Blue Staining Research Articles

The lethal fifth cardiac chamber: a rare autopsy case report of left atrial appendage aneurysm and review of literature.

Congenital left atrial appendage (LAA) aneurysm is an extremely rare condition characterized by abnormal dilatation of the LAA. We report a fatal case of a left atrial appendage aneurysm (LAAA). The presented case was a 19-year-old male who died suddenly during vigorous exercise with no relevant medical or family history. On gross examination, we observed a semi-rounded bulge at the LAA with a diameter of approximately 5cm. Sectioning the bulge revealed an aneurysmal dilatation encompassing recent and huge lamellated mural thrombi filling its cavity. Though coronaries showed no stenosis, the mid-ventricular section of the heart showed dark and whitish areas at the lateral and posterior wall of the left ventricle, which were suspicious of myocardial infarctions. Histopathological examination depicted a thinned fibrosed wall of LAA, and the mural thrombi exhibited different stages of healing, which was confirmed using Martius Scarlet Blue stain. We identified myocardial infarctions at the suspected areas in the left ventricle, confirmed by the Masson Trichrome stain. The chronic myocardial ischemia was precipitated by the LAA aneurysmal compression of the left circumflex coronary artery as it passed along the atrioventricular groove. Seven other cases who died from LAAA complications since 1922 were reviewed. Early diagnosis and adequate management of LAAA allow survival without residual complications. Yet, this article highlighted the importance of screening for cardiac diseases in those subjected to vigorous exercise.

Hypothermia regulates mitophagy and apoptosis via PINK1/Parkin-VDAC 3 signaling pathway during oxygen-glucose deprivation/recovery injury

Post-cardiac arrest brain injury (PCABI), as the main cause of high mortality and long-term disability in patients, induces mitochondrial damage and cell apoptosis. Hypothermia is well-known as an effective neuroprotective therapy, but its underlying mechanisms deserve further exploration. Previous study has demonstrated that hypothermia provides neuroprotection via increasing PINK1/Parkin-mediated mitophagy. However, whether hypothermia can regulate both apoptosis and mitophagy through the PINK1/Parkin-VDAC3 signaling pathway or not. In this study, BV2 mouse microglial cells were cultured under oxygen-glucose deprivation for 6 h following reperfusion with or without hypothermia for 2–4 h. Cell viability was examined by trypan blue stain. Mitophagy was observed by transmission electron microscope. Mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (mPTP) opening were determined respectively by JC-1 staining and BBcellProbe M61 staining using a flow cytometer. Expression of mitophagy-related proteins (Cleaved PINK1, Parkin, SQSTM1/p62, Beclin-1, LC3B II/LC3B I), apoptosis-related proteins (Bcl-2, Cytochrome C, caspase-3, cleaved caspase3) and VDAC3 were assessed using western blot analysis and quantitative real-time PCR. The interaction between Parkin and VDAC3 was confirmed by immunofluorescence colocalization. The results showed that hypothermia alleviated MMP damage, inhibited mPTP opening, then decreased cell apoptosis and activated mitophagy at 2 h after temperature intervention, which might be mediated by the PINK1/Parkin-VDAC3 signaling pathway. Moreover, the effects of hypothermia were reduced or reversed at 4 h after temperature intervention. In conclusion, the potential mechanisms of hypothermia during oxygen-glucose deprivation/recovery could be summarized as follows:1) At 2 h after temperature intervention, hypothermia provided neuroprotective effects via promoting mitophagy and reducing apoptosis through activating the PINK1/Parkin-VDAC3 signaling pathway. 2) The curative effect of hypothermia was timeliness. At 4 h after temperature intervention, hypothermia aggravated apoptosis through inhibiting Parkin recruitment to mitochondria and aggravating the release of Cyt C through open mPTP.

