Search Results (250)

This preliminary study characterises type I collagen in the digestive system of the greater weever (Trachinus draco L.) by integrating histochemical and biochemical techniques. To the best of our knowledge, this study represents the first baseline mapping of type I collagen within the gastrointestinal tract of this species. Mallory staining and indirect immunofluorescence confirmed collagen presence across the oesophagus, stomach, and intestine. The histochemical quantification of the fluorescent area (100 measurements per organ across 15 fish specimens) showed no significant differences (p = 0.1315), indicating a uniform spatial distribution. However, biochemical analysis via hydroxyproline assay and a two-way ANOVA revealed significant differences in collagen content among organs (p = 0.0308). The stomach yielded the highest concentration (4.199 µg/mg), significantly exceeding that of the intestine (1.713 µg/mg; Šídák’s post hoc, p = 0.0300). This discrepancy suggests that the higher gastric content is due to greater fibre density rather than distribution area. SDS-PAGE and Western blot confirmed protein molecular weights of 100–130 kDa, corresponding to α1 and α2 chains typical of type I collagen. The combination of these histochemical and biochemical methods effectively detects and characterises collagen in fish gastrointestinal by-products. By introducing T. draco as a novel subject in this context, these findings provide essential baseline anatomical and histological data and offer a clear scientific justification for the biotechnological valorisation of unutilised commercial fishing by-products, fully aligning with sustainable marine circular economy principles. Full article

Age-related deterioration of the dermal extracellular matrix is driven primarily by fibroblast dysfunction, leading to loss of collagen integrity, elasticity, and structural support. In aesthetic dermatology, injectable and biostimulatory interventions increasingly target qualitative dermal remodelling, with collagen reorganisation widely adopted as a histological endpoint. Picrosirius red (PSR) staining under polarised light remains the most frequently used method for visualising collagen architecture; however, its birefringence colour patterns are often misinterpreted as proxies for collagen subtype shifts, particularly between types I and III. This conceptual review examines the methodological basis of such interpretations. We summarise the biological roles of major dermal collagens and compare current histochemical, immunohistochemical, ultrastructural and molecular methods for collagen assessment. We propose an interpretative framework that separates architectural collagen remodelling from molecular collagen synthesis and addresses the temporal dissociation between early fibre reorganisation and later subtype-specific expression as a plausible explanation for between-study discrepancies. Practical guidance is provided to support responsible interpretation and reporting of PSR-based collagen analyses. PSR is best regarded as a complementary tool for assessing collagen architecture rather than a definitive method for collagen subtype identification. Full article

Cervical cancer is a common gynecological malignancy, with a 5-year survival rate of only 17% for recurrent or metastatic cases. Increased extracellular matrix stiffness, a key change in the tumor mechanical microenvironment, promotes tumor metastasis via mechanotransduction. Piezo1, a mechanosensitive cation channel, senses matrix stiffness and converts mechanical signals into intracellular chemical signals. Neutrophil extracellular traps (NETs) are overformed in tumors, but the mechanism by which matrix stiffness regulates NETs in cervical cancer remains unclear. We detected matrix stiffness and related protein expression in cervical cancer tissues using atomic force microscopy and histochemical staining. Polyacrylamide gel models were used to culture HeLa/SiHa cells, with transcriptome sequencing and ELISA to analyze IL-8 expression. NETs were induced from human peripheral blood neutrophils, and their effect on lymphatic endothelial cells was evaluated. A TC-1 mouse model was used to verify in vivo effects, and Western blot/ELISA explored the Piezo1/NF-κB pathway. Higher Young’s modulus, increased α-SMA/Collagen I expression and collagen content in metastatic cervical cancer tissues. High matrix stiffness activated Piezo1/NF-κB, upregulated IL-8, induced NETs, and enhanced lymphatic endothelial cell tube formation/migration. BAPN reduced tumor stiffness, inhibited metastasis, and decreased NETs in mice. Knocking down Piezo1 blocked NF-κB activation and IL-8 upregulation. High matrix stiffness activates Piezo1/NF-κB to promote IL-8 secretion and NETs formation, enhancing lymphangiogenesis and cervical cancer metastasis, providing a new target for advanced cervical cancer treatment. Full article

