A novel microenvironment regulated system CAR-T (MRS.CAR-T) for immunotherapeutic treatment of esophageal squamous carcinoma.

Chimeric antigen receptor T cell immunotherapy has achieved promising therapeutic effects in the treatment of hematological malignancies. However, there are still many obstacles, including on-target off-tumor antigen expression, that prevent successful application to solid tumors. We designed a tumor microenvironment (TME) regulated system chimeric antigen receptor T (MRS.CAR-T) which can only be auto-activated in the solid TME. B7-H3 was selected as the target antigen for esophageal carcinoma. An element comprising a human serum albumin (HSA) binding peptide and a matrix metalloproteases (MMPs) cleavage site was inserted between the 5' terminal signal peptide and single chain fragment variable (scFv) of the CAR skeleton. Upon administration, HSA bound the binding peptide in MRS.B7-H3.CAR-T effectively and promoted proliferation and differentiation into memory cells. MRS.B7-H3.CAR-T was not cytotoxic in normal tissues expressing B7-H3 as the antigen recognition site in the scFv was cloaked by HSA. The anti-tumor function of MRS.B7-H3.CAR-T was recovered once the cleavage site was cleaved by MMPs in the TME. The anti-tumor efficacy associated with MRS.B7-H3.CAR-T cells was improved compared to classic B7-H3.CAR-T cells in vitro and less IFN-γ was released, suggesting a treatment that may induce less extent of cytokine release syndrome-mediated toxicity. In vivo, MRS.B7-H3.CAR-T cells had strong anti-tumor activity and were safe. MRS.CAR-T represents a novel strategy to improve the efficacy and safety of CAR-T therapy in solid tumors.

High-quality color image restoration from a disturbed graded-index imaging system by deep neural networks.

Imaging transmission plays an important role in endoscopic clinical diagnosis involved in modern medical treatment. However, image distortion due to various reasons has been a major obstacle to state-of-art endoscopic development. Here, as a preliminary study we demonstrate ultra-efficient recovery of exemplary 2D color images transmitted by a disturbed graded-index (GRIN) imaging system through the deep neural networks (DNNs). Indeed, the GRIN imaging system can preserve analog images through the GRIN waveguides with high quality, while the DNNs serve as an efficient tool for imaging distortion correction. Combining GRIN imaging systems and DNNs can greatly reduce the training process and achieve ideal imaging transmission. We consider imaging distortion under different realistic conditions and use both pix2pix and U-net type DNNs to restore the images, indicating the suitable network in each condition. This method can automatically cleanse the distorted images with superior robustness and accuracy, which can potentially be used in minimally invasive medical applications.

Temporin-GHb-Derived Peptides Exhibit Potent Antibacterial and Antibiofilm Activities against Staphylococcus aureus In Vitro and Protect Mice from Acute Infectious Pneumonia.

With the continuous development of drug resistance in bacteria to traditional antibiotics, the demand for novel antibacterial agents is urgent. Antimicrobial peptides (AMPs) are promising candidates because of their unique mechanism of action and low tendency to induce drug resistance. Previously, we cloned temporin-GHb (hereafter referred to simply as "GHb") from Hylarana guentheri. In this study, a series of derived peptides were designed, namely, GHbR, GHbK, GHb3K, GHb11K, and GHbK4R. The five derived peptides had stronger antibacterial activities against Staphylococcus aureus than the parent peptide GHb and could effectively inhibit the formation of biofilms and eradicate mature biofilms in vitro. GHbR, GHbK, GHb3K, and GHbK4R exerted bactericidal effects by disrupting membrane integrity. However, GHb11K exhibited bacteriostatic efficacy with toroidal pore formation on the cell membrane. In comparison to GHbK4R, GHb3K showed much lower cytotoxicity against A549 alveolar epithelial cells, with an IC50 > 200 µM, which was much higher than its minimal inhibitory concentration (MIC = 3.1 µM) against S. aureus. The anti-infection potential of GHbK4R and GHb3K was investigated in vivo. Compared with vancomycin, the two peptides displayed significant efficacy in a mouse model of acute pneumonia infected with S. aureus. Both GHbK4R and GHb3K also had no obvious toxicity to normal mice after intraperitoneal administration (15 mg/kg) for 8 days. Our results indicate that GHb3K and GHbK4R might be promising candidates for the treatment of bacterial pneumonia infected with S. aureus.

