An indirect estimation of x-ray spectrum via convolutional neural network and transmission measurement.

OBJECTIVE: In this work, we aim to propose an accurate and robust spectrum estimation method by synergistically combining X-ray imaging physics with a convolutional neural network (CNN). Approach: The approach relies on transmission measurements, and the estimated spectrum is formulated as a convolutional summation of a few model spectra generated using Monte Carlo simulation. The difference between the actual and estimated projections is utilized as the loss function to train the network. We contrasted this approach with the weighted sums of model spectra approach previously proposed. Comprehensive studies were performed to demonstrate the robustness and accuracy of the proposed approach in various scenarios. Main results: The results show the desirable accuracy of the CNN-based method for spectrum estimation. The ME and NRMSE were -0.021 keV and 3.04% for 80kVp, and 0.006 keV and 4.44% for 100kVp, superior to the previous approach. The robustness test and experimental study also demonstrated superior performances. The CNN-based approach yielded remarkably consistent results in phantoms with various material combinations, and the CNN-based approach was robust concerning spectrum generators and calibration phantoms. Significance: We proposed a method for estimating the real spectrum by integrating a deep learning model with real imaging physics. The results demonstrated that this method was accurate and robust in estimating the spectrum, and it is potentially helpful for broad X-ray imaging tasks.

A new approach for deducing rms proton radii from charge-changing reactions of neutron-rich nuclei and the reaction-target dependence.

We report the charge-changing cross sections (σcc) of 24 p-shell nuclides on both hydrogen and carbon at about 900A MeV, of which 8,9Li, 10-12Be, 10,14,15B, 14,15,17-22N and 16O on hydrogen and 8,9Li on carbon are for the first time. Benefiting from the data set, we found a new and robust relationship between the scaling factor of the Glauber model calculations and the separation energies of the nuclei of interest on both targets. This allows us to deduce proton radii (Rp) for the first time from the cross sections on hydrogen. Nearly identical Rp values are deduced from both target data for the neutron-rich carbon isotopes; however, the Rp from the hydrogen target is systematically smaller in the neutron-rich nitrogen isotopes. This calls for further experimental and theoretical investigations.

Microbiome alteration via fecal microbiota transplantation is effective for refractory immune checkpoint inhibitor-induced colitis.

Immune checkpoint inhibitors (ICIs) target advanced malignancies with high efficacy but also predispose patients to immune-related adverse events like immune-mediated colitis (IMC). Given the association between gut bacteria with response to ICI therapy and subsequent IMC, fecal microbiota transplantation (FMT) represents a feasible way to manipulate microbial composition in patients, with a potential benefit for IMC. Here, we present a large case series of 12 patients with refractory IMC who underwent FMT from healthy donors as salvage therapy. All 12 patients had grade 3 or 4 ICI-related diarrhea or colitis that failed to respond to standard first-line (corticosteroids) and second-line immunosuppression (infliximab or vedolizumab). Ten patients (83%) achieved symptom improvement after FMT, and three patients (25%) required repeat FMT, two of whom had no subsequent response. At the end of the study, 92% achieved IMC clinical remission. 16S rRNA sequencing of patient stool samples revealed that compositional differences between FMT donors and patients with IMC before FMT were associated with a complete response after FMT. Comparison of pre- and post-FMT stool samples in patients with complete responses showed significant increases in alpha diversity and increases in the abundances of Collinsella and Bifidobacterium, which were depleted in FMT responders before FMT. Histologically evaluable complete response patients also had decreases in select immune cells , including CD8+ T cells, in the colon after FMT when compared with non-complete response patients (n = 4). This study validates FMT as an effective treatment strategy for IMC and gives insights into the microbial signatures that may play a critical role in FMT response.

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples.

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Impact of Region-of-Interest Size on Immune Profiling Using Multiplex Immunofluorescence Tyramide Signal Amplification for Paraffin-Embedded Tumor Tissues.

