Distribution of multi-level B cell subsets in thymoma and thymoma-associated myasthenia gravis.

B-cell subsets in peripheral blood (PB) and tumor microenvironment (TME) were evaluated to determine myasthenia gravis (MG) severity in patients with thymoma-associated MG (TMG) and the distribution of B cells in type B TMG. The distribution of mature B cells, including Bm1-Bm5, CD19+ and CD20+ B cells and non-switched (NSMBCs) and switched (SMBCs) memory B cells, were determined in 79 patients with thymoma or TMG. Quantitative relationships between the T and TMG groups and the TMG-low and TMG-high subgroups were determined. NSMBCs and SMBCs were compared in TME and PB. Type B thymoma was more likely to develop into MG, with types B2 and B3 being especially associated with MG worsening. The percentage of CD19+ B cells in PB gradually increased, whereas the percentage of CD20+ B cells and the CD19/CD20 ratio were not altered. The (Bm2 + Bm2')/(eBm5 + Bm5) index was significantly higher in the TMG-high than in thymoma group. The difference between SMBC/CD19+ and NSMBC/CD19+ B cell ratios was significantly lower in the thymoma than TMG group. NSMBCs assembled around tertiary lymphoid tissue in thymomas of patients with TMG. Few NSMBCs were observed in patients with thymoma alone, with these cells being diffusely distributed. MG severity in patients with TMG can be determined by measuring CD19+ B cells and Bm1-Bm5 in PB. The CD19/CD20 ratio is a marker of disease severity in TMG patients. Differences between NSMBCs and SMBCs in PB and TME of thymomas can synergistically determine MG severity in patients with TMG.

Targeting Src SH3 domain-mediated glycolysis of HSC suppresses transcriptome, myofibroblastic activation, and colorectal liver metastasis.

BACKGROUND AND AIMS: Transforming growth factor-beta 1 (TGFß1) induces HSC activation into metastasis-promoting cancer-associated fibroblasts (CAFs), but how the process is fueled remains incompletely understood. We studied metabolic reprogramming induced by TGFß1 in HSCs. APPROACHES AND RESULTS: Activation of cultured primary human HSCs was assessed by the expression of myofibroblast markers. Glucose transporter 1 (Glut1) of murine HSC was disrupted by Cre recombinase/LoxP sequence derived from bacteriophage P1 recombination (Cre/LoxP). Plasma membrane (PM) Glut1 and glycolysis were studied by biotinylation assay and the Angilent Seahorse XFe96 Analyzer. S.c. HSC/tumor co-implantation and portal vein injection of MC38 colorectal cancer cells into HSC-specific Glut1 knockout mice were performed to determine in vivo relevance. Transcriptome was obtained by RNA sequencing of HSCs and spatialomics with MC38 liver metastases. TGFß1-induced CAF activation of HSCs was accompanied by elevation of PM Glut1, glucose uptake, and glycolysis. Targeting Glut1 or Src by short hairpin RNA, pharmacologic inhibition, or a Src SH3 domain deletion mutant abrogated TGFß1-stimulated PM accumulation of Glut1, glycolysis, and CAF activation. Mechanistically, binding of the Src SH3 domain to SH3 domain-binding protein 5 led to a Src/SH3 domain-binding protein 5/Rab11/Glut1 complex that activated Rab11-dependent Glut1 PM transport under TGFß1 stimulation. Deleting the Src SH3 domain or targeting Glut1 of HSCs by short hairpin RNA or Cre recombinase/LoxP sequence derived from bacteriophage P1 recombination suppressed CAF activation in mice and MC38 colorectal liver metastasis. Multi-omics revealed that Glut1 deficiency in HSCs/CAFs suppressed HSC expression of tumor-promoting factors and altered MC38 transcriptome, contributing to reduced MC38 liver metastases. CONCLUSION: The Src SH3 domain-facilitated metabolic reprogramming induced by TGFß1 represents a target to inhibit CAF activation and the pro-metastatic liver microenvironment.

Downregulation of HMGB1 in thymoma cells affects T cell differentiation.