Betaine enhances SCAPs chondrogenic differentiation and promotes cartilage repair in TMJOA through WDR81

BackgroundThe cartilage tissue regeneration mediated with mesenchymal stem cells (MSCs) is considered as a viable strategy for temporomandibular joint osteoarthritis (TMJOA). Betaine has been confirmed to modulate the multidirectional differentiation of MSCs, while its effect on chondrogenic differentiation of Stem Cells from the Apical Papilla (SCAPs) is unknown. Here, we explored the effects and underlying mechanisms of betaine on chondrogenic differentiation of SCAPs.MethodsBetaine was added for SCAPs chondrogenic induction. The chondrogenic differentiation potential was assessed using Alcian Blue staining, Sirius Red staining and the main chondrogenic markers. In vivo cartilage regeneration effects were evaluated by the rat TMJOA model. RNA-sequencing and biological analyses were performed to select target genes and biological processes involved. The mechanism betaine acts on chondrogenic differentiation of SCAPs was further explored.ResultsBetain-treated SCAPs demonstrated stronger cartilage regeneration in vitro and promoted cartilage repair of TMJOA in vivo. Betaine enhanced the expression of WDR81 in SCAPs during chondrogenesis. WDR81 overexpression promoted chondrogenic differentiation of SCAPs, while WDR81 depletion inhibited chondrogenic differentiation. In addition, both betaine treatment and WDR81 overexpression reduced intracellular reactive oxygen species levels and increased mitochondrial membrane potential in SCAPs.ConclusionBetaine promotes SCAPs chondrogenic differentiation and provided an effective candidate for TMJOA treatment. WDR81 may serve as the potential drug target through mitophagy.

A comparative study of mitoxantrone hydrochloride injection and technetium-99 m for tracing axillary sentinel nodes in patients with breast cancer

PurposeAn earlier multicenter phase III study demonstrated the safety and efficacy of mitoxantrone hydrochloride injection for tracing (MHI) for detecting sentinel lymph nodes (SLNs) in breast cancer patients. This ancillary study was conducted to further analyze and discover differences between the two tracers that had not been reported previously.MethodsThis analysis used single-center data from a phase III trial. Patients with breast cancer were injected with both MHI and 99mTc as SLN tracers before surgery. To account for cluster effects of multiple SLNs from the same patient and further analyze the differences between the two methods and factors that could impact detection, Obuchowski’s modified McNemar test, the GLIMMIX model, and univariate and multivariate analyses were used as appropriate.ResultsIn total, 463 SLNs from 109 patients were resected, 47 of which were pathologically positive. The SLN detection rate did not significantly differ between MHI and 99mTc. MHI blue staining was an independent predictor of pathological positivity (OR 3.58, 95% CI: 1.21–10.60, p = 0.02), while the 99mTc signal was not significant in the same model (p = 0.39).ConclusionMHI was comparable to 99mTc in safety and SLN detection rates but differed in sensitivity on positive SLNs.

C-reactive protein expression in acute ischemic stroke blood clots: Implications for etiology.

C-reactive protein (CRP) is a prototypic inflammation marker, with elevated levels associated with an increased risk of cerebrovascular events. To determine whether CRP could be a useful biomarker of stroke etiology, we investigated CRP expression in acute ischemic stroke (AIS) clots from large-artery atherosclerosis (LAA), cardio-embolism (CE) and cryptogenic (Crypt) subtypes. We analysed clot samples from AIS patients (LAA, CE, Crypt; n = 50 each), collected across five stroke centres in France, Serbia, Spain, and Japan between February 2021 and February 2024, as part of the prospective Clotbase International Registry of 460 patients who underwent mechanical thrombectomy. Clot components were assessed using Martius Scarlet Blue staining. CRP expression was examined using immunohistochemistry and its co-localisation with clot components was detected using immunofluorescence. Clinical parameters were compared across etiologies. CRP expression varied significantly among clots. Most clots (65%) had minimal (⩽1%) CRP and 35% showed substantial (>1%) CRP. CE group had significantly more clots with substantial CRP than LAA and Crypt (48% vs 30% and 26%; p = 0.048). Clots with substantial CRP contained more fibrin (28.9%) than those with low CRP (20.6%; p = 0.005). Confocal microscopy showed CRP co-localised with fibrin and white blood cells (WBCs). Significantly more AIS clots of CE expressed substantial CRP compared to those of LAA and Crypt, suggesting CE strokes may be more strongly linked to inflammation. Clots with substantial CRP expression displayed significantly more fibrin compared to those with minimal CRP expression, suggesting a potential association between inflammation and fibrin-rich clots. Further study of the relationship between CRP, fibrin and WBCs in clots may improve our understanding of the processes of thrombo-inflammation.