INTRODUCTION: Representative regions of interest (ROIs) analysis from the whole slide images (WSI) are currently being used to study immune markers by multiplex immunofluorescence (mIF) and single immunohistochemistry (IHC). However, the amount of area needed to be analyzed to be representative of the entire tumor in a WSI has not been defined. METHODS: We labeled tumor-associated immune cells by mIF and single IHC in separate cohorts of non-small cell lung cancer (NSCLC) samples and we analyzed them as whole tumor area as well as using different number of ROIs to know how much area will be need to represent the entire tumor area. RESULTS: For mIF using the InForm software and ROI of 0.33 mm2 each, we observed that the cell density data from five randomly selected ROIs is enough to achieve, in 90% of our samples, more than 0.9 of Spearman correlation coefficient and for single IHC using ScanScope tool box from Aperio and ROIs of 1 mm2 each, we found that the correlation value of more than 0.9 was achieved using 5 ROIs in a similar cohort. Additionally, we also observed that each cell phenotype in mIF influence differently the correlation between the areas analyzed by the ROIs and the WSI. Tumor tissue with high intratumor epithelial and immune cells phenotype, quality, and spatial distribution heterogeneity need more area analyzed to represent better the whole tumor area. CONCLUSION: We found that at minimum 1.65 mm2 area is enough to represent the entire tumor areas in most of our NSCLC samples using mIF.

The Application Value of Metagenomic and Whole-Genome Capture Next-Generation Sequencing in the Diagnosis and Epidemiological Analysis of Psittacosis.

Background: To evaluate the value of metagenomic next-generation sequencing (mNGS) for the early diagnosis of psittacosis, and to investigate its epidemiology by whole-genome capture. Methods: Twenty-one bronchoalveolar lavage fluid (BALF) and blood samples of 16 psittacosis patients from multiple centers during August 2019 to September 2021 were analyzed retrospectively. mNGS with normal datasets (10 M 75-bp single-end reads after sequencing) and larger datasets (30 M 150-bp paired-end reads after sequencing) as well as quantitative real-time polymerase chain reaction (qPCR) were used to detect the pathogen. Also, whole-genome capture of Chlamydophila psittaci was applied to draw the phylogenetic tree. Results: mNGS successfully detected the pathogen in all 16 cases (100%), while qPCR was positive only in 5 out of 10 cases (50%), indicating a significantly higher sensitivity of mNGS than qPCR (p < 0.01). BALF-mNGS performed better than blood-mNGS (16/16 versus 3/5, p < 0.05). In addition, larger datasets (the read counts have tripled, and the base number was 12-fold larger compared to clinical mNGS with a normal dataset) of mNGS showed significantly increased contents of human DNA (p < 0.05) and decreased reads per million of the pathogen, suggesting no improvement. Whole-genome capture results of five samples (>60% coverage and >1 depth) were used to construct the phylogenetic tree. Conclusion: Significant advantages of mNGS with normal datasets were demonstrated in early diagnosing psittacosis. It is the first study to use whole-genome capture to analyze C. psittaci epidemiological information.

Comprehensive multiplexed immune profiling of the ductal carcinoma in situ immune microenvironment regarding subsequent ipsilateral invasive breast cancer risk.

BACKGROUND: Ductal carcinoma in situ (DCIS) is treated to prevent subsequent ipsilateral invasive breast cancer (iIBC). However, many DCIS lesions will never become invasive. To prevent overtreatment, we need to distinguish harmless from potentially hazardous DCIS. We investigated whether the immune microenvironment (IME) in DCIS correlates with transition to iIBC. METHODS: Patients were derived from a Dutch population-based cohort of 10,090 women with pure DCIS with a median follow-up time of 12 years. Density, composition and proximity to the closest DCIS cell of CD20+ B-cells, CD3+CD8+ T-cells, CD3+CD8- T-cells, CD3+FOXP3+ regulatory T-cells, CD68+ cells, and CD8+Ki67+ T-cells was assessed with multiplex immunofluorescence (mIF) with digital whole-slide analysis and compared between primary DCIS lesions of 77 women with subsequent iIBC (cases) and 64 without (controls). RESULTS: Higher stromal density of analysed immune cell subsets was significantly associated with higher grade, ER negativity, HER-2 positivity, Ki67 ≥ 14%, periductal fibrosis and comedonecrosis (P < 0.05). Density, composition and proximity to the closest DCIS cell of all analysed immune cell subsets did not differ between cases and controls. CONCLUSION: IME features analysed by mIF in 141 patients from a well-annotated cohort of pure DCIS with long-term follow-up are no predictors of subsequent iIBC, but do correlate with other factors (grade, ER, HER2 status, Ki-67) known to be associated with invasive recurrences.