Introduction: Thymoma is the most common anterior mediastinal tumor and is closely associated with myasthenia gravis (MG). Our previous study showed that the expression of Th17 cells increased and the expression of Treg decreased in MG-associated thymoma tissues and peripheral blood. High mobility group box 1 (HMGB1) is an inflammatory mediator and participates in the pathogenesis of various autoimmune diseases. However, its function in thymoma is still unclear. Material and methods: We first analyzed immune indices in peripheral blood of patients with MG-associated thymoma and patients with thymoma alone. Next, we explored the expression of HMGB1 in MG-associated thymoma and thymoma alone tissues. Furthermore, we transfected si-HMGB1 in thymoma cell line Thy0517 and co-cultured Thy0517 with peripheral blood mononuclear cells (PBMC). Results: In this study, the levels of IgG, C3, C4, CRP and globulins in peripheral blood of patients with MG-associated thymoma were different from those of patients with thymoma alone (p < 0.05). The expression of HMGB1 in MG-associated thymoma tissues was higher than thymoma alone. Co-culture of Thy0517 and PBMC showed that the percentage of Th17 cells in PBMC was lower than that in the control group, and the percentage of Treg cells was higher than that in the control group. Conclusions: These findings demonstrate that HMGB1 is involved in the mechanism of abnormal Th17/Treg cell differentiation in thymoma and affects the occurrence of immune abnormalities in MG-associated thymoma.

Metabolic reprogramming and its clinical implication for liver cancer.

Cancer cells often encounter hypoxic and hypo-nutrient conditions, which force them to make adaptive changes to meet their high demands for energy and various biomaterials for biomass synthesis. As a result, enhanced catabolism (breakdown of macromolecules for energy production) and anabolism (macromolecule synthesis from bio-precursors) are induced in cancer. This phenomenon is called "metabolic reprogramming," a cancer hallmark contributing to cancer development, metastasis, and drug resistance. HCC and cholangiocarcinoma (CCA) are 2 different liver cancers with high intertumoral heterogeneity in terms of etiologies, mutational landscapes, transcriptomes, and histological representations. In agreement, metabolism in HCC or CCA is remarkably heterogeneous, although changes in the glycolytic pathways and an increase in the generation of lactate (the Warburg effect) have been frequently detected in those tumors. For example, HCC tumors with activated ß-catenin are addicted to fatty acid catabolism, whereas HCC tumors derived from fatty liver avoid using fatty acids. In this review, we describe common metabolic alterations in HCC and CCA as well as metabolic features unique for their subsets. We discuss metabolism of NAFLD as well, because NAFLD will likely become a leading etiology of liver cancer in the coming years due to the obesity epidemic in the Western world. Furthermore, we outline the clinical implication of liver cancer metabolism and highlight the computation and systems biology approaches, such as genome-wide metabolic models, as a valuable tool allowing us to identify therapeutic targets and develop personalized treatments for liver cancer patients.

Improving Image Quality by Deconvolution Recovery Filter in Ultrasound Imaging.

Due to the advantages of non-radiation and real-time performance, ultrasound imaging is essential in medical imaging. Image quality is affected by the performance of the transducer in an ultrasound imaging system. For example, the bandwidth controls the pulse length, resulting in different axial resolutions. Therefore, a transducer with a large bandwidth helps to improve imaging quality. However, large bandwidths lead to increased system cost and sometimes a loss of sensitivity and lateral resolution in attenuating media. In this paper, a deconvolution recovery method combined with a frequency-domain filtering technique (DRF) is proposed to improve the imaging quality, especially for the axial resolution. In this method, the received low-bandwidth echo signals are converted into high-bandwidth signals, which is similar to the echo signals produced by a high-bandwidth transducer, and the imaging quality is improved. Simulation and experiment results show that, compared with Delay-and-sum (DAS) method, the DRF method improved axial resolution from 0.60 to 0.41 mm in simulation and from 0.62 to 0.47 mm in the tissue-mimicking phantom experiment. The contrast ratio performance is improved to some extent compared with the DAS in experimental and in-vivo images. Besides, the proposed method has the potential to further improve image quality by combining it with adaptive weightings, such as the minimum variance method.

