NPAS2, transcriptionally activated by ARRB1, promotes the malignant behaviours of lung adenocarcinoma cells and regulates the reprogramming of glucose metabolism.

Lung adenocarcinoma (LUAD) is a serious threat to public health and is accompanied by increased morbidity and mortality worldwide. Neuronal PAS domain protein2 (NPAS2) has been confirmed as an oncogene in LUAD; however, little is known about its molecular mechanism. Here, the expression level of NPAS2 was detected in LUAD cell lines and 16HBE cells. Gain- and loss-of-function experiments were performed. Cell Counting Kit-8, colony formation, flow cytometry, wound-healing and Transwell assays were conducted to assess cell proliferation, apoptosis, migration and invasion, respectively. Reprogramming of glucose metabolism was evaluated via oxygen consumption rate (OCR), complexes activities, lactic production and glucose consumption. The expression of critical proteins was examined by western blot. We demonstrated aberrant upregulation of NPAS2 and ß-arrestin-1 (ARRB1) in LUAD cell lines. ARRB1 was found to be a critical transcription factor of NPAS2 with binding sites within the promoter region of NPAS2, thereby causing its transcriptional activation. Functional experiments revealed that NPAS2 depletion significantly inhibited the malignant behaviours of A549 cells by suppressing cell proliferation, migration, invasion and epithelial-mesenchymal transition and promoting cell apoptosis. Meanwhile, NPAS2 depletion increased OCR and activities of complexes (I, II, III and V), and reduced lactic acid production and glucose uptake in A549 cells, indicating that NPAS2 depletion inhibited aerobic glycolysis, accompanied by reduced expression of glycolytic enzymes. However, the changes caused by NPAS2 knockdown were partly restored by ARRB1 overexpression. In conclusion, our study suggests that ARRB1 could transcriptionally activate NPAS2, facilitating malignant activities and glycolysis, and ultimately promoting the progression of LUAD, proving a novel therapeutic strategy for the treatment of LUAD.

A multi-task fusion model based on a residual-Multi-layer perceptron network for mammographic breast cancer screening.

BACKGROUND AND OBJECTIVE: Deep learning approaches are being increasingly applied for medical computer-aided diagnosis (CAD). However, these methods generally target only specific image-processing tasks, such as lesion segmentation or benign state prediction. For the breast cancer screening task, single feature extraction models are generally used, which directly extract only those potential features from the input mammogram that are relevant to the target task. This can lead to the neglect of other important morphological features of the lesion as well as other auxiliary information from the internal breast tissue. To obtain more comprehensive and objective diagnostic results, in this study, we developed a multi-task fusion model that combines multiple specific tasks for CAD of mammograms. METHODS: We first trained a set of separate, task-specific models, including a density classification model, a mass segmentation model, and a lesion benignity-malignancy classification model, and then developed a multi-task fusion model that incorporates all of the mammographic features from these different tasks to yield comprehensive and refined prediction results for breast cancer diagnosis. RESULTS: The experimental results showed that our proposed multi-task fusion model outperformed other related state-of-the-art models in both breast cancer screening tasks in the publicly available datasets CBIS-DDSM and INbreast, achieving a competitive screening performance with area-under-the-curve scores of 0.92 and 0.95, respectively. CONCLUSIONS: Our model not only allows an overall assessment of lesion types in mammography but also provides intermediate results related to radiological features and potential cancer risk factors, indicating its potential to offer comprehensive workflow support to radiologists.

Herbal compound cepharanthine attenuates inflammatory arthritis by blocking macrophage M1 polarization.

