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BackgroundCharacterized by an immune-suppressive tumor microenvironment (TME), pancreatic ductal adenocarcinoma (PDAC) is well-known for its poor prognosis. Tumor associated macrophages (TAMs) play a critical role in PDAC TME. An in-depth understanding of TAMs is helpful to develop new strategies for immunotherapy.MethodsA large number of single-cell RNA sequencing data and bulk RNA sequencing data of PDAC were collected for systematic bioinformatics analysis. Characterize subtypes of TAMs at single-cell resolution and its effect on prognosis. Differential gene analysis and cell-cell communication were used to describe the effect on prognosis and validated by the TCGA dataset.ResultsWe used two prognosis-favorable genes, SLC12A5 and ENPP2, to identify a benign M2-like TAMs (bM2-like TAMs), which shared similarities with C1QC + TAMs, CXCL9+ TAMs and CD169+ TAMs, by analyzing scRNA-seq data and bulk RNA data of PDAC. The bM2-like TAMs were revealed to promote T cell activation and proliferation through ALCAM/CD6 interaction. Meanwhile, the bM2-like TAMs were responsible for stroma modeling by altering αSMA+/αSMA-cell ratio. On the contrast, the rest of the M2-like TAMs were defined as malignant M2-like TAMs (mM2-like TAMs), partly overlapping with SPP1+ TAMs. mM2-like TAMs were revealed to promote tumor progression by secretion of MIF and SPP1.ConclusionOur study used two prognosis-favorable genes to divide M2-like TAMs of PDAC into anti-tumor bM2-like TAMs and pro-tumor mM2-like TAMs. The bM2-like TAMs activate T cells through ALCAM/CD6 and generate prognosis-favorable αSMA+ myofibroblasts through secreting TGFβ, which brings insight into heterogeneity of TAMs, prognosis prediction and immunotherapy of PDAC.

Abstract Engineered bacteria‐mediated antitumor approaches have been proposed as promising immunotherapies for cancer. However, the off‐target bacterial toxicity narrows the therapeutic window. Living microbes will benefit from their controllable immunogenicity within tumors for safer antitumor applications. In this study, a genetically encoded microbial activation strategy is reported that uses tunable and dynamic expression of surface extracellular polysaccharides to improve bacterial biocompatibility while retaining therapeutic efficacy. Based on screening of genes associated with Salmonella survival in macrophages, a novel attenuated Salmonella chassis strain AIS (htrA gene‐deficient) highly enriched in tumors after administration and rapidly cleared from normal organs are reported. Subsequently, an engineered bacterial strain, AISI‐H, is constructed based on the AIS strain and an optimized quorum‐sensing regulatory system. The AISI‐H strain can achieve recovery of dynamic tumor‐specific bacterial virulence through a novel HTRA‐RCSA axis‐based and quorum‐sensing synthetic gene circuit‐mediated increase in extracellular polysaccharide content. These strains act “off” in normal organs to avoid unwanted immune activation and “on” in tumors for precise tumor suppression in mice. The AISI‐H strain shows significant tumor inhibition and potent activation of anticancer immunity in a melanoma mouse model. The AISI‐H strain exhibits excellent biocompatibility. This bacterial regulation strategy expands the applications of microbe‐based antitumor therapeutics.

Abstract Background The recruitment of thermogenic adipocytes in human fat depots markedly improves metabolic disorders such as type 2 diabetes mellitus (T2DM). However, identification and quantification of thermogenic cells in human fats, especially in metabolic disorders patients, remains a major challenge. Here, we aim to provide a stringent validation of human thermogenic adipocyte signature genes, and construct transcriptome-based models to quantify the browning degree of human fats. Methods Evidence from RNA-seq, microarray analyses and experimental approaches were integrated to isolate robust human brown-like fat signature genes. Meta-analysis was employed to validate the performance of known human brown-like fat marker genes. Autoencoder was used to reveal the browning levels of human adipose samples for supervised machine learning. Ensemble machine learning was applied to devised molecular metrics for quantifying browning degree of human fats. Obesity and T2DM datasets were used to validate the performance of the molecular metrics in adipose-related metabolic disorders. Results Human brown-like adipocytes were heterogeneous populations which showed distinct transcriptional patterns and biological features. Only DHRS11, REEP6 and STX11 were robust signature genes that were consistently up-regulated in different human brown-like fats, especially in creatine-induced UCP1-independent adipocytes. The molecular metrices based on the expression patterns of the three signature genes, named human browning capacity index (HBI) and absolute HBI (absHBI), were superior to 26 traditional human brown-like fat marker genes and previously reported browning classifier in prediction of browning levels of human adipocytes and adipose tissues as well as primary cell cultures upon various physiological and pharmacological stimuli. Notably, these molecular metrics also reflected the insulin sensitivity and glucose-lipid metabolic activity of human adipose samples from obesity and T2DM patients. Conclusions In summary, this study provides promising signatures and computational tools for evaluating browning levels of human adipose samples in response to physiological and medical intervention. The metrices construction pipeline provides an alternative approach for training machine learning models using unlabeled samples.