An adaptive beamformer based on dynamic phase coherence factor for pixel-based medical ultrasound imaging.

BACKGROUND: Pixel-based beamforming realizes dynamic focusing at the pixel level with a focused beam by assuming that the received signals are composed of spherical pulses. Far-focused pixel-based (FPB) imaging was proposed to avoid artifacts around the focal depth. However, the contrast improvement is limited. OBJECTIVE: We propose an adaptive weighting method based on dynamic phase coherence factor (DPCF) to improve the image contrast while preserving the speckle pattern. METHODS: The phase variation is dynamically estimated based on the noise energy proportion of echo signals and it is used to calculate phase coherence weights for suppressing interference and preserving desired signals. A depth-dependent parameter is designed for DPCF to enhance the performance of noise and clutter suppression in the far-field region. We further use the subarray averaging technique to smooth the speckle texture. RESULTS: The proposed method was evaluated on simulated, phantom experimental, and in vivo data. Results show that, compared with the phase coherence factor (PCF) based method, DPCF respectively leads to average CR improvements by more than 60% and 24% in simulation and experiment, while obtaining an improved speckle signal-to-noise ratio. CONCLUSIONS: The proposed method is a potentially valuable approach to obtaining high-quality ultrasound images in clinical applications.

Improving axial resolution based on the deconvolution recovery method combined with adaptive weighting techniques for ultrasound imaging.

BACKGROUND: A fundamental challenge in medical ultrasound imaging is to improve the resolution accurately. Adaptive beamforming is often used to improve lateral resolution, such as minimum variance (MV) and phase coherence factor (PCF). However, it is difficult to improve the axial resolution due to the limitation of the spatial pulse length (SPL) of the transmitted signal. OBJECTIVE: A deconvolution recovery method combines two adaptive weighting techniques to improve axial resolution. METHODS: A deconvolution recovery (DR) technique is used to improve axial resolution with a shorter SPL. Then, the DR is combined with MV and PCF (DR-MVPCF) to suppress the sidelobe. The influence of different transmission modes, regularization parameters, and the estimation of point spread function are discussed on the proposed algorithm. RESULTS: In simulation, DR-MVPCF improved axial resolution from 0.41 mm (0.98 λ) to 0.09 mm (0.21 λ) compared with MV-PCF. In the water bath experiment, DR-MVPCF provided improvement of axial resolution from 0.39 mm (0.93 λ) to 0.07 mm (0.17 λ) compared with MV-PCF. In-vivo data experiment, the DR-MVPCF method increased the speckle signal-to-noise ratio and visibility of the structure while the contrast ratio and contrast-noise ratio decreased. CONCLUSIONS: The proposed method can improve the axial resolution significantly.

Minimum variance beamforming combined with covariance matrix-based adaptive weighting for medical ultrasound imaging.

BACKGROUND: The minimum variance (MV) beamformer can significantly improve the image resolution in ultrasound imaging, but it has limited performance in noise reduction. We recently proposed the covariance matrix-based statistical beamforming (CMSB) for medical ultrasound imaging to reduce sidelobes and incoherent clutter. METHODS: In this paper, we aim to improve the imaging performance of the MV beamformer by introducing a new pixel-based adaptive weighting approach based on CMSB, which is named as covariance matrix-based adaptive weighting (CMSAW). The proposed CMSAW estimates the mean-to-standard-deviation ratio (MSR) of a modified covariance matrix reconstructed by adaptive spatial smoothing, rotary averaging, and diagonal reducing. Moreover, adaptive diagonal reducing based on the aperture coherence is introduced in CMSAW to enhance the performance in speckle preservation. RESULTS: The proposed CMSAW-weighted MV (CMSAW-MV) was validated through simulation, phantom experiments, and in vivo studies. The phantom experimental results show that CMSAW-MV obtains resolution improvement of 21.3% and simultaneously achieves average improvements of 96.4% and 71.8% in average contrast and generalized contrast-to-noise ratio (gCNR) for anechoic cyst, respectively, compared with MV. in vivo studies indicate that CMSAW-MV improves the noise reduction performance of MV beamformer. CONCLUSION: Simulation, experimental, and in vivo results all show that CMSAW-MV can improve resolution and suppress sidelobes and incoherent clutter and noise. These results demonstrate the effectiveness of CMSAW in improving the imaging performance of MV beamformer. Moreover, the proposed CMSAW with a computational complexity of [Formula: see text] has the potential to be implemented in real time using the graphics processing unit.