OBJECTIVE: Cepharanthine (CEP) is a drug candidate for tumor, viral infection, and some inflammatory diseases, but its effect on rheumatoid arthritis (RA) and the underlying mechanism are incompletely understood. METHODS: CEP was administered intraperitoneally to a collagen-induced arthritis (CIA) model. Joints went radiological and histological examination and serum cytokines were examined with cytometry-based analysis. M1 macrophages were induced from THP-1 cells or mouse bone marrow-derived macrophages with LPS and IFN-γ. Bulk RNA-seq was performed on macrophage undergoing M1-polarizatioin. Western blotting was applied to determine pathways involved in monocyte chemotaxis and polarization. Glycolysis metabolites were measured by chemiluminescence while glycolytic enzymes were examined by quantitative PCR. RESULTS: We found CEP significantly ameliorated synovial inflammation and joint destruction of CIA mice. It downregulated TNF-α levels in serum and in joints. The number of M1 macrophages were reduced in CEP-treated mice. In vitro, CEP inhibited monocyte chemotaxis to MCP-1 by downregulating CCR2 and reducing ERK1/2 signaling. Additionally, CEP suppressed M1 polarization of macrophages induced by LPS and IFN-γ. Genes involved in IFN-γ signaling, IL-6-JAK/STAT3 signaling, glycolysis, and oxidative phosphorylation process were downregulated by CEP. Several enzymes critically involved in glycolytic metabolism were suppressed by CEP, which resulted in reduced citrate in M1-polarizing macrophages. The inhibitory effect of CEP on macrophage polarization might be attributed to the blockage of TLRs-MyD88/IRAK4-IRF5 signaling pathway together with suppression of overactivated glycolytic metabolism in M1-polarizing macrophages. CONCLUSION: CEP attenuated joint inflammation by suppressing monocyte chemotaxis and proinflammatory differentiation. It has the potential to be developed into a complementary or alternative therapy for RA.

Role of soluble epoxide hydrolase in the abnormal activation of fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by enigmatic pathogenesis. Polyunsaturated fatty acids (PUFAs) are implicated in RA's development and progression, yet their exact mechanisms of influence are not fully understood. Soluble epoxide hydrolase (sEH) is an enzyme that metabolizes anti-inflammatory epoxy fatty acids (EpFAs), derivatives of PUFAs. In this study, we report elevated sEH expression in the joints of CIA (collagen-induced arthritis) rats, concomitant with diminished levels of two significant EpFAs. Additionally, increased sEH expression was detected in both the synovium of CIA rats and in the synovium and fibroblast-like synoviocytes (FLS) of RA patients. The sEH inhibitor TPPU attenuated the migration and invasion capabilities of FLS derived from RA patients and to reduce the secretion of inflammatory factors by these cells. Our findings indicate a pivotal role for sEH in RA pathogenesis and suggest that sEH inhibitors offer a promising new therapeutic strategy for managing RA.

Multi-view fusion-based local-global dynamic pyramid convolutional cross-tansformer network for density classification in mammography.

Object.Breast density is an important indicator of breast cancer risk. However, existing methods for breast density classification do not fully utilise the multi-view information produced by mammography and thus have limited classification accuracy.Method.In this paper, we propose a multi-view fusion network, denoted local-global dynamic pyramidal-convolution transformer network (LG-DPTNet), for breast density classification in mammography. First, for single-view feature extraction, we develop a dynamic pyramid convolutional network to enable the network to adaptively learn global and local features. Second, we address the problem exhibited by traditional multi-view fusion methods, this is based on a cross-transformer that integrates fine-grained information and global contextual information from different views and thereby provides accurate predictions for the network. Finally, we use an asymmetric focal loss function instead of traditional cross-entropy loss during network training to solve the problem of class imbalance in public datasets, thereby further improving the performance of the model.Results.We evaluated the effectiveness of our method on two publicly available mammography datasets, CBIS-DDSM and INbreast, and achieved areas under the curve (AUC) of 96.73% and 91.12%, respectively.Conclusion.Our experiments demonstrated that the devised fusion model can more effectively utilise the information contained in multiple views than existing models and exhibits classification performance that is superior to that of baseline and state-of-the-art methods.

Risk Factors to Predict Post-Operative Organ/Space Infection After Appendectomy in the Pediatric Population: A Retrospective Case Control Analysis.

