Search Results (288)

Air pollutants can cause respiratory diseases, highlighting the need for effective preventive and therapeutic strategies. We investigated the protective effects of heat-killed Lacticaseibacillus paracasei ATG-E1 against particulate matter plus diesel exhaust particle (PM₁₀D)-induced airway inflammation. BALB/c mice were intranasally injected with PM₁₀D and treated with heat-killed L. paracasei ATG-E1 via oral gavage for 5 days. In the bronchoalveolar lavage fluid (BALF) and lungs, inflammatory mediators, immune cell subtypes, and histological changes were analyzed, while gut microbiota composition was analyzed in the cecum. Heat-killed L. paracasei ATG-E1 suppressed the infiltration of immune cells, including neutrophils, T cells, and B cells. Furthermore, it decreased various inflammatory mediators, such as C-X-C Motif chemokine ligand (CXCL)-1, macrophage inflammatory protein (MIP)-2, interleukin (IL)-1α, and tumor necrosis factor (TNF)-α, in the BALF and lung tissue, as well as serum symmetric dimethylarginine (SDMA) levels in the PM₁₀D-induced airway inflammation model. Heat-killed L. paracasei ATG-E1 also exhibited a protective effect against lung damage induced by PM₁₀D. Furthermore, heat-killed L. paracasei ATG-E1 treatment shifted the gut microbiota composition, increasing several bacterial genera. The data demonstrate that heat-killed L. paracasei ATG-E1 acts as a protective agent against air pollutant-induced lung injury, suggesting its potential as a candidate adjunctive strategy for prevention. Full article

Objective: Intracranial infection is a severe complication that can occur following neurosurgery, and early diagnosis is crucial for improving patient prognosis. In this study, we aimed to investigate, from an exploratory perspective, whether the immune microenvironment of intraoperative cerebrospinal fluid (CSF) is associated with postoperative intracranial infection (PII) in patients undergoing craniotomy for benign brain tumors. Methods: A total of 134 patients undergoing neurosurgery for benign brain tumors were included and categorized into an infection group (n = 18) and a non-infection group (n = 116). CSF samples were collected aseptically immediately after dural opening during surgery. The concentrations of 16 cytokines, including monocyte chemoattractant protein-1 (MCP-1); macrophage inflammatory protein-1α (MIP-1α) and MIP-1β; interleukin (IL)-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, and IL-17; interferon (IFN)-α and IFN-γ; tumor necrosis factor-α (TNF-α); and granulocyte colony-stimulating factor (G-CSF), were quantified using Cytometric Bead Array (CBA) technology. An independent samples t-test was used for normally distributed data, while the Mann–Whitney U test was applied for non-normally distributed data. Group comparisons were performed using independent-samples t-tests or Mann–Whitney U tests for continuous variables and χ² tests or Fisher’s exact tests for categorical variables. The Benjamini–Hochberg false discovery rate (FDR) correction was applied to all 16 cytokines to control for multiple testing. Receiver operating characteristic (ROC) curve analysis was performed to assess discriminatory capacity. Statistical significance was defined as p < 0.05. Results: PII developed in 18 of 134 patients (13.4%). Age (47.78 vs. 54.86, p = 0.028) and operative duration (390 vs. 244 min, p = 0.005) showed differences in unadjusted analyses. In the unadjusted comparisons, MCP-1 and IL-4 levels were found to be significantly lower in the infection group (MCP-1: 57.78 vs. 116.03 pg/mL, p = 0.003; IL-4: 24.38 vs. 28.18 pg/mL, p = 0.032). No cytokine remained significant after FDR correction. The ROC analysis showed that age and IL-4 demonstrated mild discriminatory performance, with AUC values of 0.665 (95% CI 0.526–0.803, p = 0.025) and 0.657 (95% CI 0.540–0.774, p = 0.032), while MCP-1 and operative duration demonstrated modest discriminatory performance, with AUC values of 0.716 (95% CI 0.595–0.838, p = 0.003) and 0.708 (95% CI 0.578–0.838, p = 0.002). Conclusions: In this study, single-point intraoperative CSF cytokines were not significantly associated with PII after stringent correction for multiple testing, and did not provide a validated clinical prediction tool. The unadjusted and direction-corrected findings for MCP-1 and IL-4 remain exploratory and require validation. Full article