Pulmonary perfusion imaging and delayed imaging to measure pulmonary capillary permeability in pulmonary contusion.

PURPOSE: Explore the application value of pulmonary perfusion imaging and delayed imaging for evaluating pulmonary capillary permeability. MATERIALS AND METHODS: After establishing a rat model of pulmonary contusion, changes in the metabolic index of technetium-99m macroaggregated albumin (99mTC-MAA) in the lungs of model rats were evaluated for two consecutive days. 99mTC-MAA metabolic indices of rat lungs with pulmonary contusion of varying severity (mild, moderate, and severe) were correlated with lung wet/dry weight ratio (W/D) and Evans blue extravasation. Finally, the method was validated in patients with pulmonary contusion and one healthy volunteer. RESULTS: The 99mTC-MAA metabolic index was 23.56% ± 2.44% in healthy control (HC) rat lung, 8.56% ± 3.42% immediately after lung contusion (d0), 8.35% ± 3.20% after 1 day (d1), and 17.45% ± 6.44% after 2 days (d2); indices at d0 and d1 were significantly higher than those at HC (P < 0.05). The metabolic index of 99mTC-MAA in lung had significant negative correlations with W/D (r = -0.8025; P = 0.0092) and Evans blue extravasation (r = -0.9356; P = 0.0002). Metabolic and oxygenation indices of 99mTC-MAA exhibited a significant positive linear correlation in patients with pulmonary contusion (r = 0.8925; P = 0.0416). CONCLUSION: Pulmonary perfusion and delayed imaging of 99mTC-MAA have potential value for evaluating pulmonary capillary permeability.

PD-L1 promotes myofibroblastic activation of hepatic stellate cells by distinct mechanisms selective for TGF-ß receptor I versus II.

Intrahepatic cholangiocarcinoma (ICC) contains abundant myofibroblasts derived from hepatic stellate cells (HSCs) through an activation process mediated by TGF-ß. To determine the role of programmed death-ligand 1 (PD-L1) in myofibroblastic activation of HSCs, we disrupted PD-L1 of HSCs by shRNA or anti-PD-L1 antibody. We find that PD-L1, produced by HSCs, is required for HSC activation by stabilizing TGF-ß receptors I (TßRI) and II (TßRII). While the extracellular domain of PD-L1 (amino acids 19-238) targets TßRII protein to the plasma membrane and protects it from lysosomal degradation, a C-terminal 260-RLRKGR-265 motif on PD-L1 protects TßRI mRNA from degradation by the RNA exosome complex. PD-L1 is required for HSC expression of tumor-promoting factors, and targeting HSC PD-L1 by shRNA or Cre/loxP recombination suppresses HSC activation and ICC growth in mice. Thus, myofibroblast PD-L1 can modulate the tumor microenvironment and tumor growth by a mechanism independent of immune suppression.

Mig-6 could inhibit cell proliferation and induce apoptosis in esophageal squamous cell carcinoma.

