Laboratory Investigation最新文献

E3 ubiquitin ligases, crucial enzymes in the ubiquitination pathway, significantly influence the development of malignant tumors, including gastric cancer (GC), by regulating the stability of oncogenic and tumor-suppressive proteins. This study employed bioinformatics analysis of public databases alongside various experimental techniques-tissue arrays, real-time reverse-transcription polymerase chain reaction, western blot, immunofluorescence, and coimmunoprecipitation-to identify and explore the role of HECW1, a pivotal NEDD4 family E3 ubiquitin ligase, in GC progression. The results demonstrated that HECW1 is markedly overexpressed in GC tissues relative to normal gastric tissues, and its elevated expression correlates with poor prognosis in GC patients. In vitro experiments revealed that HECW1 overexpression significantly enhances the metastatic capabilities of GC cells. Mechanistically, HECW1 interacts with HIPK2 to facilitate its ubiquitination and degradation, thereby activating AKT and promoting the expression of downstream epithelial mesenchymal transition-related genes. In vivo experiments confirmed HECW1's role in promoting GC cell metastasis, highlighting the HECW1-HIPK2-AKT signaling axis as critical in GC metastasis. These findings not only elucidate a novel metastasis mechanism of GC but also suggest potential molecular targets for developing new therapeutic strategies against GC.

The canonical Hippo-YAP1 signaling pathway is crucial for liver development and regeneration, but its role in repair and regeneration of intrahepatic bile duct in biliary atresia (BA) remains largely unknown. YAP1 expression in the liver tissues of patients with BA and Rhesus rotavirus-induced experimental BA mouse models were examined using quantitative reverse transcriptase-PCR and double immunofluorescence. Mouse EpCAM-expressing cell-derived liver organoids were generated and treated with Hippo-YAP1 pathway activators (Xmu-mp-1 and TRULI) or an inhibitor (Peptide17). Morphologic, immunofluorescence, RNA-seq, and bioinformatic analyses were performed. Oxidative stress in human intrahepatic biliary epithelial cells transfected with a constitutively active YAP1 (YAPS127A) plasmid was assessed using quantitative reverse transcriptase-PCR and fluorescence-activated cell sorting analysis. PRDX1 expression in BA and experimental BA mouse model livers was examined by double immunofluorescence. The mRNA expression and nuclear localization of YAP1 in EpCAM-expressing bile duct cells were increased in the livers of BA and experimental BA mouse model. Aberrant development of intrahepatic organoids, differential expression of oxidative stress response genes Sod3 and Prdx1, enrichment of oxidative stress, and mitochondrial reactive oxidative stress-associated gene sets were observed in organoids treated with the Hippo-YAP1 activator, whereas organoid development was unaffected by the addition of the Hippo-YAP1 inhibitor. Transfection with constitutively active YAP1 led to the downregulation of PRDX1 and oxidative stress in human intrahepatic biliary epithelial cells. Additionally, reduced PRDX1 expression was also observed in the bile duct of human BA and experimental BA mouse livers. In conclusion, dysregulated activation of Hippo-YAP1 signaling induces oxidative stress and impairs the development of intrahepatic biliary organoids, which indicates therapeutic strategies targeting Hippo-YAP1 signaling may offer the potential to improve biliary repair and regeneration in patients with BA.