Background/Objectives: Despite hematoxylin and eosin (H&E) staining remaining the cornerstone of histopathological diagnosis, substantial intra- and inter-laboratory variability persists. This issue is increasingly relevant in Digital Pathology, where staining inconsistency may affect whole-slide image interpretation and the performance of image analysis algorithms. In the present work, we evaluated the diagnostic adequacy and non-inferiority of a novel tabs-based H&E histochemical staining method compared with conventional liquid reagents. Methods: Fifty formalin-fixed paraffin-embedded tissue samples from routine practice were sectioned in duplicate and stained either conventionally or using H&E Stain Tabs. After slide review, 14 representative tissue samples were selected, scanned at 40× magnification, and used to generate 24 matched image pairs at different magnifications. A blind online survey was completed by 13 expert pathologists using high-quality monitors. Participants assessed overall staining preference and rated stromal, epithelial, cytoplasmic, and nuclear staining quality. Non-inferiority was tested using a predefined margin of −0.10, and paired rating differences were analyzed using the Wilcoxon signed-rank test. Results: Across 312 paired evaluations, the tabs-based method was preferred in 120 cases (38.5%), conventional staining in 118 cases (37.8%), and no preference was expressed in 74 cases (23.7%). The tabs-based method met the criterion for non-inferiority compared with standard staining (z = 2.7). Rating-scale analysis showed significantly better stromal evaluation with the tablet-based method (z = 2.638; p = 0.008), whereas no significant differences were observed for epithelial, cytoplasmic, or nuclear staining. All evaluated images were considered diagnostically adequate. Conclusions: The tabs-based H&E stain was non-inferior to the conventional method and showed particularly favorable performance in the assessment of stromal components. These findings support its potential role in improving staining reproducibility and standardization, particularly in Digital Pathology workflows where pre-analytical and analytical consistency is critical. Full article

Research on the cataloging of microstructures and chemical compound localization in peanut hulls in relation to fungal tolerance remains limited. The hull (pericarp) is the first physical interface with the soil environment and may contribute to defense against fungal invasion. Here, hull microstructure and histochemical localization of alkaloid-like compounds, cellulose, lignin, starch, and total proteins were characterized across reproductive developmental stages R3–R6 in three commercially grown cultivars (Georgia-06G, Georgia-12Y, and Georgia-18RU). Stained sections were examined by light and fluorescence microscopy, and images were quantified in Fiji-ImageJ as stained area percentage. Among the compounds studied, the highest area percentages were observed at later stages (R5 and R6). Alkaloid-like compounds, cellulose, and starch were higher at the R5 stages of G-18 (9.61 ± 0.75), G-12Y (22.96 ± 5.84), and G-06 (6.31 ± 1.13) respectively, while lignin and total proteins were highest at the R6 stage of G-18 (respectively, 14.49 ± 1.43 and 13.90 ± 1.45). The lowest histochemical presence for most metabolites occurred in the early stages (R3–R4). This indicates that hull maturation is accompanied by increased physical (sclerenchyma and lignified cells) and biochemical (alkaloid-like compounds, proteins) features consistent with protective roles. As the analysis was based on representative sections and regions of interest (ROI)-level quantification, the results are intended to guide future studies on hull-mediated defense and breeding for Aspergillus tolerance. Full article