BACKGROUND: To investigate the expression and biological functions of mitogen-induced gene 6 (Mig-6) in esophageal squamous cell carcinoma (ESCC). METHODS: The expression of Mig-6 in ESCC tissues and normal esophageal epithelial tissues were measured by immunohistochemistry (IHC) assay. MTT test was applied to detect the proliferative ability of ESCC cells after Mig-6 was upregulated by transfection. A fluid cytology assay was used to detect apoptosis of ESCC cells. Agilent whole human genome oligo microarray was used to screen different expressed genes and the possible signaling pathways which might be involved. RESULTS: The expression of Mig-6 protein was lower in ESCC tissues compared to normal esophageal epithelial tissues. Mig-6 could restrain the ESCC cell growth and induce cell apoptosis. PPAR, CAMs and MAPK signaling pathways might be involved. CONCLUSIONS: Mig-6 might be a new tumor suppressor gene and a possible target for the specific therapy of ESCC.

Covariance Matrix-Based Statistical Beamforming for Medical Ultrasound Imaging.

Medical ultrasound image quality is often limited by clutter, which is the dominant mechanism of image degradation. A variety of beamforming methods have been extensively studied to reduce clutter and, thus, enhance ultrasound image quality. This article introduces a new beamforming approach, called covariance matrix-based statistical beamforming (CMSB), to improve the image contrast and preserve the background speckle pattern while simultaneously achieving a high-resolution performance. In CMSB, adaptive selection of subarray length, diagonal reducing, and mean-to-standard-deviation ratio-based subarray averaging are inherently combined to differentiate and reduce off-axis energy effectively. Moreover, rotary averaging prior to diagonal reducing is introduced to preserve speckle statistics. Simulated, experimental, and in vivo datasets were used to evaluate the imaging performance of the proposed method. The quantitative results indicate that, compared with delay-and-sum (DAS) beamforming, CMSB leads to average improvements of 44.5% and 97.3% in lateral resolution and contrast, respectively, in phantom experiments. Our work shows that CMSB is capable of improving image resolution and contrast while maintaining the speckle reliably. Preliminary in vivo study also demonstrates that the CMSB can enhance image contrast and lesion detection.

Two types of immune infiltrating cells and six hub genes can predict the occurrence of myasthenia gravis in patients with thymoma.

Thymoma is the most common primary mass in anterior mediastinum. Although associated with low malignancy, it is often accompanied by myasthenia gravis resulting in poor prognosis. Due to the dual factors of tumor immune tolerance and autoimmune reaction, it is urgent to understand the immune status of MG with thymoma. In this study, RNA sequencing data were obtained from the TCGA and GEO cohorts to identify differentially expressed messenger RNAs and infiltrated immune cells. A total of 121 samples in TCGA and 43 samples in GEO were screened out. The infiltrated immune cells were identified by CIBERSORT, in which Tfh cells and activated DC cells were abnormal in thymoma patients. The differently expressed genes were performed by package LIMMA. The functional characteristics of differently expression genes were analyzed by GO and KEGG; one GO and seven KEGG pathways were both found in both TCGA and GEO cohorts. Meanwhile, 27 common differently expressed genes were obtained and were displayed by a Venn diagram. The TRRUST was used to screen the hub genes for the common 27 different genes and 6 genes were found. Then, PPI networks were constructed. Subsequently, the relationship between SCNAs of common genes and related immune cells tested by TIMER. Kaplan-Meier plots, ROC curve and Cox's expression model for immune infiltration and hub genes were also tested. In conclusion, we found that two types of immune infiltrated cells and six hub genes can predict the occurrence of myasthenia gravis in thymoma patients.

Imbalance of Th17 and Tregs in thymoma may be a pathological mechanism of myasthenia gravis.

An imbalance in Th17 cells and Tregs may be an important cause of the pathogenesis of thymoma with myasthenia gravis (MG). In this study, 30 patients with simple thymoma and 30 patients with thymoma with MG were analyzed. Flow cytometry analysis of Th17 and Tregs in peripheral blood revealed that the percentages of Th17 in thymoma were lower than those in thymoma with MG, while the percentages of Tregs were higher than those in simple thymoma. Serum cytokine ELISA assays showed that IL-6 levels in simple thymoma were lower than those in MG patients. Further, Th17 and Tregs levels were detected by immunohistochemical double staining of thymoma tissue; the number of positive Th17 cells in thymoma with MG was higher than that in simple thymoma, while positive Tregs showed the opposite results. RORγt protein and mRNA expression in thymoma with MG were both higher than those in simple thymoma. FOXP3 protein and mRNA expression in the thymoma with MG group were lower than those in simple thymoma. The results of coculture of thymoma cells and CD4 + T cells showed that thymoma cells could promote the differentiation of Th17 cells and inhibit the Tregs. Overall, Th17 cells and related transcription factors and cytokines in thymoma with MG patients were higher than those in thymoma patients, whereas, Tregs showed the opposite results, the mechanism may be that thymoma can secrete IL6 and IL21. These findings indicated that imbalances in Th17/Tregs may account for the pathogeny of thymoma with MG.