Abstract Live bacteria-mediated antitumor therapies mark a pivotal point in cancer immunotherapy. However, the difficulty in reconciling the safety and efficacy of bacterial therapies has limited their application. Improving bacterial tumor-targeted delivery while maintaining biosafety is a critical hurdle for the clinical translation of live microbial therapy for cancer. Here, we developed “dead” yet “functional” Salmonella-loaded macrophages using liquid nitrogen cold shock of an attenuated Salmonella typhimurium VNP20009-contained macrophage cell line. The obtained “dead” macrophages achieve an average loading of approximately 257 live bacteria per 100 cells. The engineered cells maintain an intact cellular structure but lose their original pathogenicity, while intracellular bacteria retain their original biological activity and are delay freed, followed by proliferation. This “Trojan horse”-like bacterial camouflage strategy avoids bacterial immunogenicity-induced neutrophil recruitment and activation in peripheral blood, reduces the clearance of bacteria by neutrophils and enhances bacterial tumor enrichment efficiently after systemic administration. Furthermore, this strategy also strongly activated the tumor microenvironment, including increasing antitumor effector cells (including M1-like macrophages and CD8+ Teffs) and decreasing protumor effector cells (including M2-like macrophages and CD4+ Tregs), and ultimately improved antitumor efficacy in a subcutaneous H22 tumor-bearing mouse model. The cryo-shocked macrophage-mediated bacterial delivery strategy holds promise for expanding the therapeutic applications of living bacteria for cancer.

Summary: In the tumor microenvironment (TME), tumor-associated NEs (TANs) have the potential to be protumorigenic or antitumorigenic within the TME in response to environmental cues. The diversity and plasticity of NEs (NEs) underlie the dual potential of TANs in the TME. Here, we utilized the tumor-targeting bacterium VNP20009 (VNP) to carry a plasmid expressed IFNβ (VNP-IFNβ), which can deliver IFNβ and remodel TANs to an antitumorigenic phenotype, and performed preclinical evaluations in the B16F10 lung metastasis model and the B16F10 subcutaneous xenograft model. Compared with VNP, VNP-IFNβ recruited more NEs and macrophages (Mφs) with antitumor phenotypes in lung metastases and activated dendritic cells (DCs) differentiation, which activated antitumor immune responses of CD4+ T cells, and ultimately inhibited melanoma progression. This study enriches the bacterial-mediated tumor therapy by using tumor-targeting bacteria to deliver IFNβ to the tumor site and inhibit melanoma growth and metastasis by remodeling the tumor immune microenvironment.

Summary: KRAS mutations are frequently detected in non-small cell lung cancers (NSCLCs). Although covalent KRASG12C inhibitors have been developed to treat KRASG12C-mutant cancers, effective treatments are still lacking for other KRAS-mutant NSCLCs. Thus, identifying a KRAS effector that confers poor prognosis would provide an alternative strategy for the treatment of KRAS-driven cancers. Here, we show that KRAS drives expression of deubiquitinase USP13 through Ras-responsive element-binding protein 1 (RREB1). Elevated USP13 promotes KRAS-mutant NSCLC metastasis, which is associated with poor prognosis in NSCLC patients. Mechanistically, USP13 interacts with and removes the K63-linked polyubiquitination of β-catenin at lysine 508, which enhances the binding between β-catenin and transcription factor TCF4. Importantly, we identify 2-methoxyestradiol as an effective inhibitor for USP13 from a natural compound library, and it could potently suppress the metastasis of KRAS-mutant NSCLC cells in vitro and in vivo. These findings identify USP13 as a therapeutic target for metastatic NSCLC with KRAS mutations.

