Periodic acid-Schiff Research Articles

AMPK Regulates M1 Macrophage Polarization through the JAK2/STAT3 Signaling Pathway to Attenuate Airway Inflammation in Obesity-Related Asthma.

Abstract-Obesity-related asthma is primarily characterized by nonallergic inflammation, with pathogenesis involving oxidative stress, metabolic imbalance, and immunoinflammatory mechanisms. M1 macrophages, which predominantly secrete pro-inflammatory factors, mediate insulin resistance and systemic metabolic inflammation in obese individuals. Concurrently, adenosine monophosphate-activated protein kinase (AMPK) serves as a critical regulator of intracellular energy metabolism and is closely associated with macrophage activation. However, their specific roles and associated mechanisms in obesity-related asthma remain to be explored. In this study, we investigated the macrophage polarization status and potential interventional mechanisms through obesity-related asthmatic models and lipopolysaccharide (LPS) -treated RAW264.7 cell with a comprehensive series of evaluations, including HE, PAS and Masson staining of lung histopathology, immunohistochemical staining, immunofluorescence technology, qRT-PCR, Western Blot, and ELISA inflammatory factor analysis. The results revealed M1 macrophage polarization in obesity-related asthmatic lung tissue alongside downregulation of AMPK expression. Under LPS stimulation, exogenous AMPK activation attenuated M1 macrophage polarization via the Janus kinase 2/ signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway. Additionally, in obesity-related asthmatic mice, AMPK activation was found to alleviate airway inflammation by regulating M1 macrophage polarization, the mechanism closely associated with the JAK2/STAT3 pathway. These findings not only advance our understanding of macrophage polarization in obesity-related asthma, but also provide new therapeutic targets for its treatment.

Heyingwuzi formulation alleviates diabetic retinopathy by promoting mitophagy via the HIF-1α/BNIP3/NIX axis

BACKGROUND Diabetic retinopathy (DR) is the primary cause of visual problems in patients with diabetes. The Heyingwuzi formulation (HYWZF) is effective against DR. AIM To determine the HYWZF prevention mechanisms, especially those underlying mitophagy. METHODS Human retinal capillary endothelial cells (HRCECs) were treated with high glucose (hg), HYWZF serum, PX-478, or Mdivi-1 in vitro . Then, cell counting kit-8, transwell, and tube formation assays were used to evaluate HRCEC proliferation, invasion, and tube formation, respectively. Transmission electron microscopy was used to assess mitochondrial morphology, and Western blotting was used to determine the protein levels. Flow cytometry was used to assess cell apoptosis, reactive oxygen species (ROS) production, and mitochondrial membrane potential. Moreover, C57BL/6 mice were established in vivo using streptozotocin and treated with HYWZF for four weeks. Blood glucose levels and body weight were monitored continuously. Changes in retinal characteristics were evaluated using hematoxylin and eosin, tar violet, and periodic acid-Schiff staining. Protein levels in retinal tissues were determined via Western blotting, immunohistochemistry, and immunostaining. RESULTS HYWZF inhibited excessive ROS production, apoptosis, tube formation, and invasion in hg-induced HRCECs via mitochondrial autophagy in vitro . It increased the mRNA expression levels of BCL2-interacting protein 3 (BNIP3 ), FUN14 domain-containing 1, BNIP3-like (BNIP3L , also known as NIX ), PARKIN , PTEN-induced kinase 1, and hypoxia-inducible factor (HIF )-1α . Moreover, it downregulated the protein levels of vascular endothelial cell growth factor and increased the light chain 3-II/I ratio. However, PX-478 and Mdivi-1 reversed these effects. Additionally, PX-478 and Mdivi-1 rescued the effects of HYWZF by decreasing oxidative stress and apoptosis and increasing mitophagy. HYWZF intervention improved the symptoms of diabetes, tissue damage, number of acellular capillaries, and oxidative stress in vivo . Furthermore, in vivo experiments confirmed the results of in vitro experiments. CONCLUSION HYWZF alleviated DR and associated damage by promoting mitophagy via the HIF-1α/BNIP3/NIX axis.

