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Artificial intelligence-driven image analysis has enabled significant advances in digital pathology. However, most approaches have focused on cell or organ structures. This manuscript presents a reproducible deep learning methodology for pixel-level analysis of amorphic patterns in haematoxylin and eosin-stained whole-slide histological images. This study analysed the pixel patterns in the extracellular matrix (ECM) part of connective tissue to identify differences in airway wall ECM compartments and their heterogeneity, which are microscopically similar and difficult to discern with the human eye. Through a targeted preprocessing pipeline, the deep learning model is guided to emphasise learning from pixel-level patterns in non-cellular tissue components while reducing the influence of cellular structures and artefacts. Combined with transfer learning, the model accurately distinguishes the characteristics of the airway submucosa and adventitia, achieving a test area under the curve of 0.84. Using visualisation techniques and statistical analysis, we demonstrate that random pixel imputation successfully reduces the effects of cellular structures on model learning. The framework is applied in a proof-of-principle study of lung tissue from patients with chronic obstructive pulmonary disease, illustrating how this quantitative approach can study population heterogeneity and inform novel research directions. Ultimately, this study provides an innovative and adaptable framework that unlocks the analytical potential of often-overlooked amorphic components in AI-empowered histopathology.
In pathology practice, understanding pre-analytical and analytical variables is fundamental for an accurate interpretation of the microscopic morphology of each organ. The lung is susceptible to several artifacts, which can alter the final microscopic appearance of the specimen, including (i) morphological changes ex vivo compared to the in vivo state, (ii) effects of presurgical and surgical procedures, and (iii) pre-analytical and analytical variables. In this article, the main iatrogenic artifacts described in pulmonary pathology are summarized, as well as the effects of previous biopsies, positive pressure ventilation, surgical collapse, clamping edema, consequences of surgical resection, intravascular procedures (hydrophilic polymer embolism and lipoid pneumonia due to lipid emulsions), and consequences of pathology handling. Specific artifacts are also highlighted in detail, including hook wire placement, clamping edema, cell and tissue handling, fixation delay, iatrogenic collapse, freezing of lung parenchyma, and tissue displacement. Awareness of these artifacts is crucial for proper interpretation and accurate histologic diagnosis of lung specimens.
Liver diseases constitute a major global health burden, contributing substantially to morbidity and mortality worldwide. Although effective therapeutic strategies have been developed for key risk factors, such as hepatitis C infection, therapeutic options for most other liver diseases remain limited. Noncoding RNAs (ncRNAs), which regulate diverse cellular and molecular processes, have emerged as promising therapeutic targets and biomarkers in liver diseases. In this study, we summarize the mechanisms of action and functional roles of ncRNAs, such as long ncRNAs (lncRNAs), circular RNAs (circRNAs), and PIWI-interacting RNAs (piRNAs), to improve therapeutic strategies and diagnostic precision in liver diseases, such as liver fibrosis and hepatocellular carcinoma (HCC).
Hepatocellular carcinoma (HCC) is a global malignant tumor type. Pyrroline-5-carboxylate reductase 1 (PYCR1) is a metabolic enzyme that exhibits pro-tumor properties in cancer progression. However, the exact molecular mechanism of PYCR1 in HCC progression is still unclear. CCK8, EdU, and Transwell assays were used to measure the proliferation, migration, and invasion of HCC cells, respectively. Immunostaining and flow cytometry were used to detect cellular autophagy and apoptosis. The downregulation of PYCR1 can inhibit the survival, proliferation, migration, and invasion of HCC cells. At the same time, downregulation of PYCR1 induces autophagy and subsequently activates cell apoptosis. Therefore, we pretreated HCC cells with mTOR activators or inhibitors to inhibit or promote autophagy, leading to an inhibition or an increase in apoptosis. Simultaneously, the PI3K activators or inhibitors to activate or inhibit the PI3K/AKT/mTOR pathway also lead to inhibition or activation of autophagy and apoptosis. The downregulation of PYCR1 induces autophagy-dependent apoptosis in HCC cells by inhibiting the PI3K/AKT/mTOR pathway, revealing a novel mechanistic link in HCC pathophysiology.