Stilbenes are characterized by a vinyl group connecting two benzene rings to form the basic parent nucleus. Hydrogen atoms on different positions of the benzene rings can be substituted with hydroQxyl groups. These unique structural features confer anti-inflammatory, antibacterial, antiviral, antioxidant, anticancer, cardiovascular protective, and neuroprotective pharmacological effects upon these compounds. Numerous small molecule compounds have demonstrated these pharmacological activities in recent years, including Resveratrol, and Pterostilbene, etc. Tamoxifen and Raloxifene are FDA-approved commonly prescribed synthetic stilbene derivatives. The emphasis is on the potential of these small molecules and their structural derivatives as epigenetic regulators in various diseases. Stilbenes have been shown to modulate epigenetic marks, such as DNA methylation and histone modification, which can alter gene expression patterns and contribute to disease development. This review will discuss the mechanisms by which stilbenes regulate epigenetic marks in various diseases, as well as clinical trials, with a focus on the potential of small molecule and their derivatives such as Resveratrol, Pterostilbene, and Tamoxifen.

Abstract Oncolytic bacteria are the most promising tumor target vector. Questions also remain regarding finding a balance between the therapeutic efficacy and safety of oncolytic bacteria. The critical measure of how this balance is maintained is the improvement in tumor colonization. Attenuated Salmonella typhimurium (VNP20009) as the only Salmonella strain to be evaluated in a clinical trial is a potential tumor therapeutic bacterium. A delivery system with controlled release of VNP after being loaded into neutrophils, which significantly increases the tumor‐targeting of VNP and enhances its therapeutic efficacy in a melanoma lung metastasis model is constructed. To improve the synergistic therapeutic effect, a PD1 nanobody is applied to this system (NE(PD1nb)). NE(PD1nb) activate dendritic cells (DCs) differentiation and stimulate the M1‐like differentiation of macrophages, and induce CD4+ T‐cells maturity and cytotoxic CD8+ T‐cells activation through DCs tumor antigen presentation.

Bacterial antitumor therapy has great application potential given its unique characteristics, including genetic manipulation, tumor targeting specificity and immune system modulation. However, the nonnegligible side effects and limited efficacy of clinical treatment limit their biomedical applications. Engineered bacteria for therapeutic applications ideally need to avoid their accumulation in normal organs and possess potent antitumor activity. Here, we show that macrophage-mediated tumor-targeted delivery of Salmonella typhimurium VNP20009 can effectively reduce the toxicity caused by administrating VNP20009 alone in a melanoma mouse model. This benefits from tumor-induced chemotaxis for macrophages combined with their slow release of loaded strains. Inspired by changes in the tumor microenvironment, including a decrease in intratumoral dysfunctional CD8+ T cells and an increase in PDL1 on the tumor cell surface, macrophages were loaded with the engineered strain VNP-PD1nb, which can express and secrete anti-PD1 nanoantibodies after they are released from macrophages. This novel triple-combined immunotherapy significantly inhibited melanoma tumors by reactivating the tumor microenvironment by increasing immune cell infiltration, inhibiting tumor cell proliferation, remodeling TAMs to an M1-like phenotype and prominently activating CD8+ T cells. These data suggest that novel combination immunotherapy is expected to be a breakthrough relative to single immunotherapy.

Tobacco etch virus protease (TEVp) is wildly exploited for various biotechnological applications. These applications take advantage of TEVp’s ability to cleave specific substrate sequences to study protein function and interactions. A major limitation of this enzyme is its relatively slow catalytic rate. In this study, MD simulations were conducted on TEV enzymes and known highly active mutants (eTEV and uTEV3) to explore the relationship between mutation, conformation, and catalytic function. The results suggest that mutations distant from the active site can influence the substrate-binding pocket through interaction networks. MD analysis of eTEV demonstrates that, by stabilizing the orientation of the substrate at the catalytic site, mutations that appropriately enlarge the substrate-binding pocket will be beneficial for Kcat, enhancing the catalytic efficiency of the enzyme. On the contrary, mutations in uTEV3 reduced the flexibility of the active pocket and increased the hydrogen bonding between the substrate and enzyme, resulting in higher affinity. At the same time, the MD simulation demonstrates that mutations outside of the active site residues could affect the dynamic movement of the binding pocket by altering residue networks and communication pathways, thereby having a profound impact on reactivity. These findings not only provide a molecular mechanistic explanation for the excellent mutants, but also serve as a guiding framework for rational computational design.