Background: Post-operative organ/space infection (OSI) is associated with prolonged hospital stay or re-admission. Here, we explore the predictors for OSI in pediatric patients after appendectomy. Patients and Methods: Among post-appendectomy pateints, the OSI was reviewed. A multicenter case control study was conducted to explore the risk factors for OSI among pediatric patients with appendicitis after appendectomy between January 2009 and December 2019. The potential risk factors associated with OSI were explored using multivariable logistic regression methods. Results: In the current cohort, 723 patients fulfilled the OSI criteria. According to multivariable logistic regression analysis, the occurrence of OSI was associated with complicated appendicitis (odds ratio [OR], 1.82; 95% confidence interval [CI], 1.03-3.686; p = 0.016), the lower the level of pre-operative lymphocyte-C-reactive protein (LCR; OR, 14.42; 95% CI, 1.57-73.26; p < 0.001), pan-peritonitis (OR, 4.36; 95% CI, 1.34-21.66; p = 0.006), systemic inflammatory response syndrome (SIRS; OR, 8.22; 95% CI, 1.84-49.63; p < 0.001), and abscess presentation (OR, 11.32; 95% CI, 2.03-61.86; p < 0.001). The receiver operating characteristic (ROC) curve evaluation further confirmed the relatively high accuracy of the above factors to predict OSI. Conclusions: The identified potential risk factors in the current research can be used for OSI identification in patients after appendectomy. Recognition of the risk factors may allow the choice of the treatment process more reasonably.

A20 affects macrophage polarization through the NLRP3 inflammasome signaling pathway and promotes breast cancer progression.

Breast cancer is the most common malignant tumor in females, and the majority of patients succumb to metastasis. The present study aimed to investigate the association between tumor necrosis factor alpha-induced protein 3 (A20), NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) and tumor-associated macrophage polarization, and their effects on the proliferation and metastasis of breast cancer cells. The expression of A20 in breast cancer cells was analyzed by reverse transcription-quantitative PCR (RT-qPCR) and western blotting. RT-qPCR and western blotting were also used to confirm the transfection efficiency. The viability, clone formation, migration, invasion and angiogenesis of transfected breast cancer cells were detected by Cell Counting Kit-8, colony formation, wound healing, Transwell and tube formation assays, respectively. Activated macrophages, namely M1 and M2 type macrophages, were observed by double staining immunofluorescence. The levels of M1 and M2 macrophage markers were analyzed by qPCR. The expression of angiogenesis-related proteins and NLRP3 inflammasome activation-associated proteins was detected by western blotting. The results revealed that A20 was highly expressed in breast cancer cells. Interference with A20 inhibited the proliferation, invasion, migration and angiogenesis of breast cancer cells, and inhibited the M2-like polarization of macrophages. Interference with A20 promoted the activation of the NLRP3 inflammasome. The NLRP3 inhibitor MCC950 alleviated the effect of interference with A20 to promote macrophage proliferation and recruitment, as well as M2-like polarization. In conclusion, interference with A20 inhibited macrophage proliferation and M2-like polarization through the NLRP3 inflammasome signaling pathway to inhibit breast cancer progression.

Fecal microbiota transplantation from patients with rheumatoid arthritis causes depression-like behaviors in mice through abnormal T cells activation.

Depression is common in patients with rheumatoid arthritis (RA); however, the precise mechanisms underlying a link between depression and RA remain unclear. Accumulating evidence suggests the role of gut-microbiota-brain axis in depression. In this study, we investigated whether collagen-induced arthritis (CIA) mice produce depression-like behaviors and abnormal composition of gut microbiota. Furthermore, we investigated whether fecal microbiota transplantation (FMT) from RA patients causes depression-like phenotypes in antibiotic cocktail (ABX)-treated mice. CIA mice displayed depression-like behaviors, increased blood levels of pro-inflammatory cytokine interleukin-6 (IL-6), decreased expression of synaptic proteins in the prefrontal cortex (PFC), and abnormal composition of gut microbiota. Furthermore, FMT from RA patients caused depression-like phenotypes, alterations of gut microbiota composition, increased levels of IL-6 and tumor necrosis factor-α (TNF-α), and downregulation of synaptic proteins in the PFC compared to FMT from healthy controls. There were correlations between relative abundance of microbiota and plasma cytokines, expression of synaptic proteins in the PFC or depression-like behaviors. Interestingly, FMT from RA patients induced T cells differentiation in Peyer's patches and spleen. Reduced percentage of Treg cells with an increase of Th1/Th2 index was observed in the mice after FMT from RA patients. These findings suggest that CIA mice exhibit depression-like behaviors, systemic inflammation, and abnormal composition of gut microbiota, and that FMT from RA patients produces depression-like behaviors in ABX-treated mice via T cells differentiation. Therefore, abnormalities in gut microbiota in RA patients may contribute to depression via gut-microbiota-brain axis.