Ctdsp2 Knockout Induces Zebrafish Craniofacial Dysplasia via p53 Signaling Activation

Hemifacial microsomia (HFM) is a rare congenital craniofacial deformity that significantly impacts the appearance and hearing. The genetic etiology of HFM remains largely unknown, although genetic factors are considered to be primary contributors. We previously identified CTDSP2 as a potential causative gene in HFM cases. Utilizing CRISPR/Cas9, we knocked out ctdsp2 in zebrafish and analyzed the spatiotemporal expression of ctdsp2 and neural crest cell (NCC) markers through in situ hybridization (ISH). Craniofacial cartilage and chondrocyte phenotypes were visualized using Alcian blue and wheat germ agglutinin (WGA) staining. Cell proliferation and apoptosis were assessed via immunofluorescence with PH3 and TUNEL. RNA sequencing was performed on ctdsp2−/− embryos and control siblings, followed by rescue experiments. Knockout of ctdsp2 in zebrafish resulted in craniofacial defects characteristic of HFM. We observed abnormalities in NCC apoptosis and proliferation in the pharyngeal arches, as well as impaired differentiation of chondrocytes in ctdsp2−/− embryos. RNA-Seq analysis revealed significantly higher expression of genes in the p53 signaling pathway in mutants. Furthermore, ctdsp2 mRNA injection and tp53 knockout significantly rescued pharyngeal arch cartilage dysplasia. Our findings suggest that ctdsp2 knockout induces zebrafish craniofacial dysplasia, primarily by disrupting pharyngeal chondrocyte differentiation and inhibiting NCC proliferation through p53 signaling pathway activation.

Nerve Regeneration Through Differentiation of Endometrial-Derived Mesenchymal Stem Cells into Nerve-Like Cells Using Polyacrylonitrile/Chitosan Conduit and Berberine in a Rat Sciatic Nerve Injury Model.

Nervous injuries are common in humans. One of the most advanced treatment methods is neural tissue engineering. This research aims to utilize nerve-like cells (NLCs) derived from endometrial mesenchymal stem cells (EnMSCs) on a polyacrylonitrile/chitosan (PAN/CS) scaffold, along with berberine, for the reconstruction of a rat sciatic nerve injury model. In this experimental study, EnMSCs were obtained through enzymatic digestion and identified using flow cytometry and their differentiation into adipocyte and osteoblast. PAN nanofiber scaffolds were produced through electrospinning, and EnMSCs were neurally differentiated on these scaffolds for grafting into an animal model. The expression of Nestin, Map-2, Tuj-1, and NF genes in NLCs was confirmed through RT-PCR and immunocytochemistry. Twenty-five adult male rats were used in this study, divided into 5 groups: (1) Scaffold/Cells/Berberine, (2) Scaffold/Cells, (3) Scaffold, (4) Berberine, and (5) Control. The animals were maintained for 8 weeks, and their sciatic nerve function (SFI) was assessed. Additionally, histological examinations were performed using hematoxylin/eosin, luxol fast blue staining, and immunohistochemistry. According to the results, extraction, identification, and differentiation of EnMSCs and fabrication of PAN conduit and its transplantation were successfully performed. The best behavioral performance and histology were observed in the Scaffold/Cells/Berberine group. The SFI test results were -24.08 for the Scaffold/Cells/Berberine group and -39.27 for the control group. The nerve diameter in these two groups was 591 µm and 80 µm, respectively, and the percentage of new nerve formation was 18.5% in the Scaffold/Cells/Berberine group and 0.2% in the control group. The immunohistochemistry results demonstrated that the intensity of the green color was higher in the groups with cells compared to the groups without cells. Furthermore, in the luxol staining results, all groups showed a significant improvement compared to the control group. In the Scaffold/Cells/Berberine group, fibers, and axons appeared denser, more organized, and displayed a higher intensity of blue staining. According to the results of this study, EnMSCs demonstrated efficient differentiation into NLCs. With the assistance of PAN/CS scaffolds and simultaneous administration of berberine, EnMSCs have the potential for nerve regeneration and recovery from sciatic nerve injury in the rat animal model.

Characteristics of porcine oocyte-cumulus complexes derived from various sizes of antral follicles and classified by brilliant cresyl blue staining, and developmental competence of the oocytes.