Salmonella: mediated cancer therapy has achieved remarkable anti-tumor effects in experimental animal models, but the detailed mechanism remains unsolved. In this report, the active involvement of the host immune response in this process was confirmed by comparing the tumor-suppressive effects of Salmonella in immunocompetent and immunodeficient mice bearing melanoma allografts. Since flagella are key inducers of the host immune response during bacterial infection, flagella were genetically disrupted to analyse their involvement in Salmonella-mediated cancer therapy. The results showed that flagellum-deficient strains failed to induce significant anti-tumor effects, even when more bacteria were administered to offset the difference in invasion efficiency. Flagella mainly activate immune cells via Flagellin/Toll-like receptor 5 (TLR5) signalling pathway. Indeed, we showed that exogenous activation of TLR5 signalling by recombinant Flagellin and exogenous expression of TLR5 both enhanced the therapeutic efficacy of flagellum-deficient Salmonella against melanoma. Our study highlighted the therapeutic value of the interaction between Salmonella and the host immune response through Flagellin/TLR5 signalling pathway during Salmonella-mediated cancer therapy, thereby suggesting the potential application of TLR5 agonists in the cancer immune therapy.

Abstract Inflammatory bowel disease (IBD) is a chronic and relapsing disorder for many people associated with poor health. Although there are some clinical drugs for IBD treatment, the development of effective therapeutics on IBD patients has always been necessary. Here, we show that externalized phosphatidylserine (PS) is observed on the surface of colonic capillaries. Annexin A5 (ANXA5) with high affinity for PS has a good targeting to the colon and effectively alleviates experimental colitis. In contrast, ANXA5 mutant (A5m) lacking the PS-binding ability, has no accumulation in the colon and no therapeutic effects on colitis. Mechanistic investigations indicate that ANXA5 reduces the inflammatory cell infiltration by inhibiting endothelial cell activation dependent on PS-binding ability. With the increasing of PS exposure on activated HUVECs (human umbilical vein endothelial cells), ANXA5 binding induces the internalization of TLR4 via PS-dependent endocytosis. We provide new insights on the molecular mechanism of ANXA5 for its anti-inflammatory effect. Our data suggest that PS-externalization is a potential target of ANXA5 aiming at targeted drug delivery (TDD) for IBD treatment.

Lung cancer is the common malignant tumor with the highest death rate in the world. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a potential anticancer agent induces selective apoptotic death of human cancer cells. Unfortunately, approximately half of lung cancer cell lines are intrinsically resistant to TRAIL-induced cell death. In this study, we identified RuvBL1 as a repressor of c-Jun/AP-1 activity, contributing to TRAIL resistance in lung cancer cells. Knocking down RuvBL1 effectively sensitized resistant cells to TRAIL, and overexpression of RuvBL1 inhibited TRAIL-induced apoptosis. Moreover, there was a negative correlation expression between RuvBL1 and c-Jun in lung adenocarcinoma by Oncomine analyses. High expression of RuvBL1 inversely with low c-Jun in lung cancer was associated with a poor overall prognosis. Taken together, our studies broaden the molecular mechanisms of TRAIL resistance and suggest the application of silencing RuvBL1 synergized with TRAIL to be a novel therapeutic strategy in lung cancer treatment.

The anaerobic bacterium Porphyromonas gingivalisP

Resveratrol (RSV), a health-promoting natural product, has been shown to affect various cellular processes in tumor cells. However, the specific protein targets of RSV and the mechanism of action (MOA) of its anticancer effect remain elusive. In this study, the pharmacological activity of RSV was first evaluated in A549 cells, and the results showed that RSV significantly inhibited A549 cell migration but did not affect cell viability. To elucidate the underlying mechanism, a quantitative chemical proteomics approach was employed to identify the protein targets of RSV. A total of 38 target proteins were identified, and proteomic analysis showed that the targets were mainly involved in cytoskeletal remodeling and EMT, which were verified by subsequent in vitro and in vivo assays. In conclusion, RSV inhibits A549 cell migration by binding to multiple targets to regulate cytoskeletal remodeling and suppress EMT.

Tumor associated macrophages (TAMs) promote cancer progression. Here, the author show that caspase-1 promotes TAMs differentiation by attenuating medium-chain acyl-CoA dehydrogenase activity and that inhibition of this axis results in suppression of tumour growth in a transgenic mouse model of breast cancer.

