The main olfactory bulb (MOB) hosts diverse γ-aminobutyric acid (GABA)ergic interneurons that play critical roles in sensory processing. Previous studies have reported variability in GABA immunoreactivity among cells in the granule cell layer (GCL) of the mouse MOB. However, the degree of intracellular GABA content across MOB layers and its relationship with molecular subtypes remain unclear. In this study, we conducted a quantitative analysis of GABA immunoreactivity in individual cells across all MOB layers using confocal laser scanning and electron microscopy. We found striking variability in the GABA immunoreactivity intensity among individual neurons across all layers, not limited to the GCL. Electron tomography with immunogold labeling confirmed more precisely substantial differences in intracellular GABA content, particularly in the glomerular layer and GCL. Mitral cells exhibited minimal GABA immunoreactivity, consistent with their excitatory phenotype. Co-labeling of molecular markers, including tyrosine hydroxylase, calbindin, calretinin, parvalbumin, and secretagogin, revealed that variability in GABA levels is common across all interneuron subtypes. Furthermore, a comparative analysis of the depth-dependent distribution of cells within the GCL and their GABA immunoreactivity demonstrated that more superficial regions contain stronger immunoreactive cells. This likely reflects the function of interneurons that regulate the olfactory output of tufted cells, which are projection neurons located in the superficial layers. To investigate the contribution of GABA-synthesizing enzymes, we examined the expression of glutamate decarboxylase 67 (GAD67) using GAD67-GFP knock-in mice. Although GFP intensity, reflecting GAD67 promoter activity, varied among cells throughout the MOB, it did not correlate clearly with GABA immunoreactivity. This finding suggests that GAD67 expression alone does not fully explain the observed intracellular GABA variability. Our findings show the molecular and cellular heterogeneity of GABAergic neurons in the MOB. Further integrative studies are needed to elucidate the mechanisms regulating GABA content and their functional relevance.

Temperature is a key environmental driver of hepatic physiology in ectotherms, and three-dimensional (3D) fish liver models may serve as an ethically advantageous platform to investigate warming effects under controlled conditions while reducing the need for experimental animals. Despite their increasing use in toxicology, their application to assess climate-relevant temperature effects on liver function remains limited. This study investigated the temporal effects of a warming scenario on primary hepatocyte spheroids from juvenile brown trout (Salmo trutta), a bioindicator species. The study explores how a + 3 °C increase can affect spheroid development and maintenance, as well as its impact on metabolic activity, cell proliferation and death, and morphology over time. Spheroids were maintained at 18 °C and 21 °C for 25 days and analysed at five time points using metabolic, morphometric, immunocytochemical, and ultrastructural approaches. Mitochondrial metabolic activity, assessed by resazurin reduction, showed no significant temperature-related differences. In contrast, warming accelerated spheroid formation and produced larger spheroids. Proliferative activity, assessed by proliferating cell nuclear antigen (PCNA) immunostaining, was significantly reduced at 21 °C, while caspase-3 levels remained unchanged, indicating no increase in apoptosis. The autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3A/B) showed lower immunoreactivity at 21 °C, with no temporal variation. Ultrastructural analysis revealed preserved hepatocyte integrity at both temperatures and abundant cytoplasmic dense bodies consistent with autolysosomal structures, which increased over time. Overall, a realistic warming scenario altered growth dynamics and cellular morphology. Further, this data reinforces that 3D fish liver models are viable alternative systems for assessing climate-driven effects.

CEACAM1 (CC1) is an important mediator of cell proliferation and adhesion and serves as an angiogenic factor through interaction with VEGF. Although the role of CC1 has been extensively studied in organs such as the heart, liver and lung in which CC1 is considered an important regulator of various pathological conditions, little is known about the function of CEACAM1 in the eye. In this study, we investigated the expression and function of CC1 in the retina and choroid of healthy adult mice using immunohistochemistry, fluorescence activated cell sorting (FACS) and RNA sequencing. We found CC1 to be expressed in endothelial and myeloid cells of the retina and the choroid. However, deletion of CC1 did not result in vascular abnormalities of the retina and choroid or changes in retinal myeloid cell morphology and number. Furthermore, the retinal architecture was not affected and morphometric measurements of the thickness of the inner and outer retinal layers were not altered by deletion of CC1. Accordingly, we did not observe structural or transcriptomic changes in the choroid. Our data suggest that the role of CC1 in the adult eye during steady state is attenuated or can be compensated by other molecular mediators. However, one could speculate that CC1 may become functionally relevant during pathological conditions, such as in neovascular eye diseases like proliferative diabetic retinopathy or neovascular age-related macular degeneration.