The present study sought to determine the characteristics of porcine oocyte-cumulus complexes (OCCs) derived from very small and small antral follicles (with less than 1mm and 1-3mm in diameter, respectively; VSF and SF) in comparison with controls from medium ones (with 3-6mm in diameter; MF). Additionally, the present study examined the utility of brilliant cresyl blue (BCB) staining for assessing these OCCs. The incidence of BCB- oocytes in VSF- and SF-derived OCCs was higher than that in MF-derived OCCs. Although the meiotic and developmental competences of BCB+oocytes from MF were superior to those from VSF and SF, blastocysts were successfully obtained from BCB+oocytes even derived from VSF. The mean numbers of both total and viable cumulus cells surrounding an oocyte were significantly affected not only by the origin of the OCCs, but also by the BCB status of the oocytes (largest in MF-derived OCCs containing BCB+oocytes). Although the outer and inner diameters of zona pellucida were affected by the origin of OCCs and the BCB status of oocytes (largest in MF-derived oocytes), the ooplasmic diameter of BCB+oocytes did not differ among those derived from VSF, SF, and MF. Regardless of the BCB status, the transcriptional levels of G6PD and TKT in cumulus cells decreased during follicular development from VSF to MF, whereas the RPIA mRNA level in cumulus cells of MF-derived BCB+OCCs was lower than in the others. These results underscore the utility of BCB staining for selecting MF-, SF-, and even VSF-derived OCCs containing oocytes with relatively higher meiotic and developmental competences, as well as the importance of having a sufficient number of healthy cumulus cells expressing genes related to the pentose phosphate pathway at lower levels.

Xylooligosaccharide and Akkermansia muciniphila synergistically ameliorate insulin resistance by reshaping gut microbiota, improving intestinal barrier and regulating NKG2D/NKG2DL signaling in gestational diabetes mellitus mice.

Xylooligosaccharides (XOS) ameliorate insulin resistance (IR) in gestational diabetes mellitus (GDM) probably by propagating Akkermansia muciniphila (Akk). This study aimed to investigate the effects and mechanisms of XOS, Akk and combination on IR in GDM mice/pseudo-germ-free (PGF) mice. Female mice were fed with AIN-93 (n=19) and high fat diet (HFD) (n=206). After 4weeks, HFD-fed mice were further allotted to HFD, GDM, GDM+XOS, GDM+Akk, GDM+XOS+Akk, GDM+PGF, GDM+PGF+XOS, GDM+PGF+Akk, and GDM+PGF+XOS+Akk groups (n≥19). GDM was induced by intraperitoneally injecting streptozotocin and PGF was established by intragastrically administrating antibiotic cocktails. XOS (500mg/kg·BW) or/and Akk (4×108 CFU) were gavaged once a day for 10days. Fasting blood glucose (FBG), insulin, oral glucose tolerance test (OGTT) and insulin signaling pathway were determined. Gut microbiota were detected by 16S rRNA sequencing and absolute quantities of Akk by qRT-PCR. Intestinal tissues were stained by Hematoxylin-Eosin and Periodic acid-Schiff-Alcian blue staining. Occludin and Zonula occludens-1 (ZO-1) in intestine, Natural killer group 2 member D (NKG2D) on intestinal epithelial lymphocytes (IELs) and NKG2D ligands (NKG2DL) on intestinal epithelial cells (IECs) were detected by Western blotting. In GDM mice, XOS, Akk and XOS+Akk reduced (p<0.05) the area under the curve of OGTT (AUC), insulin and homeostasis model assessment of insulin resistance (HOMA-IR), and increased (p<0.05) protein kinase B (Akt) phosphorylation in liver and insulin receptor substrate 1 (IRS-1) phosphorylation in muscle. Furthermore, XOS+Akk reduced (p<0.05) FBG and increased (p<0.05) Akt phosphorylation in muscle and IRS-1 phosphorylation in liver. XOS, Akk and XOS+Akk reshaped gut microbiota with XOS+Akk exhibiting the greatest effectiveness. XOS increased (p<0.05) Akk and clearance of gut microbiota abolished such effect. XOS, Akk and XOS+Akk reduced (p<0.05) the small intestine Chiu's score and the colon Dieleman's scores, increased (p<0.05) ZO-1 and Occludin, and reduced (p<0.05) NKG2D on IELs and NKG2DLs (H60, MULT-1, Rae-1ε) on IECs. Moreover, XOS+Akk reduced (p<0.05) MULT-1 in duodenum. Collectively, XOS and Akk synergistically ameliorate IR by reshaping gut microbiota, improving intestinal barrier and regulating NKG2D/NKG2DL signaling in GDM mice.

Development and Optimization of an Ex Vivo Model of Corneal Epithelium Damage with 1-Heptanol: Investigating the Influence of Donor Clinical Parameters and MSC-sEV Treatment on Healing Capacity.