Bifidobacterium longum (B. longum) could accumulate Selenium (Se) and nano-Se in the form of Se-B. longum and Nano-Se-B. longum, respectively. In this study, the effect of Nano-Se-B. longum in diabetic mice was evaluated. Physiological and metabolic parameters such as blood glucose, body weight, serum insulin level, intraperitoneal glucose tolerance test (IPGTT), food intake, water consumption and urine output were evaluated. The expression of insulin signalling pathway-related proteins was evaluated by western blotting. Haematoxylin and eosin (H&E) was used for histological examination of the liver, pancreas and kidney sections. Creatinine levels in serum (SCr) and blood urea nitrogen (BUN) were measured. Nano-Se-B. longum was the best in terms of delaying the onset of diabetes. Nano-Se-B. longum decreased blood glucose and body weight compared with those noted for the model group. IPGTT, food intake, water consumption and urine output significantly increased and serum insulin levels significantly decreased in the model group compared with those in all the Nano-Se-B. longum-treated mice. Histological results showed that the Nano-Se-B. longum-treated mice were better than the model group mice in terms of pathological changes. The expression of insulin signalling pathway-related proteins was upregulated in the Nano-Se-B. longum-treated groups. A significant increase in SCr and BUN levels was noted in the model group. This study for the first time reported the dose-dependent preventive effect of Nano-Se-B. longum on the onset of diabetes and renal damage. The mechanism may be related to changes in insulin signalling.

A novel annexin V derivative (Cys-Annexin V) with a single cysteine residue at its C-terminal has been developed and successfully labeled site-specifically with 18F-FBEM. 18F-FBEM was synthesized by coupling 18F-fluorobenzoic acid (18F-FBA) with N-(2-aminoethyl)maleimide using optimized reaction conditions. The yield of 18F-FBEM-Cys-Annexin V was 71.5% ± 2.0% (n = 4, based on the starting 18F-FBEM, non-decay corrected). The radiochemical purity of 18F-FBEM-Cys-Annexin V was >95%. The specific radioactivities of 18F-FBEM and 18F-FBEM-Cys-Annexin V were >150 and 3.17 GBq/µmol, respectively. Like the 1st generation 18F-SFB-Annexin V, the novel 18F-FBEM-Cys-Annexin V mainly shows renal and to a lesser extent, hepatobiliary excretion in normal mice. In rat hepatic apoptosis models a 3.88 ± 0.05 (n = 4, 1 h) and 10.35 ± 0.08 (n = 4, 2 h) increase in hepatic uptake of 18F-FBEM-Cys-Annexin V compared to normal rats was observed after injection via the tail vein. The liver uptake ratio (treated/control) at 2 h p.i. as measured via microPET correlated with the ratio of apoptotic nuclei in liver observed using TUNEL histochemistry, indicating that the novel 18F-FBEM-Cys-Annexin V is a potential apoptosis imaging agent.

A novel annexin A5 derivative (cys-annexin A5) with a single cysteine residue at its C-terminal has been developed and successfully labeled in high labeling yield with 99mTc by a ligand exchange reaction. Like the 1st generation 99mTc-HYNIC-annexin A5, the novel 99mTc-cys-annexin A5 derivative shows in normal mice mainly renal and, to a lesser extent, hepatobiliary excretion. In rat models of hepatic apoptosis there was 283% increase in hepatic uptake of 99mTc-cys-annexin A5 as compared to normal mice. The results indicate that the novel 99mTc-cys-annexin A5 is a potential apoptosis imaging agent.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a promising antitumor therapy against lung cancer which is currently undergoing a phase III clinical trial in China. Unfortunately some cancer patients in the clinical trial displayed resistance to TRAIL treatment. In investigating ways to overcome this resistance, we evaluated the inhibitory effect of galangin on TRAIL resistant A549 human lung adenocarcinoma cells. Here we report that, in comparison with the single agents, the combination of galangin and TRAIL markedly suppressed proliferation of A549 cells and induced apoptosis as shown by DAPI and JC-1 staining. The combination of galangin and TRAIL induced PARP cleavage, activation of caspase-8 and p38 MAPK (mitogen-activated protein kinases). These findings indicate that the combination of galangin and TRAIL may constitute a promising strategy for the treatment of lung cancer.