Postoperative cognitive dysfunction (POCD) is a common complication with no effective treatment in elderly patients. POCD, Alzheimer disease (AD), and many other cognitive diseases mostly involve neurotoxic microglia response, and recently, β2-microglobulin (B2M) has been suggested to play a pivotal role. A novel pyromeconic acid-styrene hybrid compound D30 was synthesized by our team and shown to be safe and effective in some neurodegenerative mouse models. In this study, we evaluated D30 on POCD and its potential mechanism. Fourteen- to 18-month-old male C57BL/6 mice were used to establish POCD through isoflurane anesthesia and surgery. The plasma of elderly patients was collected pre- and postoperatively. Primary mouse microglia were subjected to various stimulations in multiple experimental designs to imitate in vivo POCD-like conditions. Morris water maze, fear conditioning, western blot, immunofluorescent staining, and blood-brain barrier (BBB) permeability tests were conducted in this study. D30 administration significantly improved learning and memory in aged mice following POCD. Neurotoxic M1 microglia cells were dramatically increased following POCD, manifested as morphologically changing into fewer and shorter branches, enlarged somatic areas, and upregulated expression of iNOS and C1q. Notably, following POCD, B2M was significantly upregulated in the plasma and the brain. D30 treatment significantly suppressed these pathologic changes, by inhibiting the POCD-induced BBB breakdown while suppressing the surge of plasma B2M levels. D30 treatment suppressed POCD-induced surge of B2M and Aβ plaques in the brain and preserved adult hippocampal neurogenesis vulnerable to POCD. Furthermore, postoperative levels of B2M were significantly elevated over the preoperative levels in patients aged 80 years and over. In parallel with mouse plasma after POCD, the postoperative patient plasma was also much more effective at activating M1 microglia. Of note, this POCD plasma-induced activation of M1 microglia was largely prevented by D30 treatment. Taken together, by inhibiting the surge of plasma B2M, protecting BBB integrity, and reducing inflammatory response, D30 protected aged mice from B2M-facilitated POCD.

Olfactory receptor neurons (ORNs) in the olfactory epithelium are characterized by high regenerative capacity even after birth, but the molecular mechanisms involved in ORN regeneration remain unclear. Complement component 3 (C3) has been shown to promote tissue regeneration, so we hypothesized that C3 activates innate immunity and also promotes the regeneration of ORNs. In this study, we investigate the role of C3 in ORN regeneration. We used C3 knockout (KO) and wild-type C57BL/6J mice in this study to examine the olfactory regeneration process for 42 days after methimazole-induced olfactory disorder. To compare the regeneration process after ORN damage between C3 KO and wild-type mice, we conducted olfactory behavioral tests and immunohistologic analysis and examined growth factors and inflammatory cell induction. C3 KO mice showed delayed olfactory recovery with lower olfactory epithelial thickness. In C3 KO mice, ORN maturation was delayed in association with increased accumulation of immature ORNs. In the normal ORN regeneration process, undesirable immature ORNs are produced and eliminated by apoptosis. C3 deficiency reduced neutrophils induced during ORN regeneration, suggesting the involvement of C3 in ORN regeneration through neutrophil-dependent elimination of undesired ORNs. C3 is therefore suggested to have promoted ORN regeneration by preventing the accumulation of immature ORNs. In addition, C3 may assist ORN maturation by participating in ORN axon selection such as synaptic pruning. Our results indicate that C3, which is activated during pathogen infection, also promotes recovery from ORN damage. These findings may lead to new therapeutic strategies for olfactory disorder.

Hirschsprung disease, a congenital disease characterized by the absence of ganglion cells, presents significant surgical challenges. Addressing a critical gap in intraoperative diagnostics, we introduce transformative artificial intelligence approach that significantly enhances the detection of ganglion cells in frozen sections. The data set comprises 366 frozen and 302 formalin-fixed-paraffin-embedded hematoxylin and eosin-stained slides obtained from 164 patients from 3 centers. The ganglion cells were annotated on the whole-slide images (WSIs) using bounding boxes. Tissue regions within WSIs were segmented and split into patches of 2000 × 2000 pixels. A deep learning pipeline utilizing ResNet-50 model for feature extraction and gradient-weighted class activation mapping algorithm to generate heatmaps for ganglion cell localization was employed. The binary classification performance of the model was evaluated on independent test cohorts. In the multireader study, 10 pathologists assessed 50 frozen WSIs, with 25 slides containing ganglion cells, and 25 slides without. In the first phase of the study, pathologists evaluated the slides as a routine practice. After a 2-week washout period, pathologists re-evaluated the same WSIs along with the 4 patches with the highest probability of containing ganglion cells. The proposed deep learning approach achieved an accuracy of 91.3%, 92.8%, and 90.1% in detecting ganglion cells within WSIs in the test data set obtained from centers. In the reader study, on average, the pathologists' diagnostic accuracy increased from 77% to 85.8% with the model's heatmap support, whereas the diagnosis time decreased from an average of 139.7 to 70.5 seconds. Notably, when applied in real-world settings with a group of pathologists, our model's integration brought about substantial improvement in diagnosis precision and reduced the time required for diagnoses by half. This notable advance in artificial intelligence-driven diagnostics not only sets a new standard for surgical decision making in Hirschsprung disease but also creates opportunities for its wider implementation in various clinical settings, highlighting its pivotal role in enhancing the efficacy and accuracy of frozen sections analyses.