To develop and characterize a reproducible human corneal epithelial wound-healing model using 1-heptanol, and to investigate the healing potential of Bone Marrow-derived Mesenchymal Stromal Cell small Extracellular Vesicles (MSC-sEV) and the influence of donor characteristics on epithelial healing. Eighty-eight (n=88) human corneoscleral tissues unsuitable for transplantation were employed. Corneal epithelial damage was induced with 1-heptanol and monitored every 24 hours up to 96 hours using fluorescein and trypan blue staining. Histological assessment was performed on untreated and damaged tissues. Damaged areas were measured with FIJI software, and healing rates were calculated. MSC-sEV were isolated with size exclusion chromatography and characterized for their size, morphology and biomarkers. Their impact on healing was assessed in both in vitro scratch assays on cultured human corneal epithelial cells and on ex vivo 1-heptanol-damaged corneas. Histological analysis revealed detached corneal epithelium in the central area, while other layers remained unaffected. Healing rate peaked at 48 hours post-damage. Trypan blue and Fluorescein staining correlated and the former highlighted a higher initial healing rate than the latter. Diabetic and heart-beating brain-deceased donors showed impaired healing rates. MSC-sEV (79.8 nm, spherical bilayer, positive for TSG101, CD9, CD63, and CD81) significantly improved epithelial wound healing in both in vitro and ex vivo models. 1-heptanol effectively induces reproducible corneal epithelial damage, and the ex vivo organ-cultured human cornea heals the epithelium within 96 hours. Diabetes and donation from heart-beating brain-deceased donors reduce healing capacity. MSC-sEV boost epithelial repair in damaged corneas.

Underlying Mechanism of Fluoride Inhibits Colonic Gland Cells Proliferation by Inducing an Inflammation Response.

The integrity of colonic gland cells is a prerequisite for normal colonic function and maintenance. To evaluate the underlying injury mechanisms in colonic gland cells induced by excessive fluoride (F), forty-eight female Kunming mice were randomly allocated into four groups and treated with different concentrations of NaF (0, 25, 50, and 100 mg F-/L) for 70 days. As a result, the integrity of the colonic mucosa and the cell layer was seriously damaged after F treatment, as manifested by atrophy of the colonic glands, colonic cell surface collapse, breakage of microvilli, and mitochondrial vacuolization. Alcian blue and periodic acid Schiff staining revealed that F decreased the number of goblet cells and glycoprotein secretion. Furthermore, F increased the protein expression of TLR4, NF-κB, and ERK1/2 and decreased IL-6, interfered with NF-κB signaling, following induced colonic gland cells inflammation. The accumulation of F inhibited proliferation via the JAK/STAT signaling pathway, as characterized by decreased mRNA and protein expression of JAK, STAT3, STAT5, PCNA, and Ki67 in colon tissue. Additionally, the expression of CDK4 was up-regulated by increased F concentration. In conclusion, excessive F triggered colonic inflammation and inhibited colonic gland cell proliferation via regulation of the NF-κB and JAK/STAT signaling pathways, leading to histopathology and barrier damage in the colon. The results explain the damaging effect of the F-induced inflammatory response on the colon from the perspective of cell proliferation and provide a new idea for explaining the potential mechanism of F-induced intestinal damage.

Relationship between cognitive impairment and hippocampal iron overload: A quantitative susceptibility mapping study of a rat model.

The aim of this study was to establish an iron overload rat model to simulate the elevated iron levels in patients with thalassemia and to investigate the potential association between hippocampal iron deposition and cognition. Two groups of iron overloaded rats and one group of control rats were used for this study. The Morris water maze (MWM) was used to test spatial reference memory indicated by escape latency time and number of MWM platform crossings. The magnetic susceptibility value of the hippocampal tissue, a measure of iron deposition, was assessed by quantitative susceptibility mapping (QSM) and was correlated with spatial reference memory performance. The iron content in hippocampal tissue sections of the rats were assessed using diaminobenzidine (DAB)-enhanced Perl's Prussian blue (PPB) staining. The rat groups with iron overload including the Group H and Group L had higher hippocampal magnetic susceptibility values than the control rat group, i.e., Group D. In addition, the iron overloaded groups had longer MWM escape latency than the control group, and reduced number of MWM platform crossings. There was a positive correlation between the mean escape latency and the mean hippocampal magnetic susceptibility value, a negative correlation between the number of platform crossings and the mean hippocampal magnetic susceptibility value, and a negative correlation between the number of platform crossings and the latent escape time in Group H and Group L. This rat model simulating iron overload in thalassemia showed hippocampal iron overload being associated with impairment of spatial reference memory. QSM could be used to quantify brain iron overload in vivo, highlighting its potential clinical application for assessing cognitive impairment in patients with thalassemia.