Retinyl esters are the major chemical forms of vitamin A stored in the liver, and can be delivered to peripheral tissues for conversion into biologically active forms. The function and regulation of the hepatic genes that are potentially involved in catalyzing the hydrolysis of retinyl esters remain unclear. Here we show that two lipid hydrolytic genes, pancreatic-related protein 2 (mPlrp2) and procolipase (mClps), expressed specifically in the mouse pancreas, are associated with the ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). Light illumination deficiency or administration of 5′-AMP elevated the ratio of AdoMet to AdoHcy and induced the expression in the liver of mPlrp2 and mClps, which was blocked by all-trans retinoic acid. Mice fed a vitamin A-free diet exhibited increased activation of hepatic mPlrp2 and mClps expression, which was associated with increased methylation of histone H3K4 residues located near the mPlrp2 and mClps promoters. Inhibition of hepatic mPlrp2 and mClps expression by a methylase inhibitor, methylthioadenosine, markedly decreased plasma retinol levels in these mice. The activated hepatic stellate cell (HSC)-T6 cell line specifically expressed mClps and mPlrp2. Inhibition of mClps gene expressions by short hairpin RNA (shRNA) decreased hydrolysis of retinyl esters in the HSC-T6 cell line. These data suggest that the conditional expression of mPlrp2 and mClps is involved in the hydrolysis of retinyl esters in the mouse liver.

Background: Although domain IV of annexin A5 (anxA5) may be less effective in binding phosphatidylserine (PS), the four domains together may guarantee the maximum binding of anxA5 to the PS membrane. Additionally, previous research has shown that annexin mutants lacking one or more domain(s) have different biological activities compared to the wild-type. The present research mainly aims to study the role of domain IV in the crucial PS-binding function of anxA5. Methods: The domain IV-truncated anxA5 protein was constructed and purified. Isothermal titration calorimetry, flow cytometry and activated partial thromboplastin time were adopted to examine the function of domain IV in anxA5-PS binding directly or indirectly. Results: The domain IV-truncated form of anxA5 is impaired in binding PS liposome and apoptotic cells, and anticoagulation activity. The mutant cannot bind calcium, but binds PS only in the presence of calcium. Conclusions: Truncation of domain IV of anxA5 destroys its calcium-binding ability and impairs its PS-binding activity. Truncation of domain IV may induce conformation change of anxA5 or reduce the hydrophobic interactions between protein and membrane, which may explain the decrease of PS-binding affinity of the mutant.

Background: Andrographolide (ADR), the main active component of Andrographis paniculata, displays anticancer activity in various cancer cell lines, among which leukemia cell lines exhibit the highest sensitivity to ADR. In particular, ADR was also reported to have reduced drug resistance in multidrug resistant cell lines. However, the mechanism of action (MOA) of ADR’s anticancer and anti-drug-resistance activities remain elusive. Methods: In this study, we used the MV4-11 cell line, a FLT3 positive acute myeloid leukemia (AML) cell line that displays multidrug resistance, as our experimental system. We first evaluated the effect of ADR on MV4-11 cell proliferation. Then, a quantitative proteomics approach was applied to identify differentially expressed proteins in ADR-treated MV4-11 cells. Finally, cellular processes and signal pathways affected by ADR in MV4-11 cell were predicted with proteomic analysis and validated with in vitro assays. Results: ADR inhibits MV4-11 cell proliferation in a dose- and time-dependent manner. With a proteomic approach, we discovered that ADR inhibited fatty acid synthesis, cellular iron uptake and FLT3 signaling pathway in MV4-11 cells. Conclusions: ADR inhibits MV4-11 cell proliferation through inhibition of fatty acid synthesis, iron uptake and protein synthesis. Furthermore, ADR reduces drug resistance by blocking FLT3 signaling.

The artemisinin compounds, which are well-known for their potent therapeutic antimalarial activity, possess in vivo and in vitro antitumor effects. Although the anticancer effect of artemisinin compounds has been extensively reported, the precise mechanisms underlying its cytotoxicity remain under intensive study. In the present study, a high-throughput quantitative proteomics approach was applied to identify differentially expressed proteins of HCT116 colorectal cancer cell line with artesunate (ART) treatment. Through Ingenuity Pathway Analysis, we discovered that the top-ranked ART-regulated biological pathways are abrogation of fatty acid biosynthetic pathway and mitochondrial dysfunction. Subsequent assays showed that ART inhibits HCT116 cell proliferation through suppressing the fatty acid biosynthetic pathway and activating the mitochondrial apoptosis pathway. In addition, ART also regulates several proteins that are involved in NF-κB pathway, and our subsequent assays showed that ART suppresses the NF-κB pathway. These proteomic findings will contribute to improving our understanding of the underlying molecular mechanisms of ART for its therapeutic cytotoxic effect towards cancer cells.