Pulmonary fibrosis (PF) is a chronic and progressive interstitial lung disease characterized by excessive extracellular matrix (ECM) deposition and remodeling of lung structures. Currently, no effective clinical treatments exist to reverse or halt the progression of PF. Consequently, there is an urgent need to identify novel therapeutic strategies for this complex disease. Stem cells, renowned for their self-renewal and multipotent differentiation capabilities, have garnered considerable attention for their potential therapeutic applications, particularly in regenerative medicine. The primary stem cell types investigated for PF treatment include mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), and lung progenitor cells. Preclinical studies have demonstrated that these stem cells can reduce inflammation, modulate immune responses, and promote the repair of damaged lung tissue. Although stem cell transplantation shows promise in PF treatment, challenges such as safety, quality control, and therapeutic efficacy remain unresolved. Recent studies have highlighted that stem cells interact with and modify their transplanted environment, influencing their structural properties and chemical composition. These interactions strongly influence stem cell survival, phenotype, and therapeutic efficacy. Understanding these dynamics will inform the development of new strategies to improve the effectiveness of stem cell therapies for PF.
Due to the difficulty in early diagnosis and the lack of treatment for advanced disease, the mortality rate of hepatocellular carcinoma (HCC) is high, and the 5-year overall survival rate is low at present. SLC1A4 is a neutral amino acid transporter, but its regulatory role and mechanism in HCC are still unclear. Through analyzing the TCGA database and clinical tissue specimens, this study uncovered the high expression of SLC1A4 in tumor tissues of HCC. Worse more, a high level of SLC1A4 may lead to a poor prognosis of HCC. Mechanically, silencing SLC1A4 inhibited the phosphorylation activation of AKT by suppressing the ubiquitin modification of AKT at lysine 63 and amino acid influx represented by D-serine, decreasing the protein level of β-catenin in the cell nucleus and suppressing the transcriptional activity of c-Myc and EpCAM promoters. As a result, silencing SLC1A4 inhibited the proliferation, migration, and stemness of hepatic cancer cells, which was successfully reversed by the introduction of exogenous AKT. Moreover, epithelial-mesenchymal transition (EMT) in vitro and metastasis potential in vivo of hepatic cancer cells was suppressed by the downregulated SLC1A4 level. In conclusion, SLC1A4 promotes the malignant transformation of HCC through activating signal transduction mediated by AKT. The findings in this study suggested that SLC1A4 may be a diagnostic indicator for the early HCC and a therapeutic target for the advanced HCC.
[This retracts the article DOI: 10.1155/2020/6403012.].
Immune thrombocytopenia (ITP) is a complex autoimmune disorder characterized by accelerated destruction of peripheral platelets and impaired megakaryopoiesis. While the cellular effectors, dysregulated T cells, hyperactive B cells and phagocytic macrophages are well characterized, the upstream epigenetic mechanisms orchestrating this multicellular immune network remain largely elusive. This review explores the hypothesis that microRNAs (miRNAs) may serve as critical architects of immune dysregulation and bone marrow failure in ITP. We evaluate the clinical utility of circulating miRNAs as non-invasive biomarkers for diagnosis, risk stratification and predicting response to steroid and thrombopoietin receptor agonists therapies. Finally, we address current translational difficulties, such as data fragmentation and pre-analytical variables. We propose a roadmap for integrating functional validation with multi-omics, utilizing miRNA-based approaches to facilitate and advance precision medicine in ITP.
This study aimed to explore the regulatory role of sterol regulatory element-binding protein 1 (SREBP1) in the antioxidant enzyme B166 and to examine the prognostic and therapeutic potential of B166 in glioblastoma multiforme (GBM). RNA-seq data from TCGA and GTEX databases were analyzed to explore the relationship between B166 and GBM, as well as between B166 and SREBP1. Functional experiments were performed to determine whether SREBPs could regulate B166 expression level and to identify the major regulator of B166. Immunofluorescence staining was performed to investigate the cellular distribution of B166 in GBM. Quantitative reverse transcription polymerase chain reaction was employed to determine the regulatory effects of SREBP1-a. CellROX Deep Red dye was applied to detect mitochondrial reactive oxygen species (ROS) levels. The results revealed that B166 protein was significantly upregulated in GBM tissues compared with lower-grade gliomas and normal brain tissues (p<0.0001). The B166 expression level was elevated through SREBP1-a-mediated regulation in GBM, simultaneously decreasing endogenous ROS levels to maintain cellular redox homeostasis and preserve normal mitochondrial function. Reducing SREBP1 level decreased both RNA and protein expression levels of B166. Overexpression of the active form of SREBP1-a elevated the protein levels of B166 in both the mitochondria and nucleus. Pharmacological suppression of SREBP1 or genetic inhibition of B166 disrupted redox homeostasis, resulting in elevated oxidative stress and rapid cell death. In conclusion, B166 may serve as a promising therapeutic target for GBM. Its overexpression could enhance ROS elimination, highlighting its potential therapeutic benefits for GBM patients.