Dilated and soft attention-guided convolutional neural network for breast cancer histology images classification.

Breast cancer is one of the most common types of cancer in women, and histopathological imaging is considered the gold standard for its diagnosis. However, the great complexity of histopathological images and the considerable workload make this work extremely time-consuming, and the results may be affected by the subjectivity of the pathologist. Therefore, the development of an accurate, automated method for analysis of histopathological images is critical to this field. In this article, we propose a deep learning method guided by the attention mechanism for fast and effective classification of haematoxylin and eosin-stained breast biopsy images. First, this method takes advantage of DenseNet and uses the feature map's information. Second, we introduce dilated convolution to produce a larger receptive field. Finally, spatial attention and channel attention are used to guide the extraction of the most useful visual features. With the use of fivefold cross-validation, the best model obtained an accuracy of 96.47% on the BACH2018 dataset. We also evaluated our method on other datasets, and the experimental results demonstrated that our model has reliable performance. This study indicates that our histopathological image classifier with a soft attention-guided deep learning model for breast cancer shows significantly better results than the latest methods. It has great potential as an effective tool for automatic evaluation of digital histopathological microscopic images for computer-aided diagnosis.

Effect of inward rectifier potassium 2.1 channel on the osteogenic differentiation of human dental follicle cells and its mechanism.

OBJECTIVES: This study aims to explore the effect of inward rectifier potassium (Kir) 2.1 channel on the osteogenic differentiation of human dental follicle cells (hDFCs) and its mechanism. METHODS: hDFCs were isolated and cultured, and their source was verified by flow cytometry. Osteogenic differentiation ability of hDFCs was evaluated by osteogenic induction. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to detect the gene expression of Kir2.1 gene (KCNJ2) in hDFCs. Real-time quantitative PCR (RT-qPCR) was performed to detect the expression of the Kir2.1 gene (KCNJ2) in hDFCs before and after osteogenic induction. Patch clamp technique was conducted to record the membrane potential changes of hDFCs before and after osteogenic induction. Moreover, the effect on the osteogenic differentiation of hDFCs was confirmed by increasing the concentration of extracellular potassium ions (50 mmol·L-1). Kir2.1 channel blockers cesium chloride (CsCl) and C19H20CINO (ML133) were applied to determine the effect of the Kir2.1 potassium channel on the osteogenic differentiation of hDFCs. At the same time, RT-qPCR was used to observe the expression changes of osteogenic differentiation related genes Runx related transcription factor 2 (Runx2) and osteocalcin (OCN) before and after the two intervention measures. Calcium imaging was performed to observe the effect of membrane potential hyperpolarization caused by decreased extracellular potassium level (2 mmol·L-1) on intracellular calcium concentration. RESULTS: RT-PCR results showed that hDFCs expressed the Kir2.1 channel gene (KCNJ2). The RT-qPCR results showed that the KCNJ2 gene expression in hDFCs was upregulated 7 days after osteogenic induction. The patch clamp results showed that the membrane potential of hDFCs hyperpolarized to (-47±5.2) mV from (-12±3.2) mV. Alizarin red and alkaline phosphatase staining results showed that increasing the concentration of the extracellular potassium or blocking the function of the Kir2.1 channel significantly inhibited the osteogenic mineralization ability of hDFCs. The membrane potential hyperpolarization increased the intracellular calcium concentration in hDFCs. CONCLUSIONS: Membrane potential hyperpolarization mediated by the Kir2.1 channel plays an important role in the osteogenic differentiation of hDFCs.

Cinacalcet Targets the Neurokinin-1 Receptor and Inhibits PKCδ/ERK/P65 Signaling to Alleviate Dextran Sulfate Sodium-Induced Colitis.