BACKGROUND: Schistosomiasis remains a major public health concern affecting billions of people around the world. Currently, praziquantel is the only drug of choice for treatment of human schistosomiasis. The emergence of drug resistance to praziquantel in schistosomes makes the development of novel drugs an urgent task. Thioredoxin glutathione reductase (TGR) enzymes in Schistosoma mansoni and some other platyhelminths have been identified as alternative targets. The present study was designed to confirm the existense and the potential value of TGR as a target for development of novel antischistosomal agents in Schistosoma japonicum, a platyhelminth endemic in Asia. METHODS AND FINDINGS: After cloning the S. japonicum TGR (SjTGR) gene, the recombinant SjTGR selenoprotein was purified and characterized in enzymatic assays as a multifunctional enzyme with thioredoxin reductase (TrxR), glutathione reductase (GR) and glutaredoxin (Grx) activities. Immunological and bioinformatic analyses confirmed that instead of having separate TrxR and GR proteins in mammalian, S. japonicum only encodes TGR, which performs the functions of both enzymes and plays a critical role in maintaining the redox balance in this parasite. These results were in good agreement with previous findings in Schistosoma mansoni and some other platyhelminths. Auranofin, a known inhibitor against TGR, caused fatal toxicity in S. japonicum adult worms in vitro and reduced worm and egg burdens in S. japonicum infected mice. CONCLUSIONS: Collectively, our study confirms that a multifunctional enzyme SjTGR selenoprotein, instead of separate TrxR and GR enzymes, exists in S. japonicum. Furthermore, TGR may be a potential target for development of novel agents against schistosomes. This assumption is strengthened by our demonstration that the SjTGR is an essential enzyme for maintaining the thiol-disulfide redox homeostasis of S. japonicum.

BACKGROUND: Blood vessels in tumors express higher level of aminopeptidase N (APN) than normal tissues. Evidence suggests that the CNGRC motif is an APN ligand which targets tumor vasculature. Increased expression of APN in tumor vascular endothelium, therefore, offers an opportunity for targeted delivery of NGR peptide-linked drugs to tumors. METHODS/PRINCIPAL FINDINGS: To determine whether an additional cyclic CNGRC sequence could improve endothelial cell homing and antitumor effect, human plasminogen kringle 5 (hPK5) was modified genetically to introduce a CNGRC motif (NGR-hPK5) and was subsequently expressed in yeast. The biological activity of NGR-hPK5 was assessed and compared with that of wild-type hPK5, in vitro and in vivo. NGR-hPK5 showed more potent antiangiogenic activity than wild-type hPK5: the former had a stronger inhibitory effect on proliferation, migration and cord formation of vascular endothelial cells, and produced a stronger antiangiogenic response in the CAM assay. To evaluate the tumor-targeting ability, both wild-type hPK5 and NGR-hPK5 were (99 m)Tc-labeled, for tracking biodistribution in the in vivo tumor model. By planar imaging and biodistribution analyses of major organs, NGR-hPK5 was found localized to tumor tissues at a higher level than wild-type hPK5 (approximately 3-fold). Finally, the effects of wild-type hPK5 and NGR-modified hPK5 on tumor growth were investigated in two tumor model systems. NGR modification improved tumor localization and, as a consequence, effectively inhibited the growth of mouse Lewis lung carcinoma (LLC) and human colorectal adenocarcinoma (Colo 205) cells in tumor-bearing mice. CONCLUSIONS/SIGNIFICANCE: These studies indicated that the addition of an APN targeting peptide NGR sequence could improve the ability of hPK5 to inhibit angiogenesis and tumor growth.

BACKGROUND: Schistosomiasis japonica is a zoonotic parasitic disease and oral vaccine delivery system would be benefit for prevention of this disease. Although attenuated salmonella has been used as an antigen expression vector for oral vaccine development, the membrane-bound vacuoles in which bacteria reside hinders the presentation of expressed heterologous antigens to the major histocompatibility complex (MHC) molecules. The present work used an attenuated Salmonella typhimurium strain VNP20009 to secretory expression of Sj23LHDGST bivalent antigen from Schistosoma japonicum and tested the protective efficacy against S. japonicum infection in orally immunized mice. METHODOLOGY/PRINCIPAL FINDINGS: Promoters (nirB or pagC) were used to express the antigen (Sj23LHDGST) and the Salmonella type III or α-hemolysin secretion system was employed to secrete it. The immunoblotting analysis and fluorescent microscopy revealed that the antigen was effectively expressed and delivered to the cytosol of macrophages in vitro. Among recombinant vaccine strains, an engineered VNP20009 which expressed the antigen by nirB promoter and secreted it through type III secretion system (nirB-sopE(1-104)-Sj23LHD-GST) efficiently protected against S. japonicum infection in a mouse model. This strain elicited a predominantly IgG(2a) antibody response and a markedly increase in the production of IL-12 and IFN-γ. The flow cytometric analysis demonstrated that this strain caused T cell activation as evidenced by significantly increased expression of CD44 and CD69. CONCLUSION/SIGNIFICANCE: Oral delivery of antigen by nirB-driven Salmonella typhimurium type III secretion system is a novel, safe, inexpensive, efficient and convenient approach for schistosome vaccine development.