Butterfly wing color patterns are determined in pupal wing tissues, in which the prospective eyespot focus functions as a developmental organizer. Here, we investigated the microscopic structures of pupal wing tissues containing an eyespot organizer in line with the physical distortion hypothesis. Histochemical staining revealed that the pupal cuticle and epidermis were wavy and thin at 6 h but smooth and thick at 12 h postpupation. The eyespot organizer was associated with the thickest cuticle layer, called the cuticle focal spot. Transmission election microscopy (TEM) revealed that the intervening space (IVS) between the cuticle layer and the cellular apical end was wide at 6 h but narrow at 12 h postpupation. The spatial relationship between cuticle thickness and IVS width was indicative of mechanical buckling of the region adjacent to the cuticle focal spot. Live in vivo imaging revealed that the IVS at and near the eyespot organizer trapped orange fluorescent protein (OFP) injected into hemolymph. Dynamic distortions of the pupal wing epidermis and cuticular surface were detected in live individuals over time. These results suggest that physical distortions of the wing tissue induced by differential cuticle synthesis and subsequent buckling may function as mechanical morphogenic signals from eyespot organizers. Full article

Research on mule and donkey muscle composition remains limited despite their global importance as working equids. The objective of this study is to identify Mammoth donkey jacks with higher percentage of fast twitch fibers for racing mule production. A total of 33 animals were biopsied; however, only 12 samples were suitable for analysis, including racing mules (n = 7) and male Mammoth donkeys (n = 5). Animals were sedated with detomidine (10 µg/kg body weight) and butorphanol (20 µg/kg body weight). Middle gluteal muscle biopsies were collected using a 6 mm Bergström biopsy needle at a site located 20 cm dorsocaudal to the tuber coxae at a 45° angle to the base of the tail. Collection depth was 7.5 cm in adult mules and 5 cm in donkeys. Samples were prepared aseptically, anesthetized subcutaneously with lidocaine hydrochloride, and frozen in liquid nitrogen. Histochemical analysis included myosin adenosine triphosphatase (ATPase) staining at pH 9.5, 4.6, and 4.3. Fibers were classified as Type I, Type IIA, or Type IIB, and CSA measurements were obtained using NIH ImageJ software. Statistical analysis included group contrasts, summarized as mean ± SD with 95% confidence intervals, while Bayesian ANOVA outputs were presented as exploratory evidence summaries. Type IIA fibers were greater in mules (47.84 ± 7.30%) than donkeys (38.47 ± 4.48%). Results suggest that differences in equid muscle architecture may be associated with variation in Type IIA fiber composition related to work or use. Full article

Palmitic acid (PA), the most abundant saturated fatty acid in the human body, is implicated in lipotoxicity under hyperlipidemic conditions, with potential consequences for bone metabolism. To investigate its impact on developing bone tissue, this study used an ex vivo organotypic embryonic chick femur model, exposing femora to control (0 µM), low (50 µM), and high (200 µM) PA concentrations. A multimodal approach, integrating microtomographic, histochemical, ultrastructural, and gene expression analyses, was used to assess tissue architecture, matrix composition, mineralization, and molecular adaptations. PA exposure significantly reduced longitudinal femoral growth, as evidenced by decreased femoral length and tissue volume. Gene expression analysis revealed reduced expression of selected osteogenic differentiation-related markers, including RUNX2, BMP2, and SPP1. However, COL1A2 expression was upregulated, correlating with increased collagenous matrix deposition and enhanced mineralization in PA-treated groups. Alcian blue staining further suggested reduced proteoglycan-rich cartilage matrix, particularly at 200 µM PA. Additionally, PA modulated the expression of both pro-inflammatory and anti-inflammatory mediators, along with increased autophagy-associated responses, as suggested by the upregulation of autophagy-related genes and the presence of autophagosomes and autolysosomes. These findings indicate that PA does not simply exert a deleterious effect on bone tissue but rather redirects the developmental trajectory of the organotypic femur by reducing longitudinal growth while promoting collagen-rich matrix maturation and mineral compaction. This response may involve altered cartilage-associated endochondral processes, fatty-acid-driven metabolic adaptation, osteoblast/osteocyte maturation, and autophagy-associated matrix processing under lipid-enriched conditions. Full article