Golgi scaffold protein PAQR3 as a candidate suppressor of gastric cardia adenocarcinoma via regulating TGF-ß/Smad pathway.

OBJECTIVES: To investigate the function of PAQR3 in gastric cardia adenocarcinoma (GCA) and understand the possible mechanism of PAQR3 in regulating epithelial-mesenchymal transition (EMT). METHODS: We detected PAQR3 protein in 146 GCA tissues and paired normal adjacent tissues (PNTs) specimens using immunohistochemical analysis, and explored its clinical significance. The expression levels of PAQR3 protein in 20 GCA tissues, their paired PNTs, HGC27, SGC7901, and GES-1 cells were analyzed by Western blot. Wild-type PAQR3 was overexpressed in HGC27 cells. The effects of PAQR3 overexpression on the function of HGC27 cells and its underlying mechanisms were then analyzed through a series of cell and molecular biology experiments. RESULTS: PAQR3 was significantly down-regulated in GCA tissues when compared with paired PNTs (p < 0.0001). The expression level of PAQR3 in GCA tissues was significantly negatively correlated with Helicobacter pylori infection (p = 0.000), venous invasion (p = 0.000), invasion depth (p = 0.000), lymph node metastasis (p = 0.022), tumor stage (p = 0.000), and patient survival (p = 0.009). Downregulation of PAQR3 was highly correlated with increased EMT signature and activated TGF-ß/Smad pathway in GCA tissues. Overexpression of PAQR3 in HGC27 cells negatively regulates its cellular functions, such as cell proliferation and migration, and suppresses EMT. Mechanistically, overexpression of PAQR3 significantly down-regulates the protein expression levels of TGF-1, p-Smad2, and p-Smad3 in HGC27 cells. CONCLUSION: PAQR3 was significantly down-regulated in GCA tissues, HGC27, and SGC7901 cells. PAQR3 significantly inhibits the proliferation, migration, and invasion of HGC27 cells. Mechanistically, PAQR3 can inhibit the EMT process in HGC27 cells by regulating TGF-ß/Smad signaling pathway.

Complex amplitude field reconstruction in atmospheric turbulence based on deep learning.

In this paper, we use deep neural networks (DNNs) to simultaneously reconstruct the amplitude and phase information of the complex light field transmitted in atmospheric turbulence based on deep learning. The results of amplitude and phase reconstruction by four different training methods are compared comprehensively. The obtained results indicate that the training method that can more accurately reconstruct the complex amplitude field is to input the amplitude and phase pattern pairs into the neural network as two channels to train the model.

Circulating methylated THBS1 DNAs as a novel marker for predicting peritoneal dissemination in gastric cancer.

OBJECTIVES: Thrombospondin 1 (THBS1) is known to play a key role in tumor metastasis, and aberrant DNA methylation is one of the mechanisms regulating THBS1. The present study investigated whether methylated THBS1 in circulating cell-free DNA from preoperative peritoneal lavage fluid (PPLF) and peripheral blood could be used as a potential biomarker for predicting peritoneal dissemination in gastric cancer (GC) patients. METHODS: The status of THBS1 methylation was detected by quantitative methylation-specific PCR (MSP) in tumor tissues, paired PPLF, and serum from 92 GC patients. The correlation between methylated THBS1 levels and peritoneal dissemination of GC was studied, and its diagnostic value for predicting peritoneal dissemination was clarified by the receiver operating characteristic (ROC) curve. RESULTS: Aberrant THBS1 methylation in tumor tissues was significantly higher than that in paracancerous normal tissues (p < 0.0001). No THBS1 methylation was found in 40 healthy controls, and partial methylation was detected in 3 of 48 patients with chronic non-atrophic gastritis. The frequency of THBS1 methylation in pairing PPLF and serum from 92 GC patients was 52.2% (48/92) and 58.7% (54/92), respectively. The results of methylated THBS1 in pairing PPLF and serum were similar to those of tumor tissues. Aberrant THBS1 methylation in tumor tissues and pairing PPLF or serum was closely related to peritoneal dissemination, tumor progression, and poor prognosis (all p < 0.0001). CONCLUSION: Circulating methylated THBS1 DNAs in PPLF/serum may predict peritoneal dissemination, a potential poor prognostic factor for GC patients.