Mitochondrial dysfunction is not merely a byproduct of transformation but a driver of tumorigenesis, metastasis, and therapeutic resistance. Recent advancements in intercellular communication have identified Extracellular Vesicles (EVs) or exosomes as critical mediators that bridge the gap between the tumor and its microenvironment (TME). These EVs contain a complex repertoire of bioactive cargo, including proteins, lipids, and RNAs. Among the class of RNAs, small non-coding RNAs, microRNAs (miRNAs), are the most abundantly expressed bioactive compounds that are selectively packaged and delivered to recipient cells. EV-delivered miRNAs can target nuclear-encoded mitochondrial genes and have also been reported to localize to mitochondria (mitomiRs), where they function as post-transcriptional regulators of bioenergetic and mitochondrial dynamic adaptations that support tumor progression. This review explores the “EV-miRNA-Mitochondria Axis”, delineating the molecular mechanisms by which EV-carried miRNAs reprogram the “Mitochondrial Information Processing System” (MIPS) - a signaling network where mitochondria integrate metabolic cues (e.g., ROS, calcium flux) to dictate critical biological outcomes, such as immune regulation and cell survival. We summarized specific sorting machineries (e.g., hnRNPA2B1, Lupus La) that package oncogenic miRNAs into EVs and how these cargoes hijack mitochondrial function upon delivery. Specifically, we discussed how EV-miRNAs induce metabolic shifts, manipulate mitochondrial dynamics (fission/fusion), and inhibit the intrinsic apoptosis to drive cancer progression. Finally, we highlighted the dual utility of these EV-miRNAs as drivers of pathogenesis and promising non-invasive biomarkers for early diagnosis, prognostic and therapeutic monitoring. Full article

Protein biomarkers can be used for monitoring the occurrence and development of diseases. Accurate, sensitive, and low-cost methods for protein detection can facilitate therapeutic intervention, improve clinical outcome, and reduce economic pressure for patients. Molecularly imprinted polymers (MIPs) have been considered as a type of biomimetic materials for developing biosensing technologies due to their advantages of high stability, low preparation cost, and good reusability over classical biometric recognition elements such as antibodies and aptamers. Electrochemical biosensors have become the most promising technology in sensing applications in view of their high sensitivity, fast response speed, cost-effectiveness, good stability, and ease of miniaturization. Efforts have been made to develop various electrochemical biosensors for protein detection with MIPs as recognition elements. This article provides an overview of the progress in molecular imprinting methods for the design and application of electrochemical protein biosensors. The strategies for imprinting and removing templates and preparing MIPs-modified sensing electrodes are comprehensively discussed. Finally, the challenges and future perspectives of protein-imprinted electrodes are addressed. This work will contribute to the development of innovative analytical devices based on MIPs for monitoring and managing various diseases by determining protein biomarkers. Full article

Background: Fracture healing requires a coordinated inflammatory response, and its dysregulation, as seen in polytrauma, can impair bone regeneration. Human mesenchymal stem cells (hMSCs) play a central role in fracture repair through osteogenic differentiation and also via their secretome, which regulates local inflammation, angiogenesis, and tissue regeneration. Interleukin-6 (IL-6), an early pro-inflammatory cytokine, contributes to fracture healing by promoting MSC recruitment and osteogenic differentiation, whereas bone morphogenetic protein-2 (BMP-2) is a key osteoinductive factor that drives bone formation. However, the combined effects of IL-6 and BMP-2 on the hMSC secretome remain poorly understood. Methods: We cultured hMSCs in osteogenic media supplemented with recombinant IL-6 (1–20 ng/mL) alone or combined with recombinant BMP-2 (1 ng/mL) on tissue culture plastic (TCP) and within gelatin methacryloyl (GelMA) hydrogels of low (~3 kPa), medium (~15 kPa), and high (~30 kPa) stiffness. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity and calcium deposition; cytokine profiling was performed using a multiplex antibody array. Results: When cultured on TCP, IL-6 suppressed ALP activity by day 21. Co-treatment with IL-6 and BMP-2 induced a dysregulated secretome with concurrent upregulation of pro-inflammatory markers (MIP-1α, TNF-α, and GM-CSF) and anti-inflammatory mediators (IL-10, TGF-β1, and VEGF). This hyperinflammatory response was attenuated when hMSCs were encapsulated in GelMA, with high-stiffness gels most effectively suppressing pro-inflammatory chemokines and medium-stiffness gels yielding the highest ALP activity. Conclusions: These findings suggest that mechanically tuned GelMA hydrogels modulate immune and osteogenic responses of hMSCs in vitro, warranting further investigation in the context of scaffold design for fracture care. Full article