Transcriptome and metabolome analysis of the developmental changes in Cynanchum thesioides anther

Cynanchum thesioides, a xerophytic species utilized both as a medicinal herb and a food source, plays a significant role in arid and desert ecosystem management. Its inflorescence is an umbellate cyme, each carrying nearly a thousand flowers; however, its fruiting rate remains remarkably low. The normal development of the anther is a necessary prerequisite for plants to produce seeds. However, our understanding of the anther development process in Cynanchum thesioides remains limited. To better understand the pollen development process in Cynanchum thesioides, the stages of pollen development were determined through paraffin sectioning, and observations were made on the distribution characteristics of polysaccharides and lipid droplets in the pollen development of Cynanchum thesioides using Periodic Acid-Schiff stain (PAS) and 0.5% Sudan Black B tissue staining. Concurrently, the gene expression patterns and metabolite profiles were delineated across various developmental stages of Cynanchum thesioides anthers (T1: microspore stage, T2: tetrad stage, T3: mononuclear stage, and T4: maturation stage). The findings revealed that Cynanchum thesioides pollen is in an aggregate form. Polysaccharides gradually accumulate during maturation and lipid droplets form a surrounding membrane, thereby preventing pollen dispersion. Furthermore, transcriptomic and metabolomic analyses across distinct developmental phases uncovered a plethora of differentially expressed genes and metabolites associated with the flavonoid biosynthesis pathway. Flavonoid levels exhibited dynamic changes concurrent with anther development, aligning with the gene regulatory patterns of the corresponding biosynthetic pathways. The study identified 63 differentially accumulated flavonoid compounds and 21 differentially expressed genes associated with flavonoid biosynthesis. Weighted gene co-expression network analysis revealed six MYB and ten bHLH transcription factors as key candidates involved in flavonoid biosynthesis, with CtbHLH (Cluster-6587.1050) and CtMYB (Cluster-6587.31743) specifically regulating structural genes within the pathway. These findings underscore the pivotal role of flavonoid biosynthesis in anther development of Cynanchum thesioides. In conclusion, this research offers a comprehensive insight into the anther development process in Cynanchum thesioides.

Effect of ‘Procumbenase’ a serine protease from Tridax procumbens aqueous extract on wound healing: A scar free healing of full thickness wounds

Wound healing involves several cellular and molecular pathways. Tridax procumbens activates genetic pathways with antibacterial, antioxidant, anticancer, and anti-inflammatory properties, aiding wound healing. This study purified Procumbenase, a serine protease from T. procumbens extract, using gel filtration (Sephadex G-75) and ion exchange (CM-Sephadex C-50) chromatography. Characterization involved analyses of protease activity, RP-HPLC, SDS-PAGE, gelatin zymogram, PAS staining, mass spectrometry, and circular dichroism. Optimal pH and temperature were determined. Protease type was identified using inhibitors. Wound-healing potential was evaluated through tensile strength, wound models, hydroxyproline estimation, and NIH 3T3 cell scratch analysis. In incision wound rat models, Procumbenase increased tensile strength on day 14 more than saline and Povidone‑iodine. It increased wound contraction by 89 % after 10 days in excision wound models, attaining full contraction by day 15 and closure by day 21. Scarless wound healing was enhanced by 18 days of epithelialization against 22 and 21 days for saline and povidone‑iodine. Procumbenase increased hydroxyproline concentration 2.53-fold (59.93 ± 2.89 mg/g) compared to saline (23.67 ± 1.86 mg/g). In NIH 3 T3 cell scratch assay, Procumbenase increased migration by 60.93 % (50 μg) and 60.57 % (150 μg) after 48 h. Thus, Procumbenase is the primary bioactive molecule in T. procumbens, demonstrates scar-free wound healing properties.

Neuronal ceroid lipofuscinosis in a Schapendoes dog is caused by a missense variant in CLN6.

Neuronal ceroid lipofuscinosis (NCL) is a group of neurodegenerative disorders that occur in humans, dogs, and several other species. NCL is characterised clinically by progressive deterioration of cognitive and motor function, epileptic seizures, and visual impairment. Most forms present early in life and eventually lead to premature death. Typical pathological changes include neuronal accumulation of autofluorescent, periodic acid-Schiff- and Sudan black B-positive lipopigments, as well as marked loss of neurons in the central nervous system. Here, we describe a 19-month-old Schapendoes dog, where clinical signs were indicative of lysosomal storage disease, which was corroborated by pathological findings consistent with NCL. Whole genome sequencing of the affected dog and both parents, followed by variant calling and visual inspection of known NCL genes, identified a missense variant in CLN6 (c.386T>C). The variant is located in a highly conserved region of the gene and predicted to be harmful, which supports a causal relationship. The identification of this novel CLN6 variant enables pre-breeding DNA-testing to prevent future cases of NCL6 in the Schapendoes breed, and presents a potential natural model for NCL6 in humans.