Chemotherapy-induced fatigue (CIF) remains a clinically challenging condition with limited therapeutic options. This study aimed to elucidate the therapeutic potential and underlying mechanisms of the multiherbal Xinlikang (XLK) capsule against CIF using an integrated strategy that combined network pharmacology prediction with experimental validation. A murine CIF model was established using 5-fluorouracil (5-FU). XLK was administered at various doses to evaluate its efficacy through comprehensive assessments, including behavioral tests (weight-bearing swimming, tail suspension, and grip strength), histopathology (hematoxylin-eosin [H&E] and periodic acid-Schiff [PAS] staining), and metabolic indices (lactate and ATP levels). To investigate the mechanisms, an integrated network pharmacology approach was employed to identify bioactive components of XLK, predict their potential targets, and construct a "component-target-pathway" network. Core signaling pathways implicated in CIF were prioritized via protein-protein interaction (PPI) and KEGG enrichment analyses. Key predictions were subsequently verified by Western blot analysis. XLK treatment significantly ameliorated fatigue-like behaviors, improved muscle glycogen storage, and restored lactate and ATP homeostasis in CIF mice (all p  < 0.05). Network pharmacology predicted that the anti-CIF effect of XLK was closely associated with the regulation of energy metabolism-related pathways, particularly the PI3K/AKT-mTOR-FoxO signaling axis. Experimental validation confirmed that XLK significantly modulated the expression and phosphorylation levels of key proteins (e.g., p-PI3K, p-AKT, and p-mTOR) within this pathway in the skeletal muscle or relevant tissues of CIF model mice (all p  < 0.05). XLK enhances cellular energy homeostasis by regulating the PI3K/AKT-mTOR-FoxO signaling axis, thereby alleviating CIF. These findings provide a mechanistic rationale for the clinical application of XLK against CIF.
Colorectal cancer (CRC) remains a formidable global health challenge, characterized by uncontrolled cell proliferation and significant socioeconomic burden. Projections anticipate a substantial increase in new cases, straining healthcare systems worldwide. CRC, the most common histological subtype, originates from gland cells lining the colon and rectum, often preceded by benign polyps. Its development is complex, influenced by sporadic, familial, and inherited factors. Sporadic cases, accounting for 70%-80%, are linked to genetic mutations and epigenetic alterations, with lifestyle factors like physical inactivity, diet, obesity, smoking, and alcohol consumption playing significant roles. Familial cases (25%) suggest shared environmental influences, while inherited conditions (5%) like familial adenomatous polyposis (FAP) and Lynch syndrome involve germline mutation with high lifetime CRC risk. The molecular pathogenesis of CRC involves genomic instability, notably chromosomal instability (CIN), microsatellite instability (MSI), and the CpG island methylator phenotype (CIMP). Mitochondria, beyond ATP production, critically influence cellular processes. The mitochondrial metabolic theory (MMT) proposes impaired oxidative phosphorylation (OxPhos) drives genomic instability. While the Warburg effect is recognized, many tumors retain functional oxidative metabolism, often relying heavily on OxPhos. Key genetic mutations like KRAS and BRAF significantly impact CRC, influencing mitochondrial metabolism and tumor progression, and are associated with different prognostic outcomes. Emerging therapeutic strategies target metabolic vulnerabilities to overcome chemoresistance. Inhibition of mitochondrial genes and key enzymes, along with modulating calcium homeostasis, shows promise. Preclinical research highlights sorghum extracts as potential therapeutic agents. High-phenolic sorghum bran extracts demonstrate anticancer action by suppressing proliferation, inducing apoptosis, and inhibiting migration and invasion. These effects involve targeting specific cancer pathways and influencing proteins like those in the Ras-ERK pathway, β-catenin, cMyc, cyclin D1, and surviving. Specific sorghum compounds like anthocyanidins, luteolin, and quercetin exhibit cytotoxicity against CRC cells, including those with KRAS and BRAF mutations. Further research is crucial to translate these preclinical findings into clinical applications, understand bioavailability, and account for individual differences in gut microbiota.