Effector memory cytotoxic CD3+/CD8+/CD45RO+ T cells are predictive of good survival and a lower risk of recurrence in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) with high tumour-infiltrating lymphocytes (TILs) has been associated with a promising prognosis. To better understand the prognostic value of immune cell subtypes in TNBC, we characterised TILs and the interaction between tumour cells and immune cell subtypes. A total of 145 breast cancer tissues were stained by multiplex immunofluorescence (mIF), including panel 1 (PD-L1, PD-1, CD3, CD8, CD68 and CK) and panel 2 (Foxp3, Granzyme B, CD45RO, CD3, CD8 and CK). Phenotypes were analysed and quantified by pathologists using InForm software. We found that in the ER-negative (ER <1% and HER2-negative) group and the ER/PR-low positive (ER 1-9% and HER2-negative) group, 11.2% and 7.1% of patients were PD-L1+ by the tumour cell score, 29.0% and 28.6% were PD-L1+ by the modified immune cell score and 30.8% and 32.1% were PD-L1+ by the combined positive score. We combined ER-negative and ER/PR-low positive cases for the survival analysis since a 10% cut-off is often used in clinical practice for therapeutic purposes. The densities of PD-L1+ tumour cells (HR: 0.366, 95% CI: 0.138-0.970; p = 0.043) within the tumour compartment and CD3+ immune cells in the total area (tumour and stromal compartments combined) (HR: 0.213, 95% CI: 0.070-0.642; p = 0.006) were favourable prognostic biomarkers for overall survival (OS) in TNBC. The density of effector/memory cytotoxic T cells (CD3+CD8+CD45RO+) in the tumour compartment was an independent prognostic biomarker for OS (HR: 0.232, 95% CI: 0.086-0.628; p = 0.004) and DFS (HR: 0.183, 95% CI: 0.1301-0.744; p = 0.009) in TNBC. Interestingly, spatial data suggested that patients with a higher density of PD-L1+ tumour cells had shorter cell-cell distances from tumour cells to cytotoxic T cells (p < 0.01). In conclusion, we found that phenotyping tumour immune cells by mIF is highly informative in understanding the immune microenvironment in TNBC. PD-L1+ tumour cells, total T cells and effector/memory cytotoxic T cells are promising prognostic biomarkers in TNBC.

MicroRNA-140-3p represses the proliferation, migration, invasion and angiogenesis of lung adenocarcinoma cells via targeting TYMS (thymidylate synthetase).

MicroRNA (miR)-140-3p has been proved to repress lung adenocarcinoma (LUAD), and our study aims to further evaluate the mechanism. Bioinformatic analyses were performed. The viability, proliferation, migration, invasion and angiogenesis of transfected LUAD cells were all determined via Cell Counting Kit-8, colony formation, Scratch, Transwell, and tube formation assays. The targeting relationship between miR-140-3p and thymidylate synthetase (TYMS) was confirmed by dual-luciferase reporter assay. Relative expressions of miR-140-3p, TYMS, epithelial-to-mesenchymal transition- (E-cadherin, N-cadherin, vimentin), angiogenesis- (vascular endothelial growth factor (VEGF)), and apoptosis-related factors (cleaved caspase-3, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax)) were quantified by quantitative real-time polymerase chain reaction or Western blot. TYMS was high-expressed yet miR-140-3p was low-expressed in LUAD cells. Upregulation of miR-140-3p inhibited TYMS expression, viability, colony formation, migration, invasion, and tube length within LUAD cells, while downregulation of miR-140-3p did oppositely. Silenced TYMS, the downstream target gene of miR-140-3p, reversed the effects of miR-140-3p downregulation on TYMS expression, cell viability, colony formation, migration, invasion, and tube length as well as the metastasis-, apoptosis- and angiogenesis-related proteins in LUAD cells. Upregulation of miR-140-3p inhibited the proliferation, migration, invasion and angiogenesis of LUAD cells via targeting TYMS.