Left atrial remodeling (LAR) critically contributes to the progression of heart failure (HF) and the development of atrial fibrillation (AF) following myocardial infarction (MI). The protein α2δ1, primarily known for its role in neuropathic pain, is abundantly expressed in atrial tissue, but its involvement in post-MI LAR remains unclear. Here, LAR models were established in rats post-MI, and atrial hypertrophy was induced in HL-1 cells using angiotensin II (AngII). The role of α2δ1 in atrial hypertrophy was examined through treatment with either the α2δ1 inhibitor gabapentin or a C-terminal interfering peptide (α2δ1 CT-pep). A significant upregulation of α2δ1 expression was observed in the left atrium (LA) of MI rats and in AngII-treated HL-1 cells. Western blot analysis revealed increased α2δ1 levels in membrane fractions and decreased levels in the cytoplasmic fractions compared to controls. Both gabapentin and α2δ1 CT-pep treatment significantly reduced HL-1 cell hypertrophy and inhibited CAMKII and HDAC4 phosphorylation. Co-immunoprecipitation assays demonstrated an interaction between α2δ1 and GluN1, which was enhanced by AngII stimulation. Inhibition of α2δ1 attenuated the α2δ1-GluN1 interaction and reduced GluN1 translocation to the plasma membrane. In MI-induced HF rats, gabapentin treatment diminished atrial hypertrophy, suppressed AF inducibility and duration, and decreased membrane-associated α2δ1 and GluN1 levels. These findings suggest that the C-terminal domain of α2δ1 may contribute to left atrial hypertrophy in chronic ischemic heart failure and is associated with altered membrane GluN1 abundance and p-CAMKII/p-HDAC4 signaling. α2δ1 may therefore represent a potential therapeutic target for left atrial remodeling in ischemic heart failure.

The Magadi tilapia thrives in arguably the most extreme aquatic environment on earth for fish, the hot springs of Lake Magadi in Kenya with its severe water chemistry: pH 10, alkalinity 300 mEq·L-1. This fish is 100% ureotelic yet has an osmoregulation pattern typical of marine teleosts, although the dominant water anion is HCO3- rather than Cl-. The gills must actively export base equivalents (HCO3- + CO32-) and Na+ against strong electrochemical gradients, yet simultaneously take up Cl-, for which a hypothetical "Laurent model" based on ionocyte structure alone was proposed. This model has been tested using immunohistochemistry to characterize ionocyte types based on ion transport protein expression patterns [CFTR anion channel, Na+:K+:2Cl- cotransporter (NKCC)/Na+:Cl- cotransporter (NCC), Na+:HCO3- co-transporter (NBC), Na+/K+-ATPase (NKA), and urea transporter (UT)]. A typical "seawater ionocyte" (Type IV) with apical CFTR and basolateral NKCC1 and NKA, is present validating key elements of the model. A "freshwater ionocyte" (Type II) is also present (apical NCC, weaker NKA and strong NBCe1 basolateral staining). A third Type I ionocyte with only NKA staining was also identified. An acid excreting Type III ionocyte (apical NHE3 and basolateral NKA) was not present. The Magadi tilapia is unusual in having co-expression of both Type IV and Type II ionocytes, which are typically associated with Cl- excretion and uptake, respectively. Instead, we propose Type IV ionocytes are involved in base equivalent and Na+ excretion and Type II ionocytes in Cl- uptake. In these ureotelic fishes, the UT occurs only in lamellar pavement cells.

The heart of Phasmida (stick insects) remains poorly understood, particularly regarding its overall physiology and associated cells. Here, we integrate the morphology of the heart and associated cells in Cladomorphus phyllinus with transcriptomic profiling of corresponding structures in Cladomorphus trimariensis. The heart is a tubular structure composed of cardiomyocytes physically associated with alary muscles, with a location and structure similar to that described in other insects. Numerous pericardial cells are organized in cord-like aggregations around the heart, displaying abundant membrane invaginations and an extensive endocytic and lysosomal machinery, consistent with their role in hemolymph filtration. Given their abundance, these cells likely represent the main source of the numerous transcripts related to endocytosis, vesicular trafficking and lysosome-mediated digestion. Hemocytes are also present around the heart, often forming aggregations indicative of immune responses. The transcriptomic analysis indicates immune activity, including melanization and antibacterial defense, predominantly driven by the hemocytes, with the pericardial cells providing a supportive role. Our results confirm that heart and associated cells act not only in hemolymph propulsion, but also as a site involved in maintaining hemolymph homeostasis and immune defense. This work provides an integrated morphological and functional framework for the circulatory system of Phasmida, expanding comparative perspectives across insects.