ONYCHOMYCOSIS: UNRAVELING THE ROLE OF DERMATOPHYTES IN NAIL INFECTIONS

Objective: This study aimed to identify pathogenic fungi that cause onychomycosis and evaluate their clinical presentations and risk factors to enhance management through precise diagnosis and targeted treatment. Methods: The fungi were identified by macroscopic and microscopic morphological features using techniques such as slide cultures, Potassium Hydroxide (KOH) mounts, culture isolation, and Lacto Phenol Cotton Blue (LPCB) staining. Results: 110 nail clippings from patients clinically suspected of onychomycosis were analyzed; 27 had dermatophytic infections. Toenails were involved in 33.8% of the cases, and Distal and Lateral Subungual Onychomycosis (DLSO) was the primary clinical presentation. Trauma was the most common predisposing factor (19%). Conclusion: This study identified dermatophytes as the prime cause of onychomycosis, chiefly affecting toenails with DLSO as the predominant form, and recognized trauma as a key risk factor. Precise identification using traditional diagnostic methods is crucial for targeted treatment and better patient results.

The mechanism of Bovis Culus Sativus protecting BBB damage in stroke: Insights from network pharmacology, bioinformatics, and experiments.

Bovis calculus (BC) has a medicinal history of over 2000 years in treating stroke in China. Bovis Culus Sativus (BCS) has similar pharmacological effects to BC. Due to the scarcity of BC, BCS is often used as a substitute for BC in clinical practice for treating stroke in traditional Chinese medicine. This study aims to investigate the inhibitory effect of BCS on blood-brain barrier (BBB) damage following stroke, and to elucidate the molecular basis of BCS neuroprotection through network pharmacology and bioinformatics. The contents of bilirubin and bile acids in BCS were quantified using HPLC. A cerebral ischemia-reperfusion injury (CIRI) rat model was established to assess neurological function, cerebral infarction, pathological damage, and Evans Blue staining. R language was used to analyze GEO public data to identify therapeutic targets for ischemic stroke. Public databases and literature were utilized to screen for active components of BCS, and the Swiss Target Prediction database was used to predict the active drug targets. Network pharmacology analysis was conducted on drug and disease targets, followed by immune infiltration and molecular docking of key targets. Finally, ELISA, RT-PCR, Western blot, IHC, and TEM were employed to validate the effectiveness of the targets. The content of bile acids and bilirubin in the tested BCS was 6.9% and 37.89%, respectively. The study showed that BCS reduced neurological function scores and cerebral infarction rates in stroke rats, prevented Evans Blue leakage, and mitigated histopathological damage in the ischemic brain region. Additionally, BCS improved the structural and functional integrity of the BBB, enhancing the expression of Occludin, ZO-1, and Claudin-5 while downregulating the expression of MDR1, aquaporin-4, MMP-9, and MMP2. Bioinformatics and network pharmacology analyses indicated that the therapeutic effects of BCS in stroke are primarily associated with the inhibition of inflammatory pathways, including TNF, NFKB, and MAPK. ELISA, RT-PCR, and Western blot results further confirmed that BCS significantly suppressed neuroinflammation in stroke rats. BCS shows promising efficacy against ischemic stroke, maintaining the function and structural integrity of the BBB. Its protective effect on the BBB may be related to the inhibition of the TNF-NFκB-MAPK signaling pathways.

Recurrent bacterial pneumonia in Irish Wolfhounds - histopathology and characterization of bronchial cartilage changes.