Immune Phenotype and Response to Neoadjuvant Therapy in Triple-Negative Breast Cancer.

PURPOSE: Increasing tumor-infiltrating lymphocytes (TIL) is associated with higher rates of pathologic complete response (pCR) to neoadjuvant therapy (NAT) in patients with triple-negative breast cancer (TNBC). However, the presence of TILs does not consistently predict pCR, therefore, the current study was undertaken to more fully characterize the immune cell response and its association with pCR. EXPERIMENTAL DESIGN: We obtained pretreatment core-needle biopsies from 105 patients with stage I-III TNBC enrolled in ARTEMIS (NCT02276443) who received NAT from Oct 22, 2015 through July 24, 2018. The tumor-immune microenvironment was comprehensively profiled by performing T-cell receptor (TCR) sequencing, programmed death-ligand 1 (PD-L1) IHC, multiplex immunofluorescence, and RNA sequencing on pretreatment tumor samples. The primary endpoint was pathologic response to NAT. RESULTS: The pCR rate was 40% (42/105). Higher TCR clonality (median = 0.2 vs. 0.1, P = 0.03), PD-L1 positivity (OR: 2.91, P = 0.020), higher CD3+:CD68+ ratio (median = 14.70 vs. 8.20, P = 0.0128), and closer spatial proximity of T cells to tumor cells (median = 19.26 vs. 21.94 µm, P = 0.0169) were associated with pCR. In a multivariable model, closer spatial proximity of T cells to tumor cells and PD-L1 expression enhanced prediction of pCR when considered in conjunction with clinical stage. CONCLUSIONS: In patients receiving NAT for TNBC, deep immune profiling through detailed phenotypic characterization and spatial analysis can improve prediction of pCR in patients receiving NAT for TNBC when considered with traditional clinical parameters.

High-dose irradiation in combination with non-ablative low-dose radiation to treat metastatic disease after progression on immunotherapy: Results of a phase II trial.

AIM: To report early findings from a phase II trial of high-dose radiotherapy (HD-RT) with or without low-dose RT (LD-RT) for metastatic cancer. METHODS: Eligible patients had metastatic disease that progressed on immunotherapy within 6 months. Patients were given either HD-RT (20-70 Gy total; 3-12.5 Gy/f), or HD-RT + LD-RT (0.5-2 Gy/f up to 1-10 Gy total) to separate lesions, with continued immunotherapy. Radiographic response was assessed per RECIST 1.1 and Immune-Related Response Criteria (irRC). Primary endpoints: (1) 4-month disease control (DCR, complete/partial response [CR/PR] or stable disease [SD]) or an overall response (ORR, CR/PR) at any point in ≥10% of patients, per RECIST 1.1; (2) dose-limiting toxicity within 3 months not exceeding 30%. Secondary endpoint was lesion-specific response. RESULTS: Seventy-four patients (NSCLC, n = 38; melanoma n = 21) were analyzed (39 HD-RT and 35 HD-RT + LD-RT). The median follow-up time was 13.6 months. The primary endpoint was met for 72 evaluable patients, with a 4-month DCR of 42% (47% [16/34] vs. 37% [14/38] in HD-RT + LD-RT vs. HD-RT, P = 0.38), and 19% ORR at any time (26% [9/34] vs. 13% [5/38] in HD-RT + LD-RT vs. HD-RT, P = 0.27). Three patients had toxicity ≥grade 3. LD-RT lesion response (53%) was improved compared to nonirradiated lesions in HD-RT + LD-RT (23%, P = 0.002) and HD-RT (11%, P < 0.001). T- and NK cell infiltration was enhanced in lesions treated with LD-RT. CONCLUSIONS: HD-RT plus LD-RT safely improved lesion-specific response in patients with immune resistant solid tumors by promoting infiltration of effector immune cells into the tumor microenvironment.