Autophagy is a cellular process essential for maintaining homeostasis and supporting development. Herein, we aimed to evaluate the effects of gestational protein restriction (GPR) on tooth germ autophagy. CAG-RFP-EGFP-LC3 female mice were mated and randomly assigned to normal protein or low-protein groups. Embryos were collected, and autophagy flux in the molar tooth germ was quantified. At the bud stage, GPR induced a decrease in autolysosomes in the condensed mesenchyme, alongside an increase in autophagosomes in the dental epithelium. At the cap stage, an increase in lysosome markers was observed in the dental papilla, while a decrease in autolysosomes was observed in enamel organ. By the bell stage, dental papilla exhibited a decrease in autolysosomes and an increase in lysosome markers. Similarly, inner enamel epithelium showed reduced autolysosomes and elevated lysosome markers. Bud stage was the most significantly affected; we further quantified apoptosis and mitosis, revealing impaired cell proliferation and enhanced apoptosis. Our data suggest that GPR block or delay acidification of autophagic vesicles, potentially impairing the recycling of cellular components critical for tooth germ proliferation and differentiation.

The present study investigates the antennae and brain of the immature stages of the caddisfly Hydropsyche pellucidula (Andersen, and Klubnes 1834) using scanning and transmission electron microscopy as well as fluorescence microscopy. The larval antenna is unsegmented and bears two long, articulated trichoid sensilla and two large, non-articulated basiconic sensilla, all with an internal structure typical of mechanoreceptors. No changes were detected between the larval stages examined, but larval sensilla differ completely from the adult sensilla previously described in the same species, which include several chemoreceptors (e.g. trichoid, pseudoplacoid, pseudoplacoid, chaetoid, coronary, and styloconic sensilla). A preliminary account of larval brain anatomy reveals a small central body without columnar elements, mushroom bodies without calyces, and no antennal lobes in any larval stage. This condition differs markedly from the pupal brain, which-similar to the adult brain-shows a central body with a vague fan-shaped structure, mushroom bodies with small calyces, and well-structured antennal lobes containing few but relatively large glomeruli. Such dramatic change is similar to what occurs in other closely related holometabolous insects, such as Lepidoptera, but it is more pronounced, probably because of the ecological differences between larvae and adults, as also observed in other aquatic insects such as mosquitoes. The results of this research shed light on overlooked aspects of caddisfly biology. Moreover, they may enhance our understanding of the evolution of insect olfaction, since caddisflies are among the most important orders of aquatic insects and the closest relatives of Lepidoptera, a key model system in insect chemical ecology.

The urinary bladder is innervated by two anatomically distinct populations of sensory neurons originating from dorsal root ganglia (DRG): one from the thoracolumbar (TL) and another from the lumbosacral (LS) spinal region. While we previously characterized the morphology and distribution of sensory endings arising from LS DRG, the terminals of TL afferents remain undefined. In this study, we employed anterograde neuronal tracing to map these endings in mice. Following bilateral injection of dextran biotin into T12-L3 DRG and a 7-9-day recovery, whole bladders were processed to visualize spinal afferent axons and their calcitonin gene-related peptide (CGRP) immunoreactivity. We identified four morphological types of sensory endings, similar to those described for LS DRG: simple and complex types in both the sub/urothelium and the detrusor muscle. However, their distribution was fundamentally different. Most TL afferents (79%) terminated within the sub/urothelium, with a minor proportion (21%) in the detrusor. This pattern is the inverse of LS innervation, which predominantly targets the detrusor (81%). Furthermore, nearly all TL endings (96%) were peptidergic (CGRP +). This stark anatomical and neurochemical segregation suggests that TL and LS spinal afferent populations are functionally specialized. We propose that the dense TL innervation of the sub/urothelium may constitute a protective surveillance system sensitive to chemical irritation and inflammation, whereas the predominant LS innervation of the detrusor is likely specialized for detecting mechanical stretch during bladder filling.