High expression of KITLG is a new hallmark activating the MAPK pathway in type A and AB thymoma.

BACKGROUND: KIT proto-oncogene ligand (KITLG) is a pleiotropic factor which is found in diverse cancers and is involved in cell proliferation, differentiation, and survival. However, the value of KITLG in thymoma remains unclear. METHODS: A total of 121 thymoma samples from The Cancer Genome Atlas Thymoma (TCGA-THYM) dataset were used to analyze KITLG related genome-wide expression profiles, and microRNA profiles and methylation alterations and a GEO dataset-GSE29695, including 37 samples was used as verification. For cell-based studies, specific small interfering RNA targeting KITLG or a KITLG overexpression vector were used to clarify the changes of the MAPK pathway in an AB thymoma cell line Thy0517. RESULTS: Both datasets showed that high expression of KITLG was significantly associated with type A and AB thymoma. Through multiomic analysis of the TCGA-THYM, it was found that with the high expression of KITLG, there were 220 upregulated and 72 downregulated genes at the mRNA level, 79 positive and 78 negative miRNAs, 28 hypermethylation and 163 hypomethylation regions. In the thymoma cell line Thy0517, it was found that the expression of GRB2 and the phosphorylation levels of BRAF, MEK1/2, and ERK1/2 in the MAPK pathway were positively correlated with the change in KITLG. CONCLUSIONS: High expression of KITLG is a new hallmark of WHO type A and AB thymomas in which it might play a critical role through the activation of the MAPK signaling pathway. Additionally, it is hoped that KITLG will become a potential target for the diagnosis of type A and AB thymoma through further research in the future. KEY POINTS: SIGNIFICANT FINDINGS OF THE STUDY: KIT proto-oncogene ligand (KITLG) is a new hallmark of type A and AB thymomas which induce a series of aberrant alteration of mRNA, miRNA and DNA methylation. The expression of KITLG is significantly higher in type A and AB than other subtypes of thymoma. WHAT THIS STUDY ADDS: KITLG activated the MAPK signaling pathway to promote type A and AB thymoma which might be a potential diagnostic biomarker or target.

Protein diaphanous homolog 1 (Diaph1) promotes myofibroblastic activation of hepatic stellate cells by regulating Rab5a activity and TGFß receptor endocytosis.

TGFß induces the differentiation of hepatic stellate cells (HSCs) into tumor-promoting myofibroblasts but underlying mechanisms remain incompletely understood. Because endocytosis of TGFß receptor II (TßRII), in response to TGFß stimulation, is a prerequisite for TGF signaling, we investigated the role of protein diaphanous homolog 1 (known as Diaph1 or mDia1) for the myofibroblastic activation of HSCs. Using shRNA to knockdown Diaph1 or SMIFH2 to target Diaph1 activity of HSCs, we found that the inactivation of Diaph1 blocked internalization and intracellular trafficking of TßRII and reduced SMAD3 phosphorylation induced by TGFß1. Mechanistic studies revealed that the N-terminal portion of Diaph1 interacted with both TßRII and Rab5a directly and that Rab5a activity of HSCs was increased by Diaph1 overexpression and decreased by Diaph1 knockdown. Additionally, expression of Rab5aQ79L (active Rab5a mutant) increased whereas the expression of Rab5aS34N (inactive mutant) reduced the endosomal localization of TßRII in HSCs compared to the expression of wild-type Rab5a. Functionally, TGFß stimulation promoted HSCs to express tumor-promoting factors, and α-smooth muscle actin, fibronection, and CTGF, markers of myofibroblastic activation of HSCs. Targeting Diaph1 or Rab5a suppressed HSC activation and limited tumor growth in a tumor implantation mouse model. Thus, Dipah1 and Rab5a represent targets for inhibiting HSC activation and the hepatic tumor microenvironment.

