratitis.

ABSTRACTDespite the apparent progress in reproductive technologies in wild ruminant species, healthy live births have been limited. Acquiring a sound knowledge of the molecular basis of most functional aspects of spermatozoa will improve the effectiveness of reproductive techniques and optimise conservation programs for threatened species. CatSper channels, opioid receptors and CD44 are involved in sperm capacitation of humans and domestic animals, but their presence in wild ruminants is yet undisclosed. The aim of this study was to determine the presence and localisation of CatSper 1–4, μ, δ and κ‐opioid receptors and CD44 in three wild ruminant species spermatozoa (aoudad [n = 5], Iberian ibex [n = 5], mouflon [n = 5]), which show different resistance to freezing‐thawing processes. Western blotting (WB) and immunocytochemistry (ICC) performed with commercially available antibodies revealed that aoudad, Iberian ibex and mouflon are equipped with the aforementioned channels and receptors, sharing localisation with other domestic animals’ spermatozoa but presenting species‐particularities. WB revealed homogeneous results in CatSper 1, Catsper 2, Catsper 3 and CatSper 4 among the spermatozoa of the three species, unlike μ, δ and κ‐opioid and CD44 receptors that showed substantial inter‐species differences in the number of bands. ICC showed inter‐species differences in the location of CatSper 1–4, μ, δ and κ‐opioid and CD44 receptors. Data confirmed their presence and putative role on sperm function in wild ruminant species. Inter‐species differences in the location of CatSper 1–4, μ, δ and κ‐opioid and CD44 receptors might underlie the variable response to reproductive technologies in these species.

Loss of nuclear BRCA1-associated protein 1 (nBAP1) expression is strongly linked to monosomy 3 in uveal melanoma. While fine-needle aspiration (FNA) aids diagnosis, the prognostic value of nBAP1 immunocytochemistry (ICC) is still being investigated. This study examines the correlation between nBAP1 loss on ICC and fluorescence insitu hybridization (FISH) findings, as well as its clinical impact. Intraocular FNA cytology specimens with clinical concern for uveal melanoma from April 2015 to March 2023 with available nBAP1 ICC, FISH results, and clinical follow-up were examined. Two independent reviewers, blinded to the cytogenetic results, interpreted the nBAP1 ICC as either loss or retained. Statistical analysis using Fisher's exact test was utilized to evaluate the relationship between nBAP1 loss on ICC and FISH findings. Kaplan-Meier survival plots were constructed to examine the metastasis-free survival. Among the 79 cases included in the study, 86.1% (68/79) showed nBAP1 loss. Approximately 63.2% of patients with nBAP1 loss had monosomy 3, whereas none with nBAP1 retained had monosomy 3 (p < 0.001). Of the nBAP1 loss cases, 41.2% (28/68) had monosomy 3 alone, while 22.1% (15/68) had both monosomy 3 and 6p gain. Patients with nBAP1 loss had a significantly higher risk of metastasis (p ≤ 0.04), with a 5-year metastasis-free survival of 57.1% versus 100% in nBAP1 retained cases. BAP1 ICC is a cost-effective prognostic tool in uveal melanoma FNAs, strongly correlating with monosomy 3 and poor outcomes. Integrating BAP1 ICC with FISH enhances risk stratification, enabling earlier identification of high-risk patients and guiding treatment decisions.

Abstract Inflammatory breast cancer (IBC) is a highly aggressive and lethal type of breast cancer. Furthermore, patients with the triple-negative breast cancer subtype have tumors that lack the estrogen receptor, progesterone receptor, and HER2 expression. These tumors display greater invasiveness, and consequently, patients exhibit increased metastasis. Due to the lack of targeted therapies, IBC patients typically undergo multimodality therapy with cytotoxic chemotherapy like taxanes and anthracyclines, radiation, and surgery. There is an urgent need for selective therapies for IBC and natural products have gained significant attention for their potential in breast cancer therapy. In our laboratory, we work with Ergosterol Peroxide (EP), a bioactive compound extracted from the Ganoderma lucidum mushroom. Our studies indicate that EP is a unique endoperoxide that upon entrance into the cancerous cell, deploys its warhead via selective interactions with intracellular proteins, without any effect in non-cancerous cells. The Valosin-containing protein (VCP) / Ankyrin repeat and zinc finger domain-containing protein 1 (ANKZF1) complex plays a vital role in maintaining cellular proteostasis and ensuring proper protein synthesis and degradation processes. ANKZF1 interacts with VCP and translocates to the mitochondria under cellular stress conditions. We believe that EP affects this complex to induce cancer cell death. We hypothesize that EP targets the VCP/ANKZF1 complex, impairing the ability of IBC cells to clear damaged proteins and causing mitochondria dysfunction to induce IBC cell death. To validate the interruption of VCP/ANKZF1 interactions, we used Proximity Ligation Assays (PLA) with antibodies for VCP and ANKZF1 in the IBC cell model, SUM149. Veh, EP, and NMS 873 (VCP inhibitor, positive control) were administered, to detect changes in PLA signals for VCP and ANKZF1. To understand the VCP/ANKZF1 spatial relationship, under Veh, EP, and H2O2 (positive control) effects in SUM149, we used a colocalization analysis by Immunocytochemistry (ICC). Results were visualized with Gen 5 Image Prime 3.15 program in Cytation C10 Confocal Imaging Reader. The PLA preliminary results indicate that EP affects protein-protein interactions, since the intensity of green fluorescence, which indicates protein activity, is lower in the treated cells. Especially when compared to the positive control - NMS 873 the protein inhibitor. The preliminary results of ICC demonstrated that when SUM149 cells are treated with EP compound, VCP displays greater fluorescence intensity, while ANKZF1 fluorescence is diminished. In addition, cells treated with EP display a nucleus with greater size when compared to the Veh. Furthermore, ANKZF1 looks to be co-localizing with the nucleus, while VCP has an around and near localization of the nuclei when compared with the Veh. In general, EP affects VCP/ANKZF1 spatial relationship and interaction in IBC cells. Further research is needed to comprehensively understand the intricate mechanisms through which EP exerts its antineoplastic effects in IBC cells. Citation Format: Luz V. Arroyo-Cruz, Adriana Y. Aponte-Ramos, Michelle M. Martínez-Montemayor. Ergosterol Peroxide affects the VCP/ANKZF1 complex spatial relationship and interaction in IBC cells [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-12-22.

Background: Fibrosis involves excessive extracellular matrix (ECM) deposition, with matrix metalloproteinase-9 (MMP-9) playing a critical role. Natural compounds targeting MMP-9 may offer therapeutic potential. Objective: This study evaluated the antifibrotic effect of Allium sativum extract on fibroblast cells through in vitro and in silico approaches targeting MMP-9. Methods: Human dermal fibroblasts were treated with A. sativum extract (25, 50, and 100 µg/mL) for 24 and 48 hours. Cell viability was assessed using MTT assay. MMP-9 expression was analyzed via quantitative real-time PCR (qRT-PCR) and immunocytochemistry (ICC). Major garlic compounds were docked to MMP-9 using AutoDock Vina. Results: The 50 µg/mL concentration maintained &gt;85% cell viability and significantly reduced MMP-9 gene and protein expression. S-allyl cysteine exhibited the strongest binding affinity (−6.8 kcal/mol) and formed hydrogen bonds with key residues (Glu402, Ala189, His405) of MMP-9. Conclusion: Allium sativum extract demonstrates antifibrotic activity by downregulating MMP-9 expression and inhibiting its function, supporting its potential as a natural MMP-9 inhibitor for fibrosis-related disorders.