Background/Objectives: Mucopolysaccharidosis type III (MPS III, Sanfilippo syndrome) is an autosomal recessive lysosomal storage disorder caused by deficiencies in enzymes required for heparan sulfate degradation. While primarily recognized for its devastating neurodegenerative course, the systemic extent of glycosaminoglycan (GAG) accumulation remains under-characterized. This study aims to provide a detailed multisystemic pathological mapping of MPS III to challenge the traditional “brain-only” disease paradigm and highlight the clinical relevance of extracerebral involvement. Methods: We present a comprehensive clinicopathological analysis of a 15-year-old female patient with a history of profound neuropsychomotor delay, refractory epilepsy, and spastic tetraplegia. Following her death due to terminal bronchopneumonia during palliative care, a complete forensic and pathological autopsy was conducted. Tissue samples from all major organ systems were processed using routine Hematoxylin–Eosin (HE) staining, immunohistochemical staining for CD68, and specialized histochemical stains to identify intracellular storage products. Results: Macroscopic evaluation revealed significant diffuse cerebral atrophy, meningoencephalic edema, cardiac valvulopathy with compensatory myocardial remodeling, and hepatosplenomegaly. Furthermore, erosive gastrointestinal lesions and degenerative renal changes were identified. Histopathological examination confirmed widespread cytoplasmic vacuolization across diverse cell populations, including neurons, hepatocytes, renal tubular cells, and the reticuloendothelial system. These findings demonstrate that GAG deposition is a generalized process affecting nearly every parenchymal structure. Conclusions: Although neurological decline dominates the clinical phenotype, our findings underscore that MPS III is a true systemic storage disorder. Significant involvement of the cardiovascular and visceral systems contributes to the disease’s complexity and mortality. This case reinforces the critical diagnostic value of a comprehensive autopsy in delineating the full morphological spectrum of Sanfilippo syndrome, providing essential insights for multidisciplinary management. Full article

Tracheoid idioblasts in Nepenthes are anatomically specialized cells that differ distinctly from surrounding tissues in morphology, wall structure, and staining properties. Their presence has been documented in both vegetative organs, such as roots and stems, and in highly modified carnivorous leaves that form pitchers. We tested the hypothesis that if tracheoid idioblasts function to reinforce the mechanical strength of Nepenthes pitchers or to protect them from animal damage, they exhibit a secondary cell wall composition comparable to that of sclerenchyma or xylem cells, particularly with respect to its lignin and hemicellulose components. We assessed the localization of cell wall components in gland cell walls using histochemical tests, immunolabeling, and confocal microscopy. Light and scanning electron microscopy were used to reveal the tracheoid idioblast structure. Two species were examined: Nepenthes albomarginata T.Lobb ex Lindl. and Nepenthes bicalcarata Hook. f. In both species, giant tracheoid idioblasts with helical bands of secondary wall material were found throughout the pitchers. Negative phloroglucinol, fuchsin, and safranin staining test results demonstrated the absence of lignins in the tracheoid idioblast secondary cell walls. A histochemical test showed that the wall thickenings of the tracheoid idioblasts contained polysaccharides and cellulose and were rich in unsubstituted or low-substituted xylans, resembling the secondary cell walls of sclerenchyma and xylem cells. Our results suggest that tracheoid idioblasts with a helical secondary wall that is rich in xylans but not lignified most likely function as elastic reinforcing elements that increase the mechanical integrity of the organ while maintaining its flexibility and ability to undergo reversible deformation. Furthermore, tracheoid idioblasts may provide defense against herbivory. Full article