Protein molecularly imprinted polymers (MIPs), as artificial antibodies, are promising for protein separation due to their low cost, easy preparation, and high stability, but their performance is limited by poor mass transfer, imprecise imprinting, and single interaction modes. Herein, dendritic mesoporous silica nanoparticles (DMSNs) were used as the support, and a self-designed multifunctional poly(ionic liquid) macromonomer (p(VIMCD-co-VAIM-co-VSIM-co-VVIM)) served as the functional monomer to achieve directional anchoring of cytochrome C (Cyt-C). Surface-imprinted microspheres (DMSNs@MPS@PILs-MIPs) were prepared via free-radical copolymerization for Cyt-C recognition. The DMSNs possessed interconnected mesoporous channels, good dispersibility, an average particle size of ~80 nm, and a specific surface area of 267.97 m²/g. Ionic liquid monomers were synthesized via alkylation, and the macromonomer was constructed through a two-step method. Molecular dynamics simulations and spectroscopic characterization revealed the macromonomer-stabilized Cyt-C conformation, with interactions dominated by van der Waals forces. The DMSNs@MPS@PILs-MIPs featured a thin imprinted layer (~5 nm) to reduce mass-transfer resistance. Adsorption studies showed Cyt-C adsorption followed Langmuir and pseudo-second-order models, with a maximum capacity of 383.14 mg/g and an imprinting factor of 2.17. Only 12% capacity loss occurred after repeated cycles, indicating robust regeneration stability. This study provides a feasible strategy for constructing protein surface-imprinted polymers based on multifunctional synergistic interactions and conformational stabilization. Full article

Haemoglobin (Hb) concentration is a key biomarker for several diseases. Traditional laboratory methods often have limitations due to their time-consuming nature, the need for skilled personnel, or the use of high-cost instrumentation. This work presents a sensing strategy for developing new point-of-care tests (POCTs) for Hb detection via a proof of concept. The proposed sensing approach is implemented using plasmonic plastic optical fiber (POF) sensor chips that integrate an electropolymerized molecularly imprinted polymer (eMIP) film on the plasmonic surface for Hb-selective detection. The developed sensor system demonstrates an ultra-low detection limit of 80 fM in buffer, about five orders of magnitude lower than that of other comparable Hb sensors. Selectivity tests against common interfering proteins, such as bovine serum albumin (BSA) and immunoglobulin G (IgG), confirmed high specificity towards the target analyte. Moreover, the sensor’s performance was tested using a whole-blood sample, yielding results consistent with those of standard haematology analysis. The proposed sensor system, based on simple equipment, provides a quick (about 10 min) and cost-effective (about 10 euros per chip) label-free diagnostic tool for POCTs in real-world scenarios, such as finger-prick sampling, offering a less invasive alternative to traditional laboratory methods, towards devices useful for Internet of Medical Things (IoMT). Full article

Myo-inositol-1-phosphate synthase (MIPS) catalyzes the first committed step of de novo inositol biosynthesis, yet genetic evidence suggests that Arabidopsis MIPS proteins also have catalysis-independent functions. Although moonlighting proteins are increasingly recognized, their identification and functional dissection in plants remain limited. We asked whether the catalytic outputs of MIPS can be uncoupled from its inositol-independent functions. Here, using an inositol-rescue transcriptomic strategy, we separated catalytic inositol-biosynthetic outputs from inositol-independent functions of MIPS in Arabidopsis seedlings. Exogenous inositol had little effect on the wild type but extensively reprogrammed the mips1 mips3 transcriptome without fully restoring it to the wild type state. The inositol-independent branch was associated mainly with nuclear gene-regulatory processes, with broader implications for development and immunity. By contrast, the catalytic branch was linked primarily to cellular metabolism and structural organization, with broader roles in stress responses and polar growth. These findings support a dual-function model in which Arabidopsis MIPS proteins couple cytosolic inositol biosynthesis with candidate moonlighting functions associated with nuclear gene-regulatory modules. More broadly, this work provides a framework for understanding how metabolic enzymes coordinate development and stress responses, and opens new avenues for exploring how plant gene duplication may foster functional innovation and adaptation to environmental change. Full article