Vitamin D is involved in the effects of the intestinal flora and its related metabolite TMAO on perirenal fat and kidneys in mice with DKD

BackgroundVitamin D was shown to directly exert a protective effect on diabetic kidney disease (DKD) in our previous study. However, whether it has an effect on perirenal adipose tissue (PRAT) or the intestinal flora and its metabolites (trimethylamine N-oxide, TMAO) is unclear.MethodsDKD mice were received different concentrations of 1,25-(OH)2D3 for 2 weeks. Serum TNF-α levels and TMAO levels were detected. 16S rRNA sequencing was used to analyze gut microbiota. qPCR was used to detect the expression of TLR4, NF-Κb, PGC1α, and UCP-1 in kidney and adipose tissue. Histological changes in kidney and perirenal adipose tissue were observed using HE, PAS, Masson and oil red staining. Immunofluorescence and immunohistochemistry were used to detect the expression of VDR, PGC1α, podocin, and UCP-1 in kidney and adipose tissue. Electron microscopy was used to observe the pathological changes in the kidney. VDR knockout mice were constructed to observe the changes in the gut and adipose tissue, and immunofluorescence and immunohistochemistry were used to detect the expression of UCP-1 and collagen IV in the kidney.Results1,25-(OH)2D3 could improve the dysbiosis of the intestinal flora of mice with DKD, increase the abundance of beneficial bacteria, decrease the abundance of harmful bacteria, reduce the pathological changes in the kidney, reduce fat infiltration, and downregulate the expression of TLR4 and NF-κB in kidneys. The serum TMAO concentration in mice with DKD was significantly higher than that of the control group, and was significantly positively correlated with the urine ACR. In addition, vitamin D stimulated the expression of the surface markers PGC1α, UCP-1 and VDR in the PRAT in DKD mice, and TMAO downregulated the expression of PRAT and renal VDR.ConclusionsThe protective effect of 1,25-(OH)2D3 in DKD mice may affect the intestinal flora and its related metabolite TMAO on perirenal fat and kidneys.

Patient-derived parotid gland pleomorphic adenoma organoid model and its preliminary histological and functional characterization

Objective: To establish patient-derived organoid models of pleomorphic adenomas (PA) of the parotid gland and preliminarily characterize their histology, related biomarkers and functions. Methods: Fresh tumor tissue specimens were collected from surgical procedures of Oral and Maxillofacial Department. The harvested tissues were processed and cultured in a head and neck tumor organoid culture system to establish organoid models from parotid gland pleomorphic adenomas. The in vitro growth of PA organoids was recorded by light microscopy. The successfully established organoids were passaged and cryopreserved, and the cryopreserved PA organoids were revived and re-cultured to observe their viability and organoid regeneration ability. Histological characterization, as well as characterization and detection of related markers and functional proteins, were performed on the organoids, comparing them with the patient-derived tissues. Results: The constructed organoid model of pleomorphic adenoma exhibited a dense and compact three-dimensional spherical structure. Hematoxylin and eosin staining indicated morphological similarities between the organoid and its tissue of origin. Immunohistochemistry showed positive cytoplasmic staining for Calponin, cytokeratin 7, and epithelial membrane antigen in both the organoid and the source tumor tissue, suggesting consistent histopathological characteristics between the organoid and its tissue of origin. Periodic acid-Schiff staining of the organoid showed positive staining for glycogen, with positive staining located in the interior and periphery of the organoid, indicating that the organoid possessed secretory functions like the salivary gland. Conclusions: This study successfully constructed organoids of pleomorphic adenoma derived from patient samples. This model faithfully replicates the tissue morphology and biomarkers of the source tissue and exhibits biological functions associated with mucus secretion. It serves as a valuable in vitro model for studying the development and progression of salivary gland tumors.

Intraoral Salivary Gland Cystadenomas: A Case Series Study and Literature Review.

Salivary gland cystadenoma (SGCA) is a rare benign tumor that predominantly occurs in the parotid gland. SGCAs affecting the minor salivary glands are uncommon and often resemble, clinically and histopathologically, other salivary gland lesions. This study aimed to describe a series of four cases of SGCA affecting intraoral sites and performed a literature review of well-reported SGCA published in the English-language literature. SGCA cases included in this series were diagnosed in the buccal mucosa, lip, and hard palate of female patients aged between 19 and 78years. All cases underwent excisional biopsy and were histologically characterized by a multicystic growth with variable degrees of capsule formation and were lined by several types of epithelium, including some cell types that are infrequently reported in SGCA. In some cases, a small collection of lymphocytes was observed adjacent to cystic formations. All SGCA were positive for periodic acid-Schiff, and immunohistochemical reactions were positive for CK7 and p63. The follow-up time ranged widely from 3 to 53months, and to date, no recurrence has been observed. The literature review revealed a total of 33 published studies accounting for 55 SGCA cases.