Coiled-coil domain-containing 97 (CCDC97) can bind to the splicing factor SF3B complex to participate significantly in pre-mRNA splicing. However, there may be a risk of cancer development in the case of disrupted mechanism, necessitating further in-depth investigation of the underlying mechanism. The present study was conducted by searching for various databases to implement a comprehensive cancer analysis of CCDC97. Furthermore, a competitive endogenous RNA (ceRNA) network of CCDC97 was established, which was further integrated with the reported oncogene mechanisms in the CCDC family to explain the potential reasons for its abnormal expression. CCDC97 is highly expressed in various cancers and closely associated with poor prognosis. CCDC97 was also strongly associated with the infiltration of various immune cells and multiple immune checkpoints in the tumor immune microenvironment. RT-qPCR showed that CCDC97 was highly expressed in hepatocellular carcinoma (HCC) tissue samples. At the single-cell level, CCDC97 exhibited an intimate association with depleted T cells in HCC tissues. In cancers, particularly in HCC CCDC97 can serve as a promising marker for immunotherapy and prognosis in cancers, particularly in HCC. More experimental verification is needed in the future research.
Patients deficient in the peroxisomal membrane protein ACBD5 regularly exhibit a dystrophy of the retina along with decline in visual acuity. Despite the prevalent retinal phenotype, information on the pathogenesis of the retinodystrophy is limited. To gain insight into the cellular, subcellular and molecular alterations occurring in the retina, we analyzed an ACBD5-deficient mouse model by immunofluorescence microscopy, electron microscopy, full-field electroretinography (ffERG) and as well as analytical and spatial mass spectrometry (MS)-based lipidomics techniques. Histological results implied that ACBD5-deficient mice exhibit a moderate degeneration of photoreceptor, bipolar, ganglion and retinal pigment epithelial cells accompanied, however, by a prominent activation of astroglia and microglia. Reduced a- and b-wave amplitudes from ffERG point to a severe functional dysregulation of retinal signal transduction with a focus at the level of the information-processing cell of the inner retina. At the lipidome level, very long-chain polyunsaturated fatty acids (VLC-PUFA) accumulated in phosphatidylcholines from retina homogenates, most likely disrupted by a decline in peroxisome functions. Remarkably, as revealed by MALDI MS imaging, these lipidome changes affected neither the whole retina nor the photoreceptor outer segments (POS), where VLC-PUFAs display the highest concentration in phospholipids of POS membrane discs. In contrast, VLC-PUFAs in ACBD5-deficient mice consistently accumulated in the inner retinal region from the outer (OPL) to inner plexiform layer (IPL). In line with VLC-PUFA-accumulations, photoreceptor ribbon synapses in the OPL showed morphological signs of degeneration on the ultrastructural level. Hence, peroxisomal dysfunction appears to affect cell type-specific lipid homeostasis, thereby disrupting local retinal membrane physiology leading to a severe neuroinflammation of the ACBD5-deficient mouse retina.
Acinetobacter baumannii is known for its ability to invade and persist within eukaryotic cells, impacting infection outcomes and disease progression. This study investigates the role of Omp34, a key outer membrane protein (Omp), in A. baumannii interaction with epithelial cells and the protective effects of anti-Omp34 antibodies (Abs). Omp34 is a key regulator of A. baumannii epithelial cell invasion, influencing bacterial adherence, internalization, and intracellular proliferation. The presence of anti-Omp34 Abs mitigates A. baumannii-induced cellular damage and enhances bacterial clearance. The process involved the expression and purification of Omp34, which in turn induced Abs in BALB/c mice against Omp34. The acute toxicity of Omp34 was studied through a histological analysis conducted on six distinct organs in mice. HeLa cells were infected by A. baumannii ATCC 19606 and a clinical strain. Various aspects of A. baumannii behavior with HeLa cells, including HeLa cell viability, adherence, serum resistance, cell internalization, and intracellular proliferation with and without anti-Omp34 sera. Cytoskeleton inhibitors were used to study the potential roles played in the process of A. baumannii invasion by microfilaments and microtubules. Omp34 effectively triggered Ab production in mice without resulting in any toxicity. The assay for serum resistance revealed potent bactericidal and antibiofilm effects on both A. baumannii strains. Bacterial internalization was constrained when actin polymerization was inhibited. Examination under the microscope revealed instances of adherence, alterations in the cell membrane, apoptosis, vacuolization, and cell damage. HeLa cells exposed to anti-Omp34 serum showed decreased cell damage. The results provide substantial evidence of the adherence capacity of A. baumannii to proliferate in the epithelial cells. In conclusion, Omp34 plays a substantial role in regulating interactions between epithelial cells and A. baumannii, the multifaceted nature of which intricately modifies the trajectory of infection within host cells by A. baumannii.