Phosphorus deficiency in the environment induces phosphate (Pi) starvation responses of plants, in which the phosphate transporter is one of the most critical functional genes in this response mechanism. As a prevalent green manure crop in China, Vicia villosa plays a critical role in sustainable agricultural systems, and the expression of its phosphate transporter gene (VvPHT1) is modulated by soil phosphorus availability, highlighting its key adaptive function in nutrient acquisition and utilization under low-Pi conditions. Functional studies of this gene and its promoter contribute to exploring the molecular mechanisms of the tolerance of green manure crops to low phosphorus stress and to improving phosphorus-efficient V. villosa varieties. In this study, analysis of the VvPHT1 promoter sequence revealed a 1524 bp region containing multiple root-specific cis-regulatory elements, including five NODCON2GM, one NODCON1GM, six OSE2ROOTNODULE, one OSE1ROOTNODULE, and fifteen ROOTMOTIFTAPOX1 motifs. Histochemical GUS staining of transgenic Arabidopsis (Arabidopsis thaliana (L.) Heynh.) showed that the VvPHT1 promoter directed root-specific expression of the GUS reporter gene. A fusion expression vector pCAMBIA1300-VvPHT1--GFP was constructed and transformed into tobacco (Nicotiana tabacum L.) cells for subcellular localization analysis, indicating that the protein encoded by VvPHT1 was localized to the plasma membrane. To quantify its expression, VvPHT1 transcript levels in VvPHT1-overexpressing Arabidopsis (OEPHT1) lines were analyzed by quantitative real-time PCR (qRT-PCR) under different phosphorus supply conditions. The results demonstrated that under low-Pi conditions, the expression of VvPHT1 was significantly upregulated in the OEPHT1 lines compared to those of normal-Pi conditions. Furthermore, under low-Pi treatment, the OEPHT1 lines showed significantly increased fresh weight, primary root length, phosphorus content, and chlorophyll content compared to the wild-type Arabidopsis (WT), while no such differences were observed under normal-Pi conditions. In conclusion, the VvPHT1 promoter exhibits root-specific activity, and the VvPHT1 gene encodes a plasma-membrane-localized phosphate transporter that is strongly induced by phosphorus deficiency. Its overexpression enhances phosphorus uptake and plant growth under low-Pi conditions, suggesting that VvPHT1 likely functions as a high-affinity phosphate transporter involved in the adaptation to phosphorus starvation. Full article

Excessive nitrogen (N) fertilization is widely used to increase rice yield, but it often leads to lodging by weakening culm strength. This study aimed to elucidate the structural and molecular mechanisms underlying nitrogen-induced changes in culm lodging resistance in rice. Field and pot experiments with two nitrogen levels were conducted using a randomized design with three biological replicates to evaluate the effects of high nitrogen application on culm mechanical properties, secondary cell wall development, and associated metabolic pathways. Mechanical measurements and microscopic analysis revealed that high nitrogen significantly reduced culm rigidity and impaired sclerenchyma development. To investigate the underlying mechanisms, integrated transcriptomic and proteomic analyses were performed on developing internodes. Differentially expressed genes and proteins were predominantly enriched in carbohydrate metabolism and phenylpropanoid biosynthesis pathways. Notably, key enzymes involved in lignin biosynthesis were consistently downregulated at the protein level under high-nitrogen conditions. In contrast, genes and proteins related to cellulose and hemicellulose biosynthesis exhibited transient inhibition at early stages followed by recovery or upregulation at later stages. Consistent with these findings, histochemical staining and quantitative assays demonstrated a significant reduction (14–16%) in lignin content in the fourth internode, whereas cellulose content showed no substantial change. Furthermore, lignin biosynthetic genes (OsCAD2, Os4CL3, and OsCOMT) were persistently suppressed during critical stages of secondary wall formation, while cellulose synthase genes (OsCESA4, OsCESA7, and OsCESA9) displayed more variable and less sustained expression patterns. Collectively, these results demonstrate that excessive nitrogen application weakens rice culms primarily by inhibiting lignin accumulation rather than cellulose deposition. The preferential suppression of the phenylpropanoid pathway and disruption of secondary cell wall formation provide a mechanistic basis for nitrogen-induced lodging susceptibility in rice. Full article