A Potential Oncogenic Role for PFKFB3 Overexpression in Gastric Cancer Progression.

OBJECTIVES: PFKFB3 regulates glycolysis in tumor cells, might function as an oncogene, and is associated with cancer metastasis. However, its role in gastric cancer (GC) remains largely unknown. METHODS: PFKFB3 expression was assessed by immunohistochemistry (IHC) in GC tissues and paired paracancerous histological normal tissues (PCHNTs). The associations of PFKFB3 expression with clinical features and HIF-1α, Ki-67, E-cadherin, Snail, and Vimentin expression levels were assessed. A series of in vivo and in vitro experiments were performed to investigate the effects of PFKFB3 on the growth, migration, and invasion of GC cells. RESULTS: We found that PFKFB3 expression was significantly higher in GC tissues compared with PCHNTs (P = 0.000). PFKFB3 expression was positively correlated with tumor size (P = 0.000), differentiation (P = 0.025), venous invasion (P = 0.084), nerve invasion (P = 0.014), lymphatic invasion (P = 0.000), local invasion (P = 0.000), invasive depth (P = 0.000), nodal metastasis (P = 0.000), tumor-node-metastasis stage (P = 0.000), and patient survival (P = 0.000). Notably, PFKFB3 upregulation was highly correlated with increased epithelial-mesenchymal transition (EMT) in GC samples. PFKFB3 overexpression positively modulated cell proliferation, migration, and EMT in GC cells in vitro, with concomitant activation of NF-κB signaling. Administration of an NF-κB inhibitor attenuated PFKFB3-induced EMT in GC cells. PFKFB3 overexpression promoted tumor development and EMT in nude mice, which were attenuated by PFK-15, a PFKFB3 inhibitor. DISCUSSION: PFKFB3 could potentiate malignancy in GC cells through NF-κB pathway-mediated EMT, suggesting PFKFB3 represents a potential target for GC therapy.

Oxidative stress-mediated AMPK inactivation determines the high susceptibility of LKB1-mutant NSCLC cells to glucose starvation.

The liver kinase B1 (LKB1) is an important tumor suppressor and its loss-of-function mutations are observed in around 16% of non-small cell lung cancer (NSCLC) cases. One of the main functions of LKB1 is to activate AMP-activated protein kinase (AMPK) via direct phosphorylation. Under metabolic or energy stress conditions, the LKB1-AMPK axis inhibits the anabolic pathways and activates the catabolic pathways to maintain metabolic homeostasis for cell survival. In this study, we found that LKB1-mutant NSCLC cells are particularly susceptible to cell death induced by glucose starvation, but not by other forms of starvation such as amino acid starvation or serum starvation. Reconstitution of LKB1 in LKB1-mutant cells or LKB1 knockout in LKB1-wild type cells highlighted the importance of the LKB1-AMPK axis for cell survival under glucose starvation. Mechanistically, in LKB1-mutant cells, glucose starvation elicits oxidative stress, which causes AMPK protein oxidation and inactivation, and eventually cell death. Importantly, this process could be effectively reversed and rescued by 2DG (a glucose analog capable of producing NADPH, a key antioxidant), A769662 (an allosteric AMPK activator), and N-acetyl cysteine (NAC) (a ROS scavenger), indicating the presence of a vicious circle between AMPK inactivation and ROS in LKB1-mutant NSCLC cells under glucose starvation. Our study thus elucidates the critical role of redox balance in determining the susceptibility to cell death under glucose starvation in LKB1-mutant NSCLC cells. The findings from this study reveal important clues in search of novel therapeutic strategies for LKB1-mutant NSCLC by targeting glucose metabolism and redox balance.