Diagnosis values of IL-6 and IL-8 levels in serum and bronchoalveolar lavage fluid for invasive pulmonary aspergillosis in chronic obstructive pulmonary disease.

Among immunologically normal hosts, patients with chronic obstructive pulmonary disease (COPD) are considered to be at high risk of invasive pulmonary aspergillosis (IPA), and early diagnosis and treatment are the key to improving the prognosis of patients. Here we aimed to evaluate whether interleukin (IL)-6 and IL-8 might be used in the detection and diagnosis of IPA in patients with COPD. We prospectively collected 106 patients with COPD and divided them into non-IPA (n=74), probable/possible IPA (n=26) and proven IPA (n=6). Platelia Aspergillus kit was used to detect galactomannan in bronchoalveolar lavage fluid (BALF), and serum and ELISA kit was used to detect IL-6 and IL-8 levels. Diagnostic efficiency of IL-6, IL-8 and galactomannan in serum and BALF was evaluated by receiver operating characteristic curve. Compared with the non-IPA group, the proven/probable IPA group showed significantly elevated levels of IL-6 and IL-8 in both serum and BALF, which were positively correlated with galactomannan levels. The sensitivity and specificity of IL-6 for diagnosing IPA were 74.32% and 81.25% (cut-off at 92.82 pg/mL, area under the curve (AUC)=0.8366) in serum and 68.92% and 71.88% (cut-off at 229.4 pg/mL, AUC=0.7694) in BALF. The sensitivity and specificity of IL-8 for diagnosing IPA were 83.78% and 81.25% (cut-off at 93.46 pg/mL, AUC=0.8756) in serum and 85.14% and 75.00% (cut-off at 325.4 pg/mL, AUC=0.8252) in BALF. The elevated levels of IL-6 and IL-8 in patients with IPA with COPD could be used as auxiliary indicators to diagnose IPA in addition to galactomannan.

SOX4 promotes the growth and metastasis of breast cancer.

PURPOSE: Increasing evidence has shown that the transcription factor SOX4 is closely associated with the development and progression of many malignant tumors. However, the effect of SOX4 on breast cancer is unclear. In this study, we purposed to investigate the role of SOX4 in the growth and metastasis in breast cancer and the underlying mechanism. Moreover, the effect of SOX4 on cancer cell resistance to chemotherapeutic agents was also evaluated in vitro and in vivo. METHODS: We used lentivirus technique to ectopically express SOX4 in MDA-MB-231 and SUM149 cells or knockdown SOX4 in BT474 cells, and examined the effect of these changes on various cellular functions. MTT assay was used to determine the cell viability as well as resistance to chemotherapeutic agents. The regulation of SOX4 on epithelial-mesenchymal transition (EMT)-related genes was analyzed using qRT-PCR. The binding of SOX4 to the CXCR7 gene was demonstrated using chromatin immunoprecipitation assay and dual-luciferase reporter activity assay. The effect of SOX4/CXCR7 axis on metastasis was examined using Transwell migration and Matrigel invasion assays. The expression of SOX4/CXCR7 in primary tumors and metastatic foci in lymph nodes was assessed using immunohistochemistry. Cellular morphology was investigated under phase contrast microscope and transmission electron microscopy. Moreover, the effect of SOX4 on tumor growth, metastasis, and resistance to chemotherapy was also studied in vivo by using bioluminescent imaging. RESULTS: SOX4 increased breast cancer cell viability, migration, and invasion in vitro and enhanced tumor growth and metastasis in vivo. It regulated EMT-related genes and bound to CXCR7 promoter to upregulate CXCR7 transcription. Both SOX4 and CXCR7 were highly expressed in human primary tumors and metastatic foci in lymph nodes. Treatment of breast cancer cells with the CXCR7 inhibitor CCX771 reversed the SOX4 effect on cell migration and invasion. Ectopic expression of SOX4 increased the susceptibility of cells to paclitaxel. CONCLUSIONS: SOX4 plays an important role in the growth and metastasis of breast cancer. SOX4/CXCR7 may serve as potential therapeutic targets for the treatment. Paclitaxel may be a good therapeutic option if the expression level of SOX4 is high.