Unveiling the Uncommon: Gastrointestinal Basidiobolomycosis of the Colon and its Distinctive Splendore-Hoeppli Phenomenon

Background Basidiobolomycosis is a rare fungal infection caused by Basidiobolus ranarum. Case Presentation A 53-year-old man from Saudi Arabia with a known history of diverticulosis presented with severe abdominal pain and diarrhea. A CT scan revealed circumferential wall thickening of the descending and sigmoid colon with surrounding fat stranding, suggesting a diagnosis of complicated diverticulitis. Additional thick fluid was observed around the affected area. Surgical excision was pursued. A gross examination of two received large bowel segments disclosed marked ulcerated mucosa and wall thickening with exudate-covered serosal surfaces and adhesions. Microscopic examination unveiled significant infiltration by eosinophils, polymorphonuclear leukocytes, and granulomatous inflammation. Thin-walled, broad fungal hyphae of Basidiobolus, surrounded by eosinophilic material, were identified. Granulomas displayed abundant multinucleated giant cells and palisading histiocytes around central necrosis or abscess formation. Thin-walled, broad fungal hyphae of Basidiobolus, with sparse septations, are surrounded by a radiating, intensely eosinophilic cuff (Splendore-Hoeppli phenomenon). These hyphae, visible with hematoxylin and eosin staining, were further highlighted with periodic acid-Schiff and Gomori methenamine silver staining. Discussion Basidiobolomycosis may mimic neoplastic lesions. Histologically, the characteristic features include broad, thin-walled septate hyphae surrounded by eosinophilic material, a finding that is accentuated by the Splendore-Hoeppli phenomenon. Microscopic examination, along with special stains such as periodic acid-Schiff (PAS) and Gomori methenamine silver, is essential for accurate diagnosis. Conclusion Prompt recognition and appropriate antifungal therapy are vital for favorable patient outcomes. This report highlights the distinctive features of Basidiobolomycosis to raise awareness and understanding of this infrequent yet clinically significant fungal infection.

Rehmapicrogenin attenuates lipopolysaccharide-induced podocyte injury and kidney dysfunctions by regulating nuclear factor E2-related factor 2/antioxidant response element signalling.

Apoptosis and oxidative stress in kidneys are critical players in acute kidney injury (AKI). Rehmapicrogenin, a monomeric compound extracted from Rehmanniae radix, has been found to possess nitric oxide inhibitory and anti-inflammatory activities. Thus, this study aimed to investigate the roles and mechanisms of rehmapicrogenin in AKI. Lipopolysaccharide (LPS) was used to induce AKI-like conditions. Cell survival conditions were detected by cell counting kit-8 assays and flow cytometry. Several renal function markers including blood urea nitrogen, proteinuria, creatinine, and albumin were measured. Apoptosis and reactive oxygen species (ROS) production were examined by TUNEL and dihydroethidium staining, respectively. Haematoxylin-eosin staining and periodic acid-Schiff staining were conducted to assess histopathological changes. Gene expression was evaluated by western blotting, commercially available kits and immunofluorescence staining. For in vitro analysis, rehmapicrogenin inhibited the LPS-induced podocyte apoptosis by activating the Nrf2/ARE pathway. For in vivo analysis, rehmapicrogenin improved renal functions in LPS-induced mice. Additionally, rehmapicrogenin suppressed LPS-induced podocyte apoptosis and oxidative stress in kidney tissues. Mechanistically, rehmapicrogenin activated the Nrf2/ARE pathway in LPS-induced mice. Rehmapicrogenin relieves the podocyte injury and renal dysfunctions through activating the Nrf2/ARE pathway to inhibit apoptosis and oxidative stress.

Histological, biochemical and immunohistochemical assessments of Roundup®, atrazine, and 2,4-D mixtures on tissue architecture, body fluid conditions, nitrotyrosine protein and Na+/K+-ATPase expressions in the American oyster, Crassostera virginica

Pesticides are widely used to control weeds and pests in agricultural settings but harm non-target aquatic organisms. In this study, our objective was to evaluate the effect of short-term exposure (one week) to environmentally relevant concentrations of pesticides mixture (low concentration: 0.4 μg/l atrazine, 0.5 μg/l Roundup®, and 0.5 μg/l 2,4-D; high concentration: 0.8 μg/l atrazine, 1 μg/l Roundup®, and 1 μg/l 2,4-D) on tissue architecture, body fluid conditions, and 3-nitrotyrosine protein (NTP) and Na+/K+-ATPase, expressions in tissues of American oyster (Crassostrea virginica) under controlled laboratory conditions. Histological analysis demonstrated the atrophy in the gills and digestive glands of oysters exposed to pesticides mixture. Periodic acid-Schiff (PAS) staining showed the number of hemocytes in connective tissue increased in low- and high-concentration pesticides exposure groups. However, pesticides treatment significantly (P < 0.05) decreased the amount of mucous secretion in the gills and digestive glands of oysters. The extrapallial fluid (i.e., body fluid) protein concentrations and glucose levels were dropped significantly (P < 0.05) in oysters exposed to high-concentration pesticides exposure groups. Moreover, immunohistochemical analysis showed significant upregulations of NTP and Na+/K+-ATPase expressions in the gills and digestive glands in pesticides exposure groups. Our results suggest that exposure to environmentally relevant pesticides mixture causes morphological changes in tissues and alters body fluid conditions and NTP and Na+/K+-ATPase expressions in tissues, which may lead to impaired physiological functions in oysters.