Osteoarthritis (OA) is a degenerative joint disease closely linked to iron dysregulation. While the role of iron in OA is recognized, the precise mechanism of cell death remains unclear. This study aimed to elucidate how chronic low-dose iron exposure influences chondrocyte survival, iron metabolism, and ferroptosis susceptibility. The 32-day exposure effects of low-dose (20 µM) ferric ammonium citrate (FAC) treatment on the immortalized human chondrocyte cell line (C-20/A4) in terms of survival and susceptibility to ferroptosis were investigated. Cell viability, morphology, and expression of key iron regulatory genes (IRGs) and ferroptosis-related stress genes were assessed. At day-32 of FAC treatment, chondrocyte proliferation and glutathione peroxidase 4 (GPX4) expression increased relative to controls (p < 0.0001). Inhibition of GPX4 with RSL3 in FAC-treated cells resulted in significantly increased cell death (p < 0.001), indicating a possible protective role of GPX4. Iron chelation with deferoxamine conferred protection against ferroptosis. Dynamic changes in IRGs and cell cycle regulators, consistent with an adaptive strategy to resist iron overload and support survival, were observed. In-silico analysis of OA cartilage transcriptomic data identified 170 differentially expressed genes. Functional enrichment showed upregulation of ubiquitin-dependent catabolic processes and downregulation of mitotic pathways, supporting altered stress responses and cell cycle disruptions in iron-exposed chondrocytes. Chronic low-dose iron exposure induced an adaptive upregulation of GPX4, enhancing chondrocyte resistance to cell death by ferroptosis by maintaining lipid peroxide reduction. Therapeutic modulation of GPX4 and iron homeostasis may offer novel strategies for OA management where cartilage iron imbalance is implicated.

Bone regeneration remains constrained by incomplete osteogenic commitment of mesenchymal stem cells (MSCs), underscoring the need for precise lineage control. CRISPR/Cas-based epigenome editing provides programmable access to chromatin regulators without altering the DNA sequence, and catalytically inactive Cas9 (dCas9) fused to transcriptional activators, repressors, or chromatin modifiers enables locus-specific modulation of key osteogenic networks, including RUNX2, OSX, and BMP2, while suppressing inhibitory loci such as PPARG, SOST, and DKK1. Multiplex strategies further allow the concurrent activation of osteogenic genes and repression of adipogenic or Wnt antagonists, reshaping lineage allocation in vitro and in vivo. Delivery innovations-from AAV vectors and lipid nanoparticles to biomaterial scaffolds and extracellular vesicles-support local and systemic applications with increasing precision, while whole-genome chromatin profiling and high-fidelity Cas variants reduce off-target risk, and CRISPRoff/on platforms provide reversible and heritable control of transcriptional states. Proof-of-concept studies in small animals demonstrate bone repair in preclinical models, with emerging large-animal data highlighting translational potential. Remaining challenges include payload size, immunogenicity, durability of epigenetic states, GMP-grade manufacturing, and regulatory classification. Looking ahead, advances such as AI-guided gRNA libraries, mechano-responsive scaffolds, and long-term tracking of epigenetic memory may yield durable "smart" osteo-epigenetic therapies. Collectively, CRISPR/dCas9-based epigenome editing is progressing from mechanistic exploration toward clinically viable strategies for skeletal regeneration.

Temporomandibular joint osteoarthritis (TMJOA) is a degenerative joint disease characterized by the progressive deterioration of the condylar cartilage, compensatory remodeling of subchondral bone, and persistent synovial inflammation. The onset of TMJOA is accompanied by the activation of cytokines, which exacerbate the imbalance in joint homeostasis and accelerate the destruction and even irreversible degradation of joint structures. FGF19, as an important multifunctional fibroblast growth factor, has been reported to be substantially up-regulated in the cavity of osteoarthritis joint. However, its role in TMJOA progression remains unclear. In this study, we investigated its role in the condyle cartilage in TMJOA. We first established a mouse TMJOA model by unilateral anterior crossbite (UAC) and achieved overexpression of FGF19 by direct injection of adeno-associated virus carrying the Fgf19 gene. We found that FGF19 exacerbated the progressive deterioration of cartilage-subchondral bone unit in the progression of TMJOA. FGF19 accelerated the phenotypic transformation from mature cartilage to hypertrophic cartilage by reducing the expression of collagen type II (COL2A1) and aggrecan, enhancing the expression of collagen type X (COL10A1), matrix metallopeptidase 13 (MMP13), and a disintegrin and metalloproteinase with thrombospondin 5 (ADAMTS5), and exhibiting a fibrocartilage-associated change in the cartilage layer by increasing the expression of collagen type I alpha 1 and 2 (COL1A1 and COL1A2). Furthermore, FGF19 induces calcification progression of cartilage hypertrophic layer by up-regulating the expression of dentin matrix acidic phosphoprotein 1 (DMP1) via transcription factor osterix (OSX). These data provide evidence of the importance of FGF19 in the progression of TMJOA and show potential cues for interventions of cartilage disease.