Mir-29b mediates the regulation of Nrf2 on airway epithelial remodeling and Th1/Th2 differentiation in COPD rats.

COPD, or Chronic obstructive pulmonary disease, is an inflammation-related disease and lead to cachexia and muscle wasting. Altered nuclear factor erythroid 2-related factor 2 (Nrf2) expression is found in patients of COPD because it is involved in pulmonary protective effects. MiR-29b could be activated by Nrf2. We hypothesized that miR-29b might mediate the regulation of Nrf2 on Th1/Th2 differentiation and airway epithelial remodeling in COPD rats. SD rats were exposed to smoke for COPD induction. Expression of Nrf2 mRNA and miR-29b in lung tissues was quantified. Expression of Nrf2 and matrix metalloproteinase 2 (MMP2) were also detected by immunohistochemistry and western blot. Th1 markers and Th2 markers were measured by ELISA in peripheral blood. Flow cytometry was used to detect the Th1/Th2 ratio. miR-29b and Nrf2 was manipulated at mRNA level in A549 cells using transfection. Cellular growth and migration were measured in transfectants. In lung tissues of COPD rats, expression of Nrf2 and miR-29b decreased. MMP2, a target of miR-29b, had an opposite expression to miR-29b in peripheral blood. Levels of inflammatory factors and Th1/Th2 ratio increased. MiR-29b mediated the regulation of Nrf2 on remodeling of lung epithelial cells. Blocking Nrf2 expression in A549 cells led to the opposite expression of miR-29b and further decreased MMP2 production; meanwhile, cell growth and motility were improved. Different miR-29b levels affected MMP2 expression and cellular characteristics. The findings suggested that miR-29b was a regulator the pathological progress of COPD. It mediates the effect of Nrf2 on Th1/Th2 differentiation and on remodeling process of airway epithelial cells.

Screening Analysis of Sirtuins Family Expression on Anti-Inflammation of Resveratrol in Endothelial Cells.

BACKGROUND Resveratrol has been shown to possess beneficial activities including antioxidant, anti-inflammatory, and cardioprotective effects through activating a nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase family member sirtuin-1 (SIRT1) protein. The current study was undertaken to investigate the role of sirtuin family members (SIRT1-SIRT7) on the anti-inflammation activities of resveratrol in endothelial cells. MATERIAL AND METHODS Primary human umbilical vein endothelial cells (HUVECs) were pretreated with resveratrol before tumor necrosis factor (TNF)-alpha (10-20 µg/L) stimulation. Cell viability was measured using the Cell Counting Kit-8 method. Total RNA was extracted after different treatments and the NimbleGen Human 12×135K Gene Expression Array was applied to screen and analyze SIRTs expression. Quantitative real-time polymerase chain reaction and western blot were applied to verify the results of the gene expression microarrays. Reactive oxygen species (ROS) production was examined using flow cytometry analysis. RESULTS Microarray analysis showed that the expressions of SIRT1, SIRT2, SIRT3, SIRT5, SIRT6, and SIRT7 showed the tendency to increase while SIRT4 showed the tendency to decrease. SIRT1, SIRT2, SIRT5, and SIRT7 gene expression could be upregulated by pretreatment with resveratrol compared with TNF-alpha alone while there were no obvious differences of SIRT3, SIRT4, and SIRT6 expressions observed in TNF-alpha alone treated cells and resveratrol-TNF-alpha co-treated cells. Interestingly, SIRT1, SIRT2, SIRT3, SIRT4, and SIRT5 siRNA could reverse the effect of resveratrol on ROS production; SIRT1 and SIRT5 siRNA could significantly increase CD40 expression inhibited by resveratrol in TNF-a treated cells. CONCLUSIONS Our results suggest that resveratrol inhibiting oxidative stress production is associated with SIRT1, SIRT2, SIRT3, SIRT4, and SIRT5 pathways; attenuating CD40 expression was only associated with SIRT1 and SIRT5 pathways in TNF-alpha-induced endothelial cells injury.

lncRNA AWPPH promotes proliferation and inhibits apoptosis of non­small cell lung cancer cells by activating the Wnt/ß­catenin signaling pathway.