An increased incidence of bacterial pneumonia (BP) has been reported in Irish Wolfhounds (IWHs) and the disease typically recurs repeatedly. The aetiology is unclear but the development of bronchiectasis in the absence of clinical findings indicative of interstitial lung disease is common in affected IWHs. This observation led us to hypothesize that the primary cause could be at bronchial level. We evaluated bronchial cartilage structure and composition in IWHs (n=10) with previous episodes of pneumonia compared with IWH control dogs (n=5) and other large-breed control dogs (n=5) without a history of respiratory disease. Histological evaluation of lung sections was performed with haematoxylin and eosin, Masson's trichrome and Prussian blue staining as well as with immunohistochemistry (IHC) for CD3 and CD79a antigens and pancytokeratin. Bronchial cartilage proteoglycan content was evaluated in sections stained with safranin 'O' (SO) and chondrocyte apoptosis by IHC for cleaved caspase-3. Image analysis was used to measure the bronchial cartilage to lumen area ratio as well as the SO staining intensity and caspase-3-positive cell count in digital whole slide images. The most consistent histological finding in the lungs of IWHs with recurrent BP was chronic lymphoplasmacytic inflammation in the peribronchial and perivascular pulmonary interstitium, along with remodelling of the bronchial walls. The bronchial cartilage to lumen area ratio was significantly lower in all IWHs than in control dogs of other breeds (P=0.011), suggesting less cartilage support in the bronchi of the IWH breed. The SO staining intensity of the bronchial cartilage was significantly higher (P=0.008) in affected IWHs than in control dogs of other breeds. Analysis of caspase-3 immunolabelling of large bronchial cartilage revealed significantly fewer apoptotic chondrocytes in affected IWHs than in all control dogs (P=0.045). Based on these findings, altered cartilage metabolism, as a breed-related feature or secondary to chronic inflammation, may play a role in the pathogenesis of bronchiectasis development and recurrent lung infections in IWHs.

Ex Vivo Endocytoscopic Evaluation of Pancreatobiliary Cancers: A Step Toward Real-time In Vivo Diagnosis.

Endocytoscopy (EC) enables real-time diagnosis at extremely high magnification. The aim of the present study was to assess the potential of EC for the prediction of biliary malignancy. In total, 23 surgically resected cases with biliary tract cancer (BTCA, n=6), pancreatic head cancer (PCA, n=13), and non-cancerous bile duct lesion (NCBL, n=4) were enrolled in the study. The extrahepatic bile duct mucosa of the resected materials was examined ex vivo using EC. In cases of BTCA and PCA with bile duct invasion, several independent findings were identified as predictors of cancerous areas using multivariate analysis. These include, extravasation [odds ratio (OR)=15.91; 95% confidence interval (CI)=2.05-123.46], absence of fine network (OR=6.16; 95%CI=1.88-20.19), vascular dilatation (OR=4.87; 95%CI=1.56-15.25) in direct view. Additional predictors observed under methylene blue staining include anisonucleosis (OR=27.77; 95%CI=6.85-112.56), absence of large glands (OR=27.77; 95%CI=6.85-112.56), obscured nuclei (OR=7.63; 95%CI=2.23-26.17), and absence of uniform glands and small/irregular glands (OR=6.18; 95%CI=2.25-16.98). There were no differences in EC findings of non-cancerous areas between BTCA/PCA and NCBL. EC score for predicting the cancerous area (ECS-CA) was established by summing the scores (regression coefficients) of the eight EC findings described above. In the receiver operating characteristic curve analysis, the area under the curve for predicting cancerous areas using the ECS-CA was 0.960 (optimal cut-off ECS-CA, 6.1; sensitivity 91.5%; specificity, 93.6%). ECS-CA is valuable for assisting in the real-time diagnosis of biliary malignancy in vivo.

Lycopene Ameliorates Polycystic Ovary Syndrome in Rats by Inhibiting Ovarian Ferroptosis Through Activation of the AMPK/Nrf2 Pathway.

Lycopene (LYC) is an extremely powerful antioxidant with the potential to treat a range of diseases and to inhibit ferroptosis. This research aims to elucidate how LYC impacts polycystic ovarian syndrome (PCOS) and the action mechanisms. A PCOS rat model was constructed by injecting DHEA. Different doses of LYC were injected intraperitoneally in PCOS rats, the estrous cycle was recorded. The histopathological damage of ovary in PCOS rats was observed by HE staining, testosterone (T), estradiol (E2), luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels were examined by ELISA kits. Transmission electron microscopy, prussian blue staining, biochemical kits to determine ferroptosis. Immunohistochemistry and Western blot to assess the levels of ferroptosis-related and AMPK/Nrf2 pathway-related proteins to explore whether LYC affects ferroptosis in PCOS through this pathway. PCOS rats had significantly higher body weights, ovarian weights and ovarian indices, and disorganized estrous cycles, which were dose-dependently ameliorated by LYC. In addition, LYC significantly ameliorated the histopathological damage of ovary in PCOS rats and restored the normal secretion of T, E2, LH, and FSH. LYC attenuates iron deposition in PCOS ovarian tissues, reduces iron and ROS levels, and inhibits ferroptosis. Notably, LYC activated the AMPK/Nrf2 pathway, and AMPK inhibitor intervention attenuated the therapeutic effect of LYC in PCOS rats, suggesting that LYC acts through the AMPK/Nrf2 pathway. LYC attenuates estrous cycle disruption, ameliorates pathological impairments, and inhibits ferroptosis in PCOS rats by modulating the AMPK/Nrf2 pathway.