Chimeras (Holocephali) occupy a pivotal phylogenetic position, being the oldest jawed vertebrate, for understanding the evolution of hypothalamic-pituitary organization in vertebrates. However, the structural organization of the hypothalamic-pituitary axis and the anatomical relationships with the buccal lobe (BL), a unique gland thought to play a role in reproduction, remains poorly characterized. We present an anatomo-histological analysis of the chimaera Callorhinchus callorynchus using classical histology, immunohistochemistry, and RT-PCR, focus on key neuropeptides related to the control of the reproductive activity in other vertebrates: gonadotropin-releasing hormone (GnRH), secretoneurin (SN), cholecystokinin (CCK), and gonadotropic hormones. The pituitary gland displays clear regionalization into the rostral pars distalis, proximal pars distalis, and neurointermediate lobe. The BL is connected to the posterior telencephalon by paired canals containing a prominent blood vessel and bundles of neuropeptidergic fibers. Gonadotropin-releasing hormone-immunoreactive nerve cell bodies and fibers were identified in the posterior telencephalon using two antisera, with fibers projecting to the proximal pars distalis of the pituitary and the BL. Secretoneurin-immunoreactive neuronal somata and fibers were observed in the posterior telencephalon and hypothalamus, and SN-immunoreactive cells were also present within the pituitary and BL. Cholecystokinin-immunoreactive neuronal elements were detected in the hypothalamus, while CCK-immunoreactive cells were present in the pituitary, and a subset of fibers extending into the BL. RT-PCR analyses revealed the presence of Fshb and Fhb transcripts in the pituitary and BL, with a stronger apparent signal in the BL. These observations provide anatomical evidence for direct neuropeptidergic innervation of both the pituitary and BL and indicate that the BL is associated with gonadotropin subunit gene expression in C. callorynchus. This new anatomical framework of the hypothalamus-pituitary-BL axis identifies two new pathways: direct GnRH and CCK innervation, and local SN-mediated autocrine/paracrine signaling.

The posterior lobe (PL) of the pituitary contains a specialized neurovascular junction, where neurosecretory axon terminals closely associate with fenestrated capillaries to enable efficient hormone release into the circulation. The neurosecretory axon terminals and the fenestrated capillaries are supported by an outer basement membrane (BM) and an inner BM, respectively. Although BMs are critical structural components of this junction, their molecular composition and regulatory mechanisms remain poorly understood. We previously demonstrated that COL15A1, the α1 chain of collagen XV, localizes to the BMs surrounding fenestrated capillaries in the anterior lobe of the rat pituitary. In the present study, we examined the localization and regulation of COL15A1 in the rat PL. Immunohistochemical analyses revealed that COL15A1 is localized to the inner BMs of fenestrated capillaries. Combined in situ hybridization and immunohistochemistry demonstrated that Col15a1 is predominantly expressed in NG2-positive pericytes. Water deprivation, which induces arginine vasopressin (AVP) secretion, significantly decreased Col15a1 expression in the PL, whereas AVP stimulation did not affect its expression in primary cultured posterior and intermediate lobe (PIL) cells. We also found that TGFβ2 is produced by S100β-positive pituicytes and TGFβ receptor II is localized to NG2-positive pericytes in the PL. Furthermore, TGFβ2 induces Col15a1 expression in primary cultured PIL cells via TGFβ receptor II and SMAD2 signaling. Collectively, our results identify collagen XV as a key component of the inner BMs surrounding fenestrated capillaries and demonstrate that pericytes orchestrate the structural and functional organization of the neurovascular junction in the PL.