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Gliosarcoma (GS) is a rare and aggressive variant of glioblastoma multiforme (GBM), characterized by a biphasic histopathological pattern featuring both glial and mesenchymal components. Accounting for 2% of all GBM cases, this brain tumor is known for its poor prognosis, rapid progression, and resistance to conventional treatments. Despite advances in molecular profiling and multimodal treatment strategies, GS remains a therapeutic challenge due to their highly invasive nature and limited response to standard regimens. The purpose of this study was to characterize the neuroradiological, surgical, clinicopathological, and radiotherapeutic profiles of a cohort of patients affected by GS, including two recurrences of disease.
Not enough is known about how ophiopogonin D (OP-D) works and the molecular mechanisms involved in nonalcoholic steatohepatitis (NASH). This study aimed to investigate the antifibrosis effect and underlying mechanism of OP-D in NAFLD. The rats were fed a high-fat diet (HFD) to simulate NAFLD. After OP-D treatment or not, the rat serum lipid levels and inflammatory and ferroptosis-related factors were detected. Online databases and network pharmacology were used to collect the targets of OP-D against NASH. The effect of OP-D on AKT1, STAT3, ferroptotic markers, and fibrotic markers was determined using western blot or PCR assays. OP-D inhibited abnormal liver function, fibrotic markers (α-SMA/Col1α1), and ferroptosis-related factor (GPX4) in NASH rats. Network pharmacology proposed AKT1 and STAT3 as targets for OP-D against NASH. OP-D reduced the p-AKT and p-STAT3 in liver tissues. OP-D reduced the levels of AKT1, STAT3, and HIF-1α in model hepatic stellate cells. AKT1 and STAT3 overexpression reversed the antifibrosis and ferroptosis-induced effects of OP-D in model hepatic stellate cells. OP-D may alleviate fibrosis in NASH by regulating ferroptosis via the AKT1/STAT3/HIF-1α axis.
Intratumor heterogeneity (ITH) plays an important role in patients' clinical outcomes. The prognostic impact of ITH and its influencing factors is unclear in papillary thyroid carcinoma (PTC), which deserves further investigation. The Mutation Annotation Format (MAF) and clinical features were collected from The Cancer Genome Atlas Thyroid Cancer (TCGA-THCA) cohort. We first assessed the influence of ITH on the prognosis of patients. We used the Mutant Allele Tumor Heterogeneity score to evaluate and represent ITH. Then we explored the potential factors associated with ITH. Finally, we predicted possible pathways involved in ITH. Among 4 prognostic outcomes, higher ITH was mainly related to poor disease-free interval (DFI) (HR = 2.64, p = 0.01), and ITH had potential value for predicting DFI. Further, we identified BRAF mutation and thyroid differentiation score (TDS) as key factors independently influencing ITH (all p  < 0.05), especially TDS, which had a favorable ability and obtained good net benefit in predicting ITH. TDS maintained a stable negative effect on ITH. In contrast, BRAF mutation positively correlated with ITH in univariable regression (β = 0.196). Through performing sensitivity, regression, subgroup, interaction effect, and mediation analyses, we identified that TDS played a suppression effect in the impact of BRAF mutation on ITH. Finally, we revealed that the propionate metabolism pathway was most strongly associated with ITH. ITH is associated with DFI in patients with PTC and deserves more attention. TDS and BRAF mutation are the key influencing factors of ITH, but TDS exerts a suppression effect on the impact of BRAF mutation on ITH.