An agar medium-based method for screening somatic incompatibility in Agaricus bisporus

To visualize the nonself recognition reaction in the cultivated mushroom Agaricus bisporus, we developed a method using the azo dye Evans blue. The use of Evans blue highlights dead mycelial sections, which are produced following nonself recognition in the interaction zone between two individuals. This method can differentiate between distinct heterokaryons, as well as between closely related heterokaryons constructed from siblings. As it is known that co-cultivation of mixed individuals leads to reduced yield, we compared small-scale cultivation experiments to the results of our laboratory assay. Co-cultivation of strains whose interaction produced noticeable Evans blue staining also produced low yield when mixed. However, a combination that did not produce noticeable Evans blue staining still produced an incompatible-like phenotype (reduced yield) when mixed under cultivation conditions. Together, these results suggest that while our Evans blue assay can discriminate between self and nonself pairings, it alone does not encompass all aspects of this interaction. However, this method can facilitate future research into the genetics and physiology of the incompatibility phenotype in this economically important fungus.

Effects of ascorbic acid on myelination in offspring of advanced maternal age

Myelination is the process by which oligodendrocytes ensheathe axons to form myelin sheaths. Myelination is a crucial aspect of brain development and is closely associated with central nervous system abnormalities. However, previous studies have found that advanced maternal age might affect the myelination of offspring, potentially through the pathway of disrupting DNA methylation levels in the offspring's hippocampus. Current research has demonstrated that ascorbic acid can promote hydroxymethylation to reduce methylation levels in vivo. This study aims to verify the relationship between ascorbic acid and myelination, as well as the specific mechanism involved. Initially, oligodendrocyte differentiation was observed using immunofluorescence and Western blot. Myelination was assessed through Luxol Fast Blue staining, Glycine silver staining, immunofluorescence, and transmission electron microscopy. The demethylation level of oligodendrocyte progenitor cells was detected by immunofluorescence co-expression of OLIG2 and DNA hydroxylase ten-eleven translocation 1 (TET1), TET2, and TET3. Our study found that advanced maternal age could impair myelination in the hippocampus and corpus callosum of offspring. Ascorbic acid intervention may induce TET1 and TET2-mediated hydroxymethylation to ameliorate myelination disorders, promote myelination and maturation, and reverse the effects of advanced maternal age on offspring.

Omitting elective neck dissection in cT1/2N0 oral squamous cell carcinoma with sentinel lymph node metastasis: A prospective study.

To examine the distribution of non-sentinel lymph nodes (SLNs) and to determine the feasibility of omitting elective neck dissection (END) in cases of cT1/2N0 oral cancer presenting with SLN metastasis. A prospective cohort of patients with cT1/2N0 oral cancer underwent SLN biopsy using a γ-probe alongside methylene blue staining, followed by subsequent END. The primary outcome variable was non-SLN metastasis, with its predictors evaluated through logistic regression analysis. A total of 200 patients with detectable SLNs were analyzed. Logistic regression revealed a significant odds ratio of 4.28 [95% confidence interval: 2.11-14.56] for predicting non-SLN metastasis when comparing a depth of invasion greater than 4.0mm to a DOI of 4.0mm or less. Among the six cases of non-SLN metastasis, three patients with negative SLN biopsy results exhibited metastasis; one was found in ipsilateral level V and two in contralateral level Ib. In contrast, all three patients with positive SLN biopsy results had a DOI surpassing 4.0mm, presenting with at least two positive SLNs. Non-SLN metastasis was detected in ipsilateral level III for one patient and at level IV for two others. END may be judiciously omitted in cases where only one positive SLN is identified in early-stage oral cancer with a depth of invasion of≤4.0mm.