Protein recognition underpins advances in drug discovery, immunoassays, clinical diagnostics and biosensing. As a biomimetic alternative to natural receptors, molecularly imprinted polymers (MIPs) have been developed to emulate antibody–antigen complementarity by generating binding cavities that mirror the size, shape and functionality of target macromolecules through template-directed polymerization and subsequent template removal. However, protein imprinting has historically been hampered by low imprinting efficiency and limited selectivity, rendering conventional protein-imprinted polymers (PIPs) inadequate for many contemporary biomedical applications. Functional ionic liquids (ILs)—a class of designer solvents and materials distinguished by tunable structures, exceptional physicochemical properties and favorable biocompatibility—have emerged as versatile additives to address the principal limitations of traditional PIPs, including poor selectivity, sluggish mass transfer and destabilization of protein conformation. Here, we provide a systematic review of the multifaceted roles that ILs play within protein-imprinting systems, delineating their employment as template-anchoring motifs, functional monomers, cross-linkers, porogens and structural stabilizers, and evaluating the consequent effects on polymer architecture and recognition performance. We further probe the multiplicity of non-covalent interactions between ILs and template proteins—highlighting the synergistic modulation afforded by electrostatic forces, hydrogen bonding, hydrophobic interactions and π-π stacking—and consider how such interplay can be harnessed to fine-tune binding-site fidelity. Consolidating recent progress, we summarize IL-enabled PIP applications in protein-specific recognition, biosensor development and analysis of complex real-world samples, and we critically examine the prevailing technical challenges and prospects for translation. The evidence indicates that ILs, by furnishing abundant interaction sites, accelerating mass transport and stabilizing native protein conformations, can markedly enhance PIP adsorption capacity, target specificity and recyclability, positioning them as a cornerstone for next-generation protein separation and enrichment materials and paving the way toward industrial deployment of protein-imprinting technologies. Full article

Grade C molar-incisor pattern periodontitis (C-MIP) is a rapidly progressive form of periodontal disease affecting young individuals that is often linked to a highly virulent genotype of Aggregatibacter actinomycetemcomitans (Aa). Although Aa is present in the healthy oral microbiome, its transition into subgingival tissue correlates with the conversion from healthy to diseased status within the periodontal pocket. These changes may be due to immune evasion strategies attributed to Aa exotoxins. We previously demonstrated that a host cell protein, cellugyrin, plays a critical role in exotoxin internalization and subsequent cytotoxicity. Herein, we assess the contribution of cellugyrin to Aa phagocytosis and intracellular trafficking in human macrophages. Confocal imaging demonstrated that Aa co-localizes with cellugyrin. Importantly, cellugyrin-deficient macrophages exhibited a significant reduction in phagocytosed Aa. Furthermore, we analyzed the role of retrograde trafficking in Aa survival. Retro-2-mediated inhibition of this trafficking pathway resulted in increased intracellular Aa, likely due to increased survival. Collectively, our findings suggest that cellugyrin is involved in Aa phagocytosis and that retrograde trafficking may play a role in subsequent host cell clearance of Aa. Full article

Background/Objectives: Chronic obstructive pulmonary disease (COPD) is characterized by incomplete recovery of airflow blockage; however, effective therapeutic agents that can prevent lung function deterioration are limited. East Asian herbal treatments have gained attention for their potential benefits in managing COPD. This study aimed to evaluate the inhibitory effects of Gyeongok-go (GOG) on lung injury in a COPD mouse model. Methods: Lipopolysaccharide (LPS)-induced alveolar macrophage (MH-S) cells were treated with GOG (50, 100, 200, and 400 μg/mL), and analyzed using enzyme-linked immunosorbent assay (ELISA). C57BL/6 mice were challenged with cigarette smoke extract and LPS and then treated with vehicle only, dexamethasone (3 mg/kg), or GOG (100, 200, or 400 mg/kg). Bronchoalveolar lavage fluid (BALF) or lung tissues were analyzed using cytospin, ELISA, real-time PCR, flow cytometry, hematoxylin and eosin, and Masson’s trichrome staining. Results: Treatment with GOG decreased tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 expression in LPS-challenged MH-S cells. In COPD mice, GOG significantly decreased the elevated numbers of neutrophils, total cells, macrophages, and Gr-1⁺/Siglec-F, Gr-1⁺/CD11b⁺, and CD44^high/CD62L⁻ cells. It also downregulated the expression of TNF-α, IL-17A, macrophage inflammatory protein-2 (MIP2), and CXC chemokine ligand-1 in BALF. GOG also inhibited the increase in Mip2, Cox-2, and Trpv1 mRNA expression. Moreover, GOG prevented the increase in the number of total cells, neutrophils, Gr-1⁺/Siglec-F, Gr-1⁺/CD11b⁺, CD44^high/CD62L⁻, and CD21⁺/CD35⁺/B220⁺ cells in lung tissues. Notably, GOG decreased the severity of lung injury. Conclusions: Overall, these findings indicate that GOG alleviates lung injury, suggesting its potential in the treatment of COPD. Full article