Malignant solitary fibrous tumor occurring in the mediastinal pleura showing NAB2ex4-STAT6ex2 fusion and negative STAT6 immunohistochemistry: A case report.

Solitary fibrous tumor (SFT) is a rare clinical tumor, defined as a mesenchymal tumor of fibroblastic origin. A classic SFT is benign in most cases, but its clinical behavior is unpredictable. Lately, molecular analyses has discovered that almost all SFTs harbor an NAB2-STAT6 fusion gene, which is considered specific to this tumor type. Recent studies have suggested that nuclear STAT6 immunoreactivity is a highly sensitive and specific marker of SFTs and can be helpful when diagnosis is inconclusive by conventional methods. We herein report the case of a rare malignant solitary fibrous tumor occurring in the mediastinal pleura. An 82-year-old Chinese man with intermittent breathlessness was referred to our hospital. Chest CT showed a significantly enhanced irregular huge soft tissue mass in the anterior mediastinal area. After radical resection, the immunohistochemistry staining results of the sample showed that STAT6 was negative. The final diagnosis was confirmed by qualitative endpoint reverse transcriptase-polymerase chain reaction technique, showing positive NAB2ex4-STAT6ex2 fusion.

Focal Adhesion Kinase Promotes Hepatic Stellate Cell Activation by Regulating Plasma Membrane Localization of TGFß Receptor 2.

Transforming growth factor ß (TGFß) induces hepatic stellate cell (HSC) differentiation into tumor-promoting myofibroblast, although underlying mechanism remains incompletely understood. Focal adhesion kinase (FAK) is activated in response to TGFß stimulation, so it transmits TGFß stimulus to extracellular signal-regulated kinase and P38 mitogen-activated protein kinase signaling. However, it is unknown whether FAK can, in return, modulate TGFß receptors. In this study, we tested whether FAK phosphorylated TGFß receptor 2 (TGFßR2) and regulated TGFßR2 intracellular trafficking in HSCs. The FAKY397F mutant and PF-573,228 were used to inhibit the kinase activity of FAK, the TGFßR2 protein level was quantitated by immunoblotting, and HSC differentiation into myofibroblast was assessed by expression of HSC activation markers, alpha-smooth muscle actin, fibronectin, or connective tissue growth factor. We found that targeting FAK kinase activity suppressed the TGFßR2 protein level, TGFß1-induced mothers against decapentaplegic homolog phosphorylation, and myofibroblastic activation of HSCs. At the molecular and cellular level, active FAK (phosphorylated FAK at tyrosine 397) bound to TGFßR2 and kept TGFßR2 at the peripheral plasma membrane of HSCs, and it induced TGFßR2 phosphorylation at tyrosine 336. In contrast, targeting FAK or mutating Y336 to F on TGFßR2 led to lysosomal sorting and degradation of TGFßR2. Using RNA sequencing, we identified that the transcripts of 764 TGFß target genes were influenced by FAK inhibition, and that through FAK, TGFß1 stimulated HSCs to produce a panel of tumor-promoting factors, including extracellular matrix remodeling proteins, growth factors and cytokines, and immune checkpoint molecule PD-L1. Functionally, targeting FAK inhibited tumor-promoting effects of HSCs in vitro and in a tumor implantation mouse model. Conclusion: FAK targets TGFßR2 to the plasma membrane and protects TGFßR2 from lysosome-mediated degradation, thereby promoting TGFß-mediated HSC activation. FAK is a target for suppressing HSC activation and the hepatic tumor microenvironment.

A dynamic generalized coherence factor for side lobe suppression in ultrasound imaging.