AWPPH is a newly discovered long non­coding (lnc)RNA that serves an oncogenic role in the development of several types of cancer; however, its involvement in non­small cell lung cancer (NSCLC) is unknown. Therefore, the aim of the present study was to investigate the function of AWPPH in NSCLC. The results demonstrated that AWPPH expression levels were significantly upregulated in the lung tissues and serum samples of patients with NSCLC compared with in healthy controls. High expression levels of AWPPH effectively distinguished NSCLC patients from healthy controls. In addition, patients with high expression levels of AWPPH had significantly shorter survival time. AWPPH overexpression in NSCLC cells promoted proliferation and inhibited apoptosis, and activated the Wnt/ß­catenin signaling pathway, which is a classic signaling pathway involved in the development and progression of different types of cancers. Treatment with a Wnt/ß­catenin signaling pathway activator produced no significant effect on AWPPH expression. Therefore, it was concluded that lcRNA AWPPH could promote the growth of NSCLCs by activating the Wnt/ß­catenin signaling pathway.

Superior efficacy of cotreatment with BET protein inhibitor and BCL2 or MCL1 inhibitor against AML blast progenitor cells.

First-generation bromodomain extra-terminal protein (BETP) inhibitors (BETi) (e.g., OTX015) that disrupt binding of BETP BRD4 to chromatin transcriptionally attenuate AML-relevant progrowth and prosurvival oncoproteins. BETi treatment induces apoptosis of AML BPCs, reduces in vivo AML burden and induces clinical remissions in a minority of AML patients. Clinical efficacy of more potent BETis, e.g., ABBV-075 (AbbVie, Inc.), is being evaluated. Venetoclax and A-1210477 bind and inhibit the antiapoptotic activity of BCL2 and MCL1, respectively, lowering the threshold for apoptosis. BETi treatment is shown here to perturb accessible chromatin and activity of enhancers/promoters, attenuating MYC, CDK6, MCL1 and BCL2, while inducing BIM, HEXIM1, CDKN1A expressions and apoptosis of AML cells. Treatment with venetoclax increased MCL1 protein levels, but cotreatment with ABBV-075 reduced MCL1 and Bcl-xL levels. ABBV-075 cotreatment synergistically induced apoptosis with venetoclax or A-1210477 in patient-derived, CD34+ AML cells. Compared to treatment with either agent alone, cotreatment with ABBV-075 and venetoclax was significantly more effective in reducing AML cell-burden and improving survival, without inducing toxicity, in AML-engrafted immune-depleted mice. These findings highlight the basis of superior activity and support interrogation of clinical efficacy and safety of cotreatment with BETi and BCL2 or MCL1 inhibitor in AML.

Ablation and Inhibition of the Immunoproteasome Catalytic Subunit LMP7 Attenuate Experimental Abdominal Aortic Aneurysm Formation in Mice.