Superficial malignant peripheral nerve sheath tumors (SF-MPNSTs) are rare cancers and can be difficult to distinguish from spindle cell (SCM) or desmoplastic (DM) melanomas. Their biology is poorly understood. We performed whole-exome sequencing and RNA sequencing (RNA-seq) on SF-MPNST (n = 8) and compared them with cases of SCM (n = 7), DM (n = 8), and deep MPNST (D-MPNST, n = 8). Immunohistochemical staining for H3K27me3 and PRAME was also performed. SF-MPNST demonstrated intermediate features between D-MPNST and melanoma. Patients were younger than those with melanoma and older than those with D-MPNST; the outcome was worse and better, respectively. SF-MPNST tumor mutational burden (TMB) was higher than D-MPNST and lower than melanoma; differences were significant only between SF-MPNST and SCM (P = .0454) and between D-MPNST and SCM (P = .001, Dunn's Kruskal-Wallis post hoc test). Despite having an overlapping mutational profile in some common cancer-associated genes, the COSMIC mutational signatures clustered DM and SCM together with UV light exposure signatures (SBS7a, 7b), and SF- and D-MPNST together with defective DNA base excision repair (SBS30, 36). RNA-seq revealed differentially expressed genes between SF-MPNST and SCM (1670 genes), DM (831 genes), and D-MPNST (614 genes), some of which hold promise for development as immunohistochemical markers (SOX8 and PLCH1) or aids (MLPH, CALB2, SOX11, and TBX4). H3K27me3 immunoreactivity was diffusely lost in most D-MPNSTs (7/8, 88%) but showed variable and patchy loss in SF-MPNSTs (2/8, 25%). PRAME was entirely negative in the majority (0+ in 20/31, 65%), including 11/15 melanomas, and showed no significant difference between groups (P = .105, Kruskal-Wallis test). Expression of immune cell transcripts was upregulated in melanomas relative to MPNSTs. Next-generation sequencing revealed multiple differential features between SF- MPNST, D-MPNST, SCM, and DM, including tumor mutation burden, mutational signatures, and differentially expressed genes. These findings help advance our understanding of disease pathogenesis and improve diagnostic modalities.

Accurate whole-cell segmentation is essential in various biomedical applications, particularly in studying the tumor microenvironment. Despite advancements in machine learning for nuclei segmentation in hematoxylin and eosin (H&E)-stained images, there remains a need for effective whole-cell segmentation methods. This study aimed to develop a deep learning-based pipeline to automatically segment cells in H&E-stained tissues, thereby advancing the capabilities of pathological image analysis. The Cell Segmentation with Globally Optimized boundaries (CSGO) framework integrates nuclei and membrane segmentation algorithms, followed by postprocessing using an energy-based watershed method. Specifically, we used the You Only Look Once (YOLO) object detection algorithm for nuclei segmentation and U-Net for membrane segmentation. The membrane detection model was trained on a data set of 7 hepatocellular carcinomas and 11 normal liver tissue patches. The cell segmentation performance was extensively evaluated on 5 external data sets, including liver, lung, and oral disease cases. CSGO demonstrated superior performance over the state-of-the-art method Cellpose, achieving higher F1 scores ranging from 0.37 to 0.53 at an intersection over union threshold of 0.5 in 4 of the 5 external datasets, compared to that of Cellpose from 0.21 to 0.36. These results underscore the robustness and accuracy of our approach in various tissue types. A web-based application is available at https://ai.swmed.edu/projects/csgo, providing a user-friendly platform for researchers to apply our method to their own data sets. Our method exhibits remarkable versatility in whole-cell segmentation across diverse cancer subtypes, serving as an accurate and reliable tool to facilitate tumor microenvironment studies. The advancements presented in this study have the potential to significantly enhance the precision and efficiency of pathologic image analysis, contributing to better understanding and treatment of cancer.