Leucine-rich-repeat kinase 2 (LRRK2) is a multidomain protein highly expressed in immune cells and implicated in the regulation of immune functions including immune signaling, cytokine release and autophagy. LRRK2 is one of the therapeutic targets in Parkinson’s Disease (PD). Aberrant activation of LRRK2 can also contribute to intestinal inflammation, mainly in inflammatory bowel disease (IBD). Hence the modulation of LRRK2 may influence gut inflammation providing an improvement in disease outcomes. Over the years, microRNAs (miRNAs) have acquired much attention thanks to their potential as therapeutic targets in several pathological conditions, including inflammatory disorders. In this study, we aimed to examine the ability of miR-369-3p in the modulation of autophagy targeting LRRK2 expression. Bioinformatics analysis revealed that Lrrk2 is a target gene of miR-369-3p, and LRRK2 expression was increased in ulcerative colitis patients compared with that in a healthy control. In in vitro analysis, we validated that mimic transfection with miR-369-3p in Raw264.7 significantly reduced the expression of LRRK2 both in basal and inflammatory conditions. Moreover, the inhibition of LRRK2 limited the nuclear translocation of Nuclear factor kappa B (NF-κB) induced by lipopolysaccharide (LPS) stimulation. In turn, we found that, in inflammatory conditions, the intracellular increase in miR-369-3p precluded the inhibition of autophagy by LRRK2 by restoring autophagy marker light chain 3 (LC3)II/I ratio, BECLIN-1 and decreasing p62 expression. Furthermore, we detected that the upregulation of miR-369-3p decreased the release of pro-inflammatory mediators Tumor necrosis factor (TNF), C-C motif ligand 2/Monocyte chemoattractant protein-1 (CCL2/MCP-1), C-C motif ligand 3/Macrophage inflammatory protein-1 alpha (CCL3/MIP-1α) and C-C motif ligand 5/Regulated on activation, normal T-cell expressed and secreted (CCL5/RANTES) and increased the anti-inflammatory cytokine interleukin 10 (IL-10) in response to LPS. This study supports the anti-inflammatory potential of miR-369-3p in immune cells and suggests the potential of miR-369-3p as a therapeutic agent in the treatment of acute intestinal inflammatory conditions such as ulcerative colitis. Full article

Chronic alcohol exposure disrupts blood–brain barrier (BBB) integrity and promotes neuroinflammation, with P2X7 receptor (P2X7R) signaling playing a critical role. Our prior work in male mice linked P2X7R inhibition to reduced extracellular adenosine triphosphate (eATP) release, modulated extracellular vesicle (EV) cargo, and attenuated neuroinflammation in chronic intermittent ethanol (CIE)-exposed mice. However, sex-specific roles of P2X7R signaling and EV-mediated mechanisms in alcohol-induced neuroinflammation remain unclear. Male and female mice were exposed to ethanol vapor for three weeks and treated with Brilliant Blue G (BBG), a P2X7R inhibitor. Compared to their respective CIE-unexposed controls, brain gene expression of tumor necrosis factor–α (Tnf-α), interleukin-1 beta (Il-1b), interleukin-6 (Il-6), monocyte chemoattractant protein-1 (Mcp-1), and Fas ligand (Fasl) significantly increased in CIE-exposed males, while only Il-1b increased in females. P2X7R inhibition significantly reduced these cytokines. Pericyte immunostaining was decreased by CIE (indicating BBB injury) in male mice only and was restored by P2X7R inhibition with no difference between groups in females. Occludin staining (another BBB marker) did not differ between the treatment groups in male and female animals. Circulating cytokines (Macrophage inflammatory protein-1 alpha (MIP-1α), tumor necrosis factor–α (TNF-α), interleukin-1 beta (IL-1β), and interleukin-27 subunit p28/interleukin-30 (IL-27p28/IL-30) were significantly elevated in CIE-exposed males but not in females, with BBG treatment reducing cytokines in males. Circulating eATP, P2X7Rs, P-glycoprotein (P-gp), EVs, and EV-mtDNA, which we identified in our previous study, were increased in both sexes and partially decreased by P2X7R blockade. Spatial memory was impaired by CIE exposure in males but not females, and this deficit was reversed by BBG treatment. Our findings reveal sex differences in CIE-induced circulating cytokines, neuroinflammation, and memory impairment, with a stronger response in males. However, other markers of cell injury associated with CIE exposure were upregulated in both sexes; P2X7R inhibition effectively mitigated these effects, highlighting the functional relevance of targeting the P2X7R in alcohol-induced injury. Full article