Detection of carbohydrate distribution in the testis of Aceh bull using periodic acid Schiff staining

Abstract Spermatogenesis that occurred in the seminiferous tubule of the testis requires various substances, one of which is carbohydrates. Data regarding carbohydrates distribution in the testis of Aceh bull is currently not available. The objective of this study is to determine the neutral carbohydrates distribution in the seminiferous tubule and interstitial tissue of Aceh bull testis using histochemical periodic acid Schiff (PAS) staining. Five pairs adult testis of Aceh bull were use and fixed in neutral buffered formalin 10% (immersion fixation), and then processed into histological preparation to be stained using PAS staining. A positive reaction on the presence of neutral carbohydrate in the testis indicated by magenta color in the specific cells and other components of testis. According microscopic observation, neutral carbohydrates found in the spermatogonium, spermatocyte, round spermatid, elongated spermatid, basal membrane, myoid peritubular cell, and cytoplasm of Sertoli cell in seminiferous tubule. Furthermore, the specific neutral carbohydrates were observed in the round spermatid with various expression, indicated that carbohydrates (glycoprotein) have an important role for acrosome formation in the round, elongating, and elongated spermatid during spermiogenesis to produce spermatozoa. Additionally, neutral carbohydrates were also found in the interstitial tissue of testis, including Leydig cell, fibrocyte, blood vessels, connective tissue, and tunica albuginea of testis. In conclusion, the presence of neutral carbohydrate in the testis of Aceh bull indicates that this substance is required to maintain spermatogenesis and tissue structure of the testis.

Long-Term Follow-Up of Pediatric Excimer Laser-Assisted Penetrating Keratoplasty for Congenital Stromal Corneal Dystrophy.

The purpose of this study was to highlight characteristic clinical and microscopic findings and report the long-term follow-up of pediatric excimer laser-assisted penetrating keratoplasty (excimer-PKP) for congenital stromal corneal dystrophy (CSCD). A 2-year-old Greek child presented with CSCD at our department. Clinical examination showed bilateral flake-like whitish corneal opacities affecting the entire corneal stroma up to the limbus. Genetic testing identified a mutation of the decorin gene (c.962delA). The variant was not present in the parents and represented a de novo mutation. The uncorrected visual acuity was 20/100 in both eyes. Excimer-PKP (8.0/8.1 mm) was performed on the right eye at the age of 2.5 years and on the left eye at the age of 3 years. Postoperatively, alternating occlusion treatment was performed. The light microscopic examination demonstrated a disorganized extracellular matrix of the corneal stroma characterized by a prominent irregular arrangement of stromal collagen lamellae with large interlamellar clefts containing ground substance, highlighted by periodic acid-Schiff- and Alcian blue-positive reaction detecting acid mucopolysaccharides. Electron microscopy showed disorganization and caliber variation of collagen lamellae and thin filaments within an electron-lucent ground substance. The postoperative course was unremarkable. Both grafts remained completely clear 14 years postoperatively. Corneal tomography showed moderate regular astigmatism with normal corneal thickness. The corrected distance visual acuity was 20/25 in both eyes. Excimer-PKP for CSCD might be associated with excellent long-term results and a good prognosis, particularly when the primary surgery is performed at a very young age. However, this requires close postoperative follow-up examinations by an experienced pediatric ophthalmologist to avoid severe amblyopia.

Network and structural analysis of quail mucins with expression pattern of mucin 1 and mucin 4 in the intestines of the Iraqi common quail (Coturnix coturnix).