Oral delivery of proteins faces challenges due to the harsh conditions of the gastrointestinal (GI) tract, including gastric acid and intestinal enzyme degradation. Permeation enhancers are limited in their ability to deliver proteins with high molecular weight and can potentially cause toxicity by opening tight junctions. To overcome these challenges, we propose the use of montmorillonite (MMT) as an adjuvant that possesses both inflammation-oriented abilities and the ability to regulate gut microbiota. This adjuvant can be used as a universal protein oral delivery technology by fusing with advantageous binding amino acid sequences. We demonstrated that anti-TNF-α nanobody (VII) can be intercalated into the MMT interlayer space. The carboxylate groups (-COOH) of aspartic acid (D) and glutamic acid (E) interact with the MMT surface through electrostatic interactions with sodium ions (Na+). The amino groups (NH2) of asparagine (N) and glutamine (Q) are primarily attracted to the MMT layers through hydrogen bonding with oxygen atoms on the surface. This binding mechanism protects VII from degradation and ensures its release in the intestinal tract, as well as retaining biological activity, leading to significantly enhanced therapeutic effects on colitis. Furthermore, VII@MMT increases the abundance of short-chain fatty acids (SCFAs)-producing strains, including Clostridia, Prevotellaceae, Alloprevotella, Oscillospiraceae, Clostridia_vadinBB60_group, and Ruminococcaceae, therefore enhance the production of SCFAs and butyrate, inducing regulatory T cells (Tregs) production to modulate local and systemic immune homeostasis. Overall, the MMT adjuvant provides a promising universal strategy for protein oral delivery by rational designed protein. [ABSTRACT FROM AUTHOR]

Abstract Hexachlorobutadiene (HCBD) is a concerning chemical that is included in the United States Toxic Substances Control Act, and the Stockholm Convention. Knowledge of the sources of HCBD is insufficient and is pivotal for accurate inventory and implementing global action. In this study, unintentional HCBD release and source emission factors of 121 full-scale industrial plants from 12 industries are investigated. Secondary copper smelting, electric arc furnace steelmaking, and hazardous waste incineration show potential for large emission reductions, which are found of high HCBD emission concentrations of > 20 ng/g in fine particulate matter in this study. The highest HCBD emission concentration is observed for the secondary copper smelting industry (average: 1380 ng/g). Source emission factors of HCBD for the 12 industries range from 0.008 kg/t for coal fire power plants to 0.680 kg/t for secondary lead smelting, from which an estimation of approximately 8452.8 g HCBD emissions annually worldwide achieved. The carcinogenic risks caused by HCBD emissions from countries and regions with intensive 12 industrial sources are 1.0-80 times higher than that without these industries. These results will be useful for formulating effective strategies of HCBD control.

The recently proposed learning-to-learn iterative search algorithm (LISA) for multiple-input multiple-output (MIMO) detection can achieve excellent BER performance for low-order modulation signals, such as BPSK/QPSK, but its performance is degraded as the modulation order increases. This limits its application in high data rate transmission. Motivated by this observation, this paper proposes a decomposition-ordering-based LISA (DO-LISA) to improve the accuracy of LISA for higher-order QAM signals. Firstly, the detection of high-order QAM signal is transformed into the detection of QPSK signal to exploit the facility of binary classification. To further exploit the sequential features of a bidirectional long short-term memory (BiLSTM) network, channel ordering is adopted to enhance the detection accuracy. Moreover, to make the proposed method more practical, low-rank decomposition and channel pruning are adopted to accelerate and compress the neural network. Numerical experiments show that the proposed method can achieve quasi-ML BER performance. Compared with the full model, the compact model achieves $2.37\times $ compression ratio and $2.38\times $ acceleration ratio with limited accuracy loss. In addition, the proposed DO-LISA shows its good generalization ability by preserving very good BER performance both in the correlated channel condition and imperfect channel condition.