The rapid and accurate detection of SARS-CoV-2 biomarkers remains a critical requirement for effective outbreak control and decentralized diagnostics. Although RT-PCR is the current gold standard, its reliance on centralized laboratories and long processing times limits its applicability in point-of-care settings. In this context, optical biosensing platforms based on surface plasmon resonance (SPR) offer attractive features, including label-free, real-time, and quantitative detection. This study explores the use of synthetic receptors for the highly sensitive detection of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Specifically, soft molecularly imprinted polymer nanoparticles (nanoMIPs) were employed as synthetic receptors and integrated into a high-sensitivity, portable plasmonic platform based on a D-shaped plastic optical fiber (POF) SPR sensor. The nanoMIPs were selectively imprinted against the RBD, characterized by Dynamic Light Scattering (DLS), Isothermal Titration Calorimetry (ITC), and Scanning Electron Microscopy (SEM) to confirm nanoMIPs size, binding properties, and surface morphology. Next, the nanoMIPs were immobilized onto a gold-coated sensing surface, enabling enhanced specificity, affinity, and signal amplification compared to conventional biological recognition elements. The resulting RBD-SPR-nanoMIPs sensor demonstrated promising analytical performance, exhibiting high selectivity against potentially interfering proteins and an anticipated sensitivity suitable for RBD detection at femtomolar concentrations. The inherent stability of nanoMIPs suggests the potential for reusable SPR sensing platforms, paving the way for next-generation synthetic receptor-based plasmonic biosensors. Full article

The aquaporin 0 (AQP0)/major intrinsic protein of eye lens (MIP) cDNA was cloned and sequenced. Initial studies of the tissue distribution of mRNA expression proved to be incorrect. Subsequent experiments showed that AQP0 mRNA is expressed strongly in the eye with moderately strong expression in the kidneys and some expression was seen in the brain and muscle tissue, and very low expression in the esophagus/fundic stomach. Another set of PCR reactions with five times the amount of cDNA additionally showed mRNA/cDNA expression in the liver, rectal gland, and a very low level in the intestine. Sporadic expression of different pieces of AQP0 cDNA was seen in various experiments in gill and pyloric stomach. A custom polyclonal antibody was produced against a region near the C-terminal end of the AQP0 protein sequence. The antibody gave a band of around the correct size (for the AQP0 protein) on the Western blot, which also showed a few other higher-molecular-weight bands. The antibody was also used in immunohistochemistry, and in the kidney, it showed staining in the proximal II (PII), intermediate segment I (IS I), and late distal tubule (LDT) parts of the sinus zone region of nephrons as well as some staining in the bundle zone tubule segments, suggesting a role for AQP0 as a water channel. In the rectal gland, the antibody showed weak apical membrane staining in a few secretory tubules near the duct, but also somewhat stronger staining in cells appearing to connect various secretory tubules, suggesting a role in cell–cell adhesion. In the spiral valve intestine side wall and valve flap, after signal amplification, weak antibody staining was seen in the apical and lateral membranes of epithelial cells adjacent to the luminal surface. There was also some staining in the intestinal muscle. In the rectum/colon, staining was seen in a layer of cells underlying the epithelium and in some muscle layers. In the gill, there was very weak staining in secondary lamellae epithelial cells and in connective tissue surrounding blood vessels and blood sinuses. The low level of transcript expression in the rectal gland, gill, and intestinal tissues suggests caution in the interpretation of the immunohistochemical staining in these tissues. Full article