In avian and other species, mucins (MUCs) play a crucial role in the gastrointestinal tract (GIT), and constitute a large group of O-glycosylated glycoproteins, are glycoconjugate proteins. MUCs present in two forms: (1) membrane-attached on cell surfaces to repel external threats and (2) detachable, gel-forming proteins in the soluble form. In quail GIT, the specific types of MUCs that are expressed remain largely unknown. We investigated the expression of MUC1 and MUC4 MUCs in the GIT of Iraqi common quails and conducted network and structural analyses of all known MUC types across quail breeds. Histological and gene expression analyses of MUC1 and MUC4 were conducted using fresh small intestine and large intestine samples from 10 quails. Using the STRING Database, Chimera software, and PrankWeb-ligand binding site prediction tool, network and structural analyses of all reported types of quail MUCs were conducted. Most intestinal MUCs in quails were acidic, with few neutral MUCs detectable through Alcian blue and periodic acid-schiff stains. Acidic MUCs were more expressed in the duodenum, ileum, cecum, and colon, whereas neutral MUCs were more expressed in the jejunum. MUC1 and MUC4 messenger RNA expression was significantly higher in the jejunum and colon than in the duodenum and ileum. The analysis of the network revealed that MUC 1, 15, 16, and 24 formed homologous networks, while MUC 2, 4, 5, and 6 formed heterologous networks. Specific MUC combinations, including MUC5A-MUC6, MUC5A-MUC5B, and MUC5B-MUC6, show higher intermolecular hydrogen bond formation affinity. MUC15, MUC16, and MUC24 showed minimal interaction with other MUC types. Among the analyzed MUCs, MUC5B, and MUC6 had the highest probability for binding, while MUC2, MUC4, and MUC5A showed lower probabilities despite greater numbers of binding sites. This study's results offer significant insights into quails' MUCs' composition, expression, network interactions, and binding sites, advancing knowledge of MUC-related processes in gastrointestinal physiology and their potential connection to gastrointestinal diseases.

Protective effect of Albizia chinensis saponin on ethanol-induced gastric mucosal injury in rats focus on mucus and mucosal barriers

This study aims to explore the protective effect of Albizia chinensis saponin on ethanol-induced acute gastric ulcer in rats and elucidate its mechanisms. SD rats were deprived of water for 24 hours before the experiment. The control group and model group were administered water by gavage, and the positive drug group received rabeprazole sodium solution(40 mg·kg~(-1)) by gavage. The experimental groups were given different doses of Albizia chinensis saponin solution(3, 10, and 30 mg·kg~(-1)). After 30 minutes, the control group received 1.5 mL of water by gavage, while the other groups were administered an equal volume of 95% ethanol for modeling. After six hours, the rats were killed by cervical dislocation, and the stomachs were collected. The ulcer area was measured, and the ulcer index was calculated. Hematoxylin-eosin(HE) staining was performed to assess histopathological changes in gastric tissue. Periodic acid-Schiff(PAS) staining was used to evaluate the distribution of gastric mucosal surface mucus. Enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of phospholipids and aminohexose in the gastric mucosa. Western blot was performed to determine the expression levels of the bicarbonate transporter, matrix metalloproteinase, and tight junction-associated proteins in gastric tissue. Immunohistochemistry(IHC) staining was conducted to quantify the number of positive cells for secreted mucin and tight junction-associated proteins. The results showed that the gastric tissue surface of rats in the control group was smooth without ulceration, and the gastric ulcer index of rats in the model group was 35±11. Albizia chinensis saponin at doses of 3, 10, and 30 mg·kg~(-1) resulted in inhibition rates of gastric ulcer of 46%(P&lt;0.01), 85%(P&lt;0.001), and 100%(P&lt;0.001), respectively. Severe disruption of gastric mucosal structure and absence of the mucus layer were observed in the model group. Compared with the model group, the Albizia chinensis saponin group showed intact gastric mucosal surface mucus layer, significantly increased levels of phospholipids and aminohexose in the mucus, increased number of MUC5AC positive cells, and upregulated expression levels of the bicarbonate transporter SLC26A3 and CFTR. It also showed decreased phosphorylation of JNK and c-Jun, reduced expression levels of MMP-8, elevated expression of TIMP-1, and increased expression levels of Occludin and ZO-1. In conclusion, Albizia chinensis saponin enhances the function of the mucus-bicarbonate barrier by upregulating the content of MUC5AC, phospholipids, and aminohexose and increasing the expression levels of the bicarbonate transporter SLC26A3 and CFTR. Moreover, Albizia chinensis saponin exerts its protective effects on gastric ulcers by inhibiting the JNK signaling pathway to prevent excessive activation of MMP-8, thereby reducing the degradation of Occludin and ZO-1 and enhancing the mucosal barrier function. In summary, Albizia chinensis saponin exerts its anti-gastric ulcer effects by simultaneously enhancing the mucus barrier and the mucosal barrier.