Low-molecular mass protein 7 (LMP7) is a proteolytic subunit of the immunoproteasome that is involved in regulating inflammatory responses. However, the role of LMP7 in the pathogenesis of abdominal aortic aneurysm (AAA) remains unknown. In this study, ApoE knockout (KO) or LMP7/ApoE double KO (dKO) mice were infused with angiotensin II (Ang II, 1000 ng/kg per minute) for up to 28 d. We found that LMP7 expression was significantly upregulated in AAA tissues from ApoE KO mice and human patients. Moreover, Ang II infusion markedly increased the incidence and severity of AAA in ApoE KO mice, which was considerably reduced in LMP7/ApoE dKO mice. Histological alterations, including aortic wall thickening, collagen deposition, elastin fragmentation, and vascular smooth muscle cell apoptosis in AAA tissue of ApoE KO mice, were also significantly attenuated in LMP7/ApoE dKO mice. Interestingly, LMP7/ApoE dKO mice showed a marked reduction of infiltration of CD3+ T cells, especially CD4+ T cells in AAA tissues compared with ApoE KO mice. Moreover, ablation of LMP7 substantially inhibited the differentiation of CD4+ T cells into Th1 and Th17 cells by reducing the activation of multiple transcriptional factors. We also investigated the effects of an LMP7-specific inhibitor PR-957 (also known as ONX 0914) on AAA formation in ApoE KO mice. PR-957 treatment could reduce the AAA incidence and severity. In conclusion, our results provide, to our knowledge, novel evidence that ablation or pharmacological inhibition of LMP7 attenuates Ang II-induced AAA formation, and LMP7 might be a novel therapeutic target for treating AAA in humans.

Targeting nuclear ß-catenin as therapy for post-myeloproliferative neoplasm secondary AML.

Transformation of post-myeloproliferative neoplasms into secondary (s) AML exhibit poor clinical outcome. In addition to increased JAK-STAT and PI3K-AKT signaling, post-MPN sAML blast progenitor cells (BPCs) demonstrate increased nuclear ß-catenin levels and TCF7L2 (TCF4) transcriptional activity. Knockdown of ß-catenin or treatment with BC2059 that disrupts binding of ß-catenin to TBL1X (TBL1) depleted nuclear ß-catenin levels. This induced apoptosis of not only JAKi-sensitive but also JAKi-persister/resistant post-MPN sAML BPCs, associated with attenuation of TCF4 transcriptional targets MYC, BCL-2, and Survivin. Co-targeting of ß-catenin and JAK1/2 inhibitor ruxolitinib (rux) synergistically induced lethality in post-MPN sAML BPCs and improved survival of mice engrafted with human sAML BPCs. Notably, co-treatment with BET protein degrader ARV-771 and BC2059 also synergistically induced apoptosis and improved survival of mice engrafted with JAKi-sensitive or JAKi-persister/resistant post-MPN sAML cells. These preclinical findings highlight potentially promising anti-post-MPN sAML activity of the combination of ß-catenin and BETP antagonists against post-MPN sAML BPCs.

Microarray profiles reveal that circular RNA hsa_circ_0007385 functions as an oncogene in non-small cell lung cancer tumorigenesis.

OBJECTIVE: Circular RNAs (circRNAs) are a novel class of non-protein-coding RNA. Emerging evidence indicates that circRNAs participate in the regulation of many pathophysiological processes. This study aims to explore the expression profiles and pathological effects of circRNAs in non-small cell lung cancer (NSCLC). METHODS: Human circRNAs microarray analysis was performed to screen the expression profile of circRNAs in NSCLC tissue. Expressions of circRNA and miRNA in NSCLC tissues and cells were quantified by qRTPCR. Functional experiments were performed to investigate the biological functions of circRNA, including CCK-8 assay, colony formation assay, transwell assay and xenograft in vivo assay. RESULTS: Human circRNAs microarray revealed a total 957 abnormally expressed circRNAs (> twofold, P < 0.05) in NSCLC tissue compared with adjacent normal tissue. In further studies, hsa_circ_0007385 was significantly up regulated in NSCLC tissue and cells. In vitro experiments with hsa_circ_0007385 knockdown resulted in significant suppression of the proliferation, migration and invasion of NSCLC cells. In vivo xenograft assay using hsa_circ_0007385 knockdown, significantly reduced tumor growth. Bioinformatics analysis and luciferase reporter assay verified the potential target miR-181, suggesting a possible regulatory pathway for hsa_circ_0007385. CONCLUSION: In summary, results suggest hsa_circ_0007385 plays a role in NSCLC tumorigenesis, providing a potential therapeutic target for NSCLC.