Coherence-based weighting techniques have been widely studied to weight beamsummed data to improve image quality in ultrasound imaging. Although generalized coherence factor (GCF) enhances the robustness of coherence factor (CF) with preserved speckle pattern by including some incoherent components, the side lobe suppression performance is insufficient due to constant cut-off frequency M0. To address this problem, we introduced in this paper a dynamic GCF method, referred to as DGCF-C, based on the amplitude standard deviation and the convolution output of aperture data. The cut-off frequency is adaptively selected for GCF at each imaging point using the amplitude standard deviation of aperture data. Moreover, the convolution output of aperture data is used to calculate the dynamic GCF. The proposed method is evaluated in simulation and tissue-mimicking phantom studies. The image quality was analyzed in terms of resolution, contrast ratio (CR), generalized contrast-to-noise ratio (GCNR), speckle signal-to-noise ratio (sSNR), and signal-to-noise ratio (SNR). The results demonstrate that DGCF-C (Mmax=2) achieves mean resolution improvements of 35.1% in simulation, and 32.6% in experiment, compared with GCF (M0=1). Moreover, DGCF-C (Mmax=4) outperforms GCF (M0=2) with an average GCNR improvement of 13.5% and an average sSNR improvement of 15.2%, which indicates the better-preservation of speckle.

p300 Acetyltransferase Is a Cytoplasm-to-Nucleus Shuttle for SMAD2/3 and TAZ Nuclear Transport in Transforming Growth Factor ß-Stimulated Hepatic Stellate Cells.

Nuclear translocation of mothers against decapentaplegic homolog 2/3 (SMAD2/3), core transcription factors of transforming growth factor ß (TGF-ß) signaling, is critical for hepatic stellate cell (HSC) differentiation into metastasis-promoting myofibroblasts. SMAD2/3 have multiple coactivators, including WW domain-containing transcription regulator protein 1 (WWTR1 or TAZ) and p300 acetyltransferase. In the nucleus, TAZ binds to SMAD2/3 to prevent SMAD2/3 nuclear export. However, how TAZ and SMAD2/3 enter the nucleus remains poorly understood because neither contains a nuclear localization signal (NLS), an amino acid sequence tagging proteins for nuclear transport. p300 is an NLS-containing large scaffold protein, so we hypothesized that SMAD2/3 and TAZ may undergo nuclear import through complexing with p300. Coimmunoprecipitation, immunofluorescence, and nuclear fractionation assays revealed that TGF-ß1 promoted binding of SMAD2/3 and TAZ to p300 and that p300 inactivation disrupted TGF-ß1-mediated SMAD2/3 and TAZ nuclear accumulation. Deleting the p300 NLS blocked TGF-ß1-induced SMAD2/3 and TAZ nuclear transport. Consistently, p300 inactivation suppressed TGF-ß1-mediated HSC activation and transcription of genes encoding tumor-promoting factors, such as connective tissue growth factor, Tenascin C, Periostin, platelet-derived growth factor C, and fibroblast growth factor 2, as revealed by microarray analysis. Chromatin immunoprecipitation-real-time quantitative PCR showed that canonical p300-mediated acetylation of histones also facilitated transcription in response to TGF-ß1 stimulation. Interestingly, although both TGF-ß1-mediated and stiffness-mediated HSC activation require p300, comparison of gene expression data sets revealed that transcriptional targets of TGF-ß1 were distinct from those of stiffness-p300 mechanosignaling. Lastly, in tumor/HSC coinjection and intrasplenic tumor injection models, targeting p300 of activated-HSC/myofibroblasts by C646, short hairpin RNA, or cre-mediated gene disruption reduced tumor and liver metastatic growth in mice. Conclusion: p300 facilitates TGF-ß1-stimulated HSC activation by both noncanonical (cytoplasm-to-nucleus shuttle for SMAD2/3 and TAZ) and canonical (histone acetylation) mechanisms. p300 is an attractive target for inhibiting HSC activation and the prometastatic liver microenvironment.