The median nerve´s system of connective tissue distal to the pronator teres to the carpal tunnel

BackgroundConnective tissue serves a role beyond mere spatial filling. Furthermore, there is increasing evidence that connective tissue plays an important role in the pathogenesis of conditions such as carpal tunnel syndrome (CTS). According to our hypothesis, the median nerve (MN) is surrounded by a system of connective tissue distal to the pronator teres and extending up to, and including, the carpal tunnel. MethodsTo visualize the connective tissue surrounding the median nerve, we dissected the forearms of 15 body donors from pronator teres to the carpal tunnel, created plastination slices stained with Periodic Acid-Schiff (PAS), and injected ink into the seen spaces. We verified our findings with a segmentational analysis of radiological data of 10 healthy individuals. ResultsWe macroscopically describe the median nerve´s system of connective tissue (MC) distal to the pronator teres and up to and including the carpal tunnel. This system creates, connects, and separates spaces. At least from the pronator teres to the carpal tunnel it also creates subspaces from proximal to distal. For the MC, we established a mean cross-sectional area of 153.1 mm2 (SD=37.15) in the carpal tunnel. The median nerve consistently resides at the center of this MC, which further connects to flexor muscles of the forearm, and to the radius bone. In the carpal tunnel, the MC creates subspaces inside. There, it also acts as the outermost internal layer enveloping flexor tendons, and the MN. DiscussionThe term MC does not negate but orders the existence of other “connectives”, like subsynovial connective tissue, endo-, epi- or perineuria, epimysia, periostea, or peritendinea, to a hierarchy related to the median nerve. Diseases of the MN are common. Knowing the anatomy of the MC and how it relates to MN function may help clinicians recognize and understand conditions like CTS.

The effect of β-Glucan induced intestinal trained immunity against Trichinella spiralis infection

Parasitic helminth Trichinella spiralis (Ts) induce mixed Th1/Th2 response with predominant type 2 immune responses, with protective immunity mediated by interleukin (IL)-4, IL-5, and IL-13. β-Glucan (BG) has been shown to have the ability to induce trained immunity, confers non-specific protection from secondary infections. However, whether BG-induced trained immunity played a role in protective type 2 immunity against Ts infection is unclear. In this study, BG was administered five days before Ts infection to induce trained immunity. Our findings demonstrate that BG pretreatment effectively reduced the number of T. spiralis adults and muscle larvae, whereas inhibition of trained immunity abolished the effect of BG. Additionally, we observed a significant increase in goblet cells and mucus production as evidenced by Alcian blue periodic acid-Schiff staining. Furthermore, quantitative real-time PCR analysis revealed a significant upregulation of IL-4, IL-5, and IL-13 expression in response to BG. Conversely, the inhibitor of trained immunity reversed these effects, suggesting that BG-induced trained immunity confers strong protection against Ts infection. In conclusion, these findings suggest that BG-induced trained immunity may play a role in protection against infections caused by other helminths.

Smartphone-based machine learning model for real-time assessment of medical kidney biopsy

BackgroundKidney biopsy is the gold-standard for diagnosing medical renal diseases, but the accuracy of the diagnosis greatly depends on the quality of the biopsy specimen, particularly the amount of renal cortex obtained. Inadequate biopsies, characterized by insufficient cortex or predominant medulla, can lead to inconclusive or incorrect diagnoses, and repeat biopsy. Unfortunately, there has been a concerning increase in the rate of inadequate kidney biopsies, and not all medical centers have access to trained professionals who can assess biopsy adequacy in real time. In response to this challenge, we aimed to develop a machine learning model capable of assessing the percentage cortex of each biopsy pass using smartphone images of the kidney biopsy tissue at the time of biopsy. Methods747 kidney biopsy cores and corresponding smartphone macro images were collected from five unused deceased donor kidneys. Each core was imaged, formalin-fixed, sectioned, and stained with Periodic acid–Schiff (PAS) to determine cortex percentage. The fresh unfixed core images were captured using the macro camera on an iPhone 13 Pro. Two experienced renal pathologists independently reviewed the PAS-stained sections to determine the cortex percentage. For the purpose of this study, the biopsies with less than 30% cortex were labeled as inadequate, while those with 30% or more cortex were classified as adequate. The dataset was divided into training (n=643), validation (n=30), and test (n=74) sets. Preprocessing steps involved converting High-Efficiency Image Container iPhone format images to JPEG, normalization, and renal tissue segmentation using a U-Net deep learning model. Subsequently, a classification deep learning model was trained on the renal tissue region of interest and corresponding class label. ResultsThe deep learning model achieved an accuracy of 85% on the training data. On the independent test dataset, the model exhibited an accuracy of 81%. For inadequate samples in the test dataset, the model showed a sensitivity of 71%, suggesting its capability to identify cases with inadequate cortical representation. The area under the receiver-operating curve (AUC-ROC) on the test dataset was 0.80. ConclusionWe successfully developed and tested a machine learning model for classifying smartphone images of kidney biopsies as either adequate or inadequate, based on the amount of cortex determined by expert renal pathologists. The model's promising results suggest its potential as a smartphone application to assist real-time assessment of kidney biopsy tissue, particularly in settings with limited access to trained personnel. Further refinements and validations are warranted to optimize the model's performance.