Objective: Inflammatory bowel disease is an immune-mediated chronic inflammatory disease of the gastrointestinal tract for which curative drugs are currently not available. This study was performed to assess the therapeutic effects of cinacalcet on dextran sulfate sodium (DSS)-induced colitis. Methods: Primary macrophages obtained from bone marrow and the macrophage cell line RAW264.7 were used to examine the inhibitory effect of cinacalcet on cytokine production, the PKCδ/ERK/P65 signaling pathway, and NF-κB P65 translocation. Colitis was induced using DSS to assess the treatment effect of cinacalcet. Bioinformatics approaches were adopted to predict potential targets of cinacalcet, and a drug affinity responsive target stability (DARTs) assay was performed to confirm binding between cinacalcet and potential target. Results: In vivo analysis showed that cinacalcet reduced the disease activity score, prevented shortening of the colon, diminished inflammatory cell infiltration, and protected the structural integrity of the intestinal wall. Cinacalcet also reduced production of the inflammatory cytokines TNFα, IL-1ß, and IL-6 in the colon and sera of mice with DSS-induced colitis. In vitro studies revealed that cinacalcet suppressed the translocation of P65 and inhibited production of the inflammatory cytokines IL-1ß and IL-6. Mechanistic studies revealed that the target of cinacalcet was neurokinin-1 receptor (NK1R) and their binding was confirmed by a DARTs assay. Furthermore, the inhibition of NK-κB P65 activation was found to occur via the suppression of PKCδ/ERK/P65 signaling mediated by cinacalcet. Conclusion: Cinacalcet inhibits the activation of NF-κB and reduces the production of inflammatory cytokines by suppressing the PKCδ/ERK/P65 signaling pathway via targeting NK1R, suggesting that it can be used to treat inflammatory diseases, particularly colitis.

Euphorbia Factor L2 ameliorates the Progression of K/BxN Serum-Induced Arthritis by Blocking TLR7 Mediated IRAK4/IKKß/IRF5 and NF-kB Signaling Pathways.

Toll like receptor (TLR)s have a central role in regulating innate immunity and their activation have been highlighted in the pathogenesis of rheumatoid arthritis (RA). EFL2, one of diterpenoids derived from Euphorbia seeds, is nearly unknown expect for its improving effect on acute lung injury. Our present study aimed to investigate EFL2's pharmacokinetic features, its therapeutic effect on rheumatoid arthritis, and explored the potential anti-arthritic mechanisms. K/BxN serum transfer arthritis (STA) murine model was used to assess EFL2's anti-arthritic effects. We also applied UPLC-MS method to measure the concentrations of EFL2 in plasma. The inhibitory effects of this compound on inflammatory cells infiltration and activation were determined by flow cytometry analysis and quantitative real-time polymerase chain reaction (qRT-PCR) in vivo, and immunochemistry staining and ELISA in murine macrophages and human PBMCs in vitro, respectively. The mechanism of EFL2 on TLRs mediated signaling pathway was evaluated by PCR array, Western blot, plasmid transfection and confocal observation. Intraperitoneal (i.p.) injection of EFL2, instead of oral administration, could effectively ameliorate arthritis severity of STA mice. The inflammatory cells migration and infiltration into ankles were also significantly blocked by EFL2, accompanied with dramatically reduction of chemokines mRNA expression and pro-inflammatory cytokines production. In vivo PCR microarray indicated that EFL2 exerted anti-arthritis bioactivity by suppressing TLR7 mediated signaling pathway. In vitro study confirmed the inhibitory effects of EFL2 on TLR7 or TLR3/7 synergistically induced inflammatory cytokines secretion in murine macrophages and human PBMCs. In terms of molecular mechanism, we further verified that EFL2 robustly downregulated TLR7 mediated IRAK4-IKKß-IRF5 and NF-κB signaling pathways activation, and blocked IRF5 and p65 phosphorylation and translocation activity. Taken together, our data indicate EFL2's therapeutic potential as a candidate for rheumatoid arthritis and other TLR7-dependent diseases.

A lysosome targetable fluorescent probe for palladium species detection base on an ESIPT phthalimide derivative.

A novel lysosome-targetable phthalimide fluorescent probe was designed for detecting palladium based on ESIPT for signal transduction. The fluorescent probe conjugating with allylcarbamate displayed weak fluorescent due to the ESIPT process hinder by allylcarbamate. But with the addition of palladium, the ESIPT emission was recovery though the palladium-catalyzed deallylation reaction and the fluorescence intensity exhibited 40-fold enhancement at 511 nm. In addition, the probe showed excellent selectivity, high sensitivity, fast responds and low limit detection for palladium with a larger Stoke-shift. Moreover, the targetable probe was also successfully applied for detecting palladium in lysosomes of living cells. Hence, the probe though ESIPT modulation is a promising for monitoring palladium in practical samples.