Glandular trichomes (GTs) are epidermal outgrowths that function as “natural cell factories” for the synthesis of specialized metabolites. Beyond their traditional understanding, GTs on cucumber fruits can form an undesirable trait known as bloom, which negatively affects market value. However, the secretory process, metabolite profiles, and genetic regulation underlying GT development in cucumber remain largely unclear. In this study, we employed scanning electron microscopy (SEM), transmission electron microscopy (TEM), histochemical staining, multi-omics analyses, and liquid chromatography–mass spectrometry (LC-MS) to systematically investigate GT development. The secretory process was classified into four distinct stages via SEM observations: morphogenesis, active metabolism, head sunken, and metabolite release. TEM revealed progressive ultrastructural changes, including increased organelle abundance and expansion of the periplasmic space, which facilitate metabolite transport and release. This process occurs through an autonomous mechanism involving osmiophilic substances and eventual cell rupture. LC-MS analysis identified 744 metabolites belonging to 11 classes, with phenylpropanoids/polyketides—particularly flavonoids—being the most abundant. While metabolite classes are conserved between European greenhouse and North China ecotypes, specific metabolite contents vary significantly. Multi-transcriptome analysis identified 60 candidate genes associated with GT development. Among these, CsaV4_3G003418 was functionally validated through virus-induced gene silencing (VIGS) to be involved in early GT development. Collectively, this work elucidates the secretory mechanism and metabolic characteristics of cucumber GTs, providing a foundation for future functional studies and biotechnological applications of secondary metabolites. Full article

Sparassis latifolia is an edible and medicinal mushroom with significant economic value, now commercially cultivated on a large scale in China. However, current cultivars face challenges, including an extended mycelial growth period and unstable fruiting body yields. Advances in molecular breeding and functional genomics for this species are hindered by the absence of a reliable genetic transformation system. In this study, we first determined that S. latifolia is highly sensitive to carboxin and hygromycin, two selective agents commonly used in fungal genetics. We subsequently constructed a novel binary vector, pCbxHyg, harboring a carboxin resistance cassette driven by its native Pleurotus eryngii promoter and a hygromycin resistance cassette under the control of the P. eryngii Glycerol 3-phosphate dehydrogenase (GPD) gene promoter. Initial transformation attempts using Agrobacterium-mediated transformation of liquid-cultured mycelial pellets were unsuccessful. During microscopic examination, we discovered that S. latifolia mycelia produce abundant asexual chlamydospores. Using these chlamydospores as recipient material, we efficiently and reproducibly obtained transformants with the pCbxHyg vector under both carboxin and hygromycin selection. This method highlights the advantage of using asexual spores of Basidiomycetes as recipients for genetic transformation. PCR analysis confirmed the stable integration of the exogenous resistance genes into the fungal genome. The functionality of the system was further validated by transforming chlamydospores with a vector carrying a β-glucuronidase (GUS) reporter gene, whose expression was confirmed via histochemical staining of the resulting transformant mycelia. This work establishes the first successful Agrobacterium-mediated genetic transformation system for S. latifolia, providing a foundational platform for future gene function studies and molecular breeding efforts. Full article

Plant cell walls provide structural integrity and defense against biotic and abiotic stresses. In rice (Oryza sativa), xylan is the major hemicellulose, and β-xylosidase hydrolyzes xylan by removing xylose residues from non-reducing ends. We analyzed a transgenic rice line (OsXylGH3-1-FOX) that constitutively overexpresses a GH3-family β-xylosidase (Os03g0749100) under the maize ubiquitin promoter. Following inoculation with M. oryzae, OsXylGH3-1-FOX leaves exhibited increased lesion numbers and disease indices, indicating reduced resistance, whereas leaf sheaths showed fewer fungal penetrations, suggesting enhanced resistance. To investigate these organ-specific responses, we quantified cell wall components. In leaves, xylose and arabinose decreased by ~33%, and galacturonic acid (pectin) by ~50%. In leaf sheaths, xylose and arabinose were unchanged, while galacturonic acid and cellulose increased by ~50% and ~70%, respectively. Histochemical staining confirmed reduced pectin in leaves and stronger, organized cellulose and pectin in leaf sheaths. These findings suggest that decreased pectin weakens cell adhesion, facilitating pathogen ingress in leaves, whereas increased pectin and cellulose reinforce wall integrity in leaf sheaths. Thus, pectin and cellulose abundance strongly correlate with organ-specific blast resistance, while hemicellulose plays a secondary role. Full article