Macroscopic, microscopic, and immunofluorescent characterization of the Greek tortoise (Testudo graeca graeca) oropharyngeal floor with concern to its feed adaptation as a herbivorous land reptile.

The current investigation focuses on gross anatomy, light, and scanning electron microscopy (SEM) of the Testudo graeca oropharyngeal floor, with particular reference to the immunofluorescence technique to examine its tongue. The T. graeca oropharyngeal floor showed many anatomical structures: the lower rhamphotheca, paralingual ridge, lower alveolar ridge, tongue, laryngeal mound, and glottis. The lower rhamphotheca appeared as a V-shaped jaw line with a highly serrated edge and a median tomium (beak). SEM observations of the lingual apex and the lingual body showed rectangular and conical filiform papillae with porous surfaces and taste pores. Meanwhile, the lingual root had two wings that carried papillae with different shapes: dagger-shaped, conical, bifurcated, and leaf-like papillae, and these papillae lacked taste pores. The laryngeal mound had openings for the laryngeal mucus gland and its secretions. Light microscopy findings showed mucous glands in the propria submucosa and near the mucosal surface of the lingual apex. The lingual root had lingual papillae and two hyaline cartilaginous skeletons between skeletal muscles, and the lingual papillae were elongated filiform, rectangular filiform papillae, and fungiform papillae. The lamina propria constituted the core of the lingual papillae and the mucous gland, they had a positive reaction with the periodic acid schiff (PAS) reagent. The apical surface of the fungiform papillae had taste pores. Under immunofluorescence, the vimentin was detected in taste bud cells, and synaptophysin reacted to the taste buds and nerve bundles. The current study of the Greek tortoise oropharyngeal floor investigated its herbivorous eating habits using its serrated lower rhamphotheca, a large tongue with differently shaped papillae, and numerous mucous glands. RESEARCH HIGHLIGHTS: The Greek tortoise (T. graeca graeca) oropharyngeal floor showed many anatomical structures: lower rhamphotheca, paralingual ridge, lower alveolar ridge, tongue, laryngeal mound, and glottis. SEM and light microscopy observations of the tongue revealed varied types and shapes of lingual papillae with a porous surface on the tongue apex (rectangular or conical filiform papillae), on the tongue body (filiform and fungiform papillae), and on the tongue root (dagger-shaped, conical, bifurcated, and leaf-like papillae). Light microscopy findings: the lamina propria constituted the core of the lingual papillae and had numerous mucous glands that had a slightly magenta-red color with PAS reagent. The apical surface of the fungiform papillae had taste pores. Vimentin and synaptophysin gave a reaction to the taste buds.

Fungi and bacteria occupy distinct spatial niches within carious dentin.

The role of bacteria in the etiology of dental caries is long established, while the role of fungi has only recently gained more attention. The microbial invasion of dentin in advanced caries especially merits additional research. We evaluated the fungal and bacterial community composition and spatial distribution within carious dentin. Amplicon 16S rRNA gene sequencing together with quantitative PCR was used to profile bacterial and fungal species in caries-free children (n = 43) and 4 stages of caries progression from children with severe early childhood caries (n = 32). Additionally, healthy (n = 10) and carious (n = 10) primary teeth were decalcified, sectioned, and stained with Grocott's methenamine silver, periodic acid Schiff (PAS) and calcofluor white (CW) for fungi. Immunolocalization was also performed using antibodies against fungal β-D-glucan, gram-positive bacterial lipoteichoic acid, gram-negative endotoxin, Streptococcus mutans, and Candida albicans. We also performed field emission scanning electron microscopy (FESEM) to visualize fungi and bacteria within carious dentinal tubules. Bacterial communities observed included a high abundance of S. mutans and the Veillonella parvula group, as expected. There was a higher ratio of fungi to bacteria in dentin-involved lesions compared to less severe lesions with frequent preponderance of C. albicans, C. dubliniensis, and in one case C. tropicalis. Grocott's silver, PAS, CW and immunohistochemistry (IHC) demonstrated the presence of fungi within carious dentinal tubules. Multiplex IHC revealed that fungi, gram-negative, and gram-positive bacteria primarily occupied separate dentinal tubules, with rare instances of colocalization. Similar findings were observed with multiplex immunofluorescence using anti-S. mutans and anti-C. albicans antibodies. Electron microscopy showed monomorphic bacterial and fungal biofilms within distinct dentin tubules. We demonstrate a previously unrecognized phenomenon in which fungi and bacteria occupy distinct spatial niches within carious dentin and seldom co-colonize. The potential significance of this phenomenon in caries progression warrants further exploration.