Inhibition of deubiquitination by PR-619 induces apoptosis and autophagy via ubi-protein aggregation-activated ER stress in oesophageal squamous cell carcinoma.

OBJECTIVES: Targeting the deubiquitinases (DUBs) has become a promising avenue for anti-cancer drug development. However, the effect and mechanism of pan-DUB inhibitor, PR-619, on oesophageal squamous cell carcinoma (ESCC) cells remain to be investigated. MATERIALS AND METHODS: The effect of PR-619 on ESCC cell growth and cell cycle was evaluated by CCK-8 and PI staining. Annexin V-FITC/PI double staining was performed to detect apoptosis. LC3 immunofluorescence and acridine orange staining were applied to examine autophagy. Intercellular Ca2+ concentration was monitored by Fluo-3AM fluorescence. The accumulation of ubi-proteins and the expression of the endoplasmic reticulum (ER) stress-related protein and CaMKKß-AMPK signalling were determined by immunoblotting. RESULTS: PR-619 could inhibit ESCC cell growth and induce G2/M cell cycle arrest by downregulating cyclin B1 and upregulating p21. Meanwhile, PR-619 led to the accumulation of ubiquitylated proteins, induced ER stress and triggered apoptosis by the ATF4-Noxa axis. Moreover, the ER stress increased cytoplasmic Ca2+ and then stimulated autophagy through Ca2+ -CaMKKß-AMPK signalling pathway. Ubiquitin E1 inhibitor, PYR-41, could reduce the accumulation of ubi-proteins and alleviate ER stress, G2/M cell cycle arrest, apoptosis and autophagy in PR-619-treated ESCC cells. Furthermore, blocking autophagy by chloroquine or bafilomycin A1 enhanced the cell growth inhibition effect and apoptosis induced by PR-619. CONCLUSIONS: Our findings reveal an unrecognized mechanism for the cytotoxic effects of general DUBs inhibitor (PR-619) and imply that targeting DUBs may be a potential anti-ESCC strategy.

Silencing of AP-4 inhibits proliferation, induces cell cycle arrest and promotes apoptosis in human lung cancer cells.

Activating enhancer-binding protein (AP)-4 is a member of the basic helix-loop-helix transcription factors, and is involved in tumor biology. However, the role of AP-4 in human lung cancer remains to be fully elucidated. In the present study, the expression of AP-4 in human lung cancer tissues and cells was investigated by reverse transcription-quantitative polymerase chain reaction, and it was observed that the level of AP-4 was increased in tumor tissues and cells compared with their normal counterparts. AP-4 expression was knocked down by transfection with a specific small interfering RNA (siRNA) in lung cancer cells, and this indicated that siRNA-mediated silencing of AP-4 inhibited cell proliferation, arrested the cell cycle at the G0/G1 phase and induced apoptosis by modulating the expression of p21 and cyclin D1. The results of the present study suggest that AP-4 may be an oncoprotein that has a significant role in lung cancer, and that siRNA-mediated silencing of AP-4 may have therapeutic potential as a strategy for the treatment of lung cancer.

Enhancement of silencing DNA polymerase ß on the radiotherapeutic sensitivity of human esophageal carcinoma cell lines.

Human DNA polymerase ß (DNA polymeraseß (polß)) is a small monomeric protein which is essential for short-patch base excision repair (BER). It plays an important role in regulating the radiation sensitivity of tumor cells in the course of tumor radiation therapy. In this study, qRT-PCR and Western blot assays were used to quantify polß expression levels in esophageal carcinoma (EC) cells that were transfected with polß small interfering RNA (siRNA). Cell counting Kit-8 (CCK-8), flow cytometry, and Hoechst/PI stain assays were conducted to evaluate the effects of silencing polß on the radiotherapeutic sensitivity of EC cells. We found that the expression levels of polß in EC cells were significantly decreased after transfection with polß siRNA. Then, we found that polß silencing increased the sensitivity of EC cells to radiation therapy. In conclusion, our study paves the way for a better understanding of the mechanism of the polß gene in DNA repair, and we propose that RNA interference technology will have important applications in gene therapy of EC and other cancers in the future.