Differences between the intestinal microbial communities of healthy dogs from plateau and those of plateau dogs infected with Echinococcus.

OBJECTIVE: Cystic echinococcosis (CE) represents a profoundly perilous zoonotic disease. The advent of viral macrogenomics has facilitated the exploration of hitherto uncharted viral territories. In the scope of this investigation, our objective is to scrutinize disparities in the intestinal microbiotic ecosystems of canines dwelling in elevated terrains and those afflicted by Echinococcus infection, employing the tool of viral macrogenomics. METHODS: In this study, we collected a comprehensive total of 1,970 fecal samples from plateau dogs infected with Echinococcus, as well as healthy control plateau dogs from the Yushu and Guoluo regions in the highland terrain of China. These samples were subjected to viral macrogenomic analysis to investigate the viral community inhabiting the canine gastrointestinal tract. RESULTS: Our meticulous analysis led to the identification of 136 viral genomic sequences, encompassing eight distinct viral families. CONCLUSION: The outcomes of this study hold the potential to enhance our comprehension of the intricate interplay between hosts, parasites, and viral communities within the highland canine gut ecosystem. Through the examination of phage presence, it may aid in early detection or assessment of infection severity, providing valuable insights into Echinococcus infection and offering prospects for potential treatment strategies.

Cross-Phosphorylation between AgrC Histidine Kinase and the Noncognate Response Regulator Lmo1172 in Listeria monocytogenes under Benzalkonium Chloride Stress.

Benzalkonium chloride (BC) is widely used for disinfection in the food industry. However, Listeria monocytogenes strains with resistance to BC have been reported recently. In L. monocytogenes, the Agr communication system consists of a membrane-bound peptidase AgrB, a precursor peptide AgrD, a histidine kinase (HK) AgrC, and a response regulator (RR) AgrA. Our previous study showed that the agr genes are significantly upregulated by BC adaptation. This study aimed to investigate the role of the Agr system in BC resistance in L. monocytogenes. Our results showed that the Agr system was involved in BC resistance. However, a direct interaction between BC and AgrC was not observed, nor between BC and AgrA. These results indicated that BC could induce the Agr system via an indirect action. Both AgrBD and AgrC were required for growth under BC stress. Nevertheless, when exposed to BC, the gene deletion mutant ∆agrA strain exhibited better growth performance than its parental strain. The RR Lmo1172 played a role in BC resistance in the ∆agrA strain, suggesting that Lmo1172 may be an alternative to AgrA in the phosphotransfer pathway. Phosphorylation of Lmo1172 by AgrC was observed in vitro. The cognate HK Lmo1173 of Lmo1172 was not involved in BC stress, regardless of whether it was as the wild-type or the ∆agrA mutant strain. Our evidence suggests that the HK AgrC cross-phosphorylates its noncognate RR Lmo1172 to cope with BC stress when the cognate RR AgrA is absent. In vivo, further studies will be required to detect phosphotransfer of AgrC/AgrA and AgrC/Lmo1172.

Exenatide and Metformin Improve Serum Indices and Intestinal Flora in patients with Type 2 Diabetes Mellitus and Non-Alcoholic Fatty Liver Disease.

The aim of the study was to investigate th e in flue nce of Exenatide comb ined with Met formin on fasti ng blood glucose, postpr andial glucose, triglycerides, total cholesterol, alanine aminotransferase, aspartate aminotransferase, and inte s tinal flora in typ e 2 diab etes mellitus cases with non-alcoholic fatty liver disease. A total of 128 type 2 diabetes mellitus patients with non-alcoholic fatty liver disease, diagnosed from Januar y 2019 to January 2022, were included and randomly assigned to either G roup A (n=64) or Gro up B (n =64). Group A received Metformin, while Group B received Exenatide injection and Metfor min. After 24 weeks of treat ment, blood glucose indices (fasting blood glucose and postprandial glucose), blood lipid indices (triglycerides and total cholesterol), liver func tion indices (alanine aminotransferase and aspar tate aminotransferase) were all lower in Group B than in Group A (p<0.001 for all). Counts o f Escherichia coli and Enterococcus faecalis were lower in Group B than in Group A (both p<0.05), counts of Bifidobacteria and Lactobacillus were highe r i n Group B than in Grou p A (both p<0.05). Combin ation of Exenati de and Metformi n may have synergistic effects in improving metabo lic an d hepatic pa rameters, a s well as re gulat ing intestinal flora, which cou ld provide a pro misin g therapeutic option for the management of these patients.

Virome of high-altitude canine digestive tract and genetic characterization of novel viruses potentially threatening human health.

The majority of currently emerging infectious illnesses are zoonotic infections, which have caused serious public health and economic implications. The development of viral metagenomics has helped us to explore unknown viruses. We collected 1,970 canine feces from Yushu and Guoluo in the plateau region of China for this study to do a metagenomics analysis of the viral community of the canine digestive tract. Our analysis identified 203 novel viruses, classified into 11 known families and 2 unclassified groups. These viruses include the hepatitis E virus, first identified in dogs, and the astrovirus, coronavirus, polyomavirus, and others. The relationship between the newly identified canine viruses and known viruses was investigated through the use of phylogenetic analysis. Furthermore, we demonstrated the cross-species transmission of viruses and predicted new viruses that may cause diseases in both humans and animals, providing technical support for the prevention and control of diseases caused by environmental pollution viruses. IMPORTANCE Most emerging infectious diseases are due to zoonotic disease agents. Because of their effects on the security of human or animal life, agriculture production, and food safety, zoonotic illnesses and livestock diseases are of worldwide significance. Because dogs are closely related to humans and domestic animals, they serve as one of the important links in the transmission of zoonotic and livestock diseases. Canines can contaminate the environment in which humans live such as water and soil through secretions, potentially altering the human gut microbiota or causing diseases. Our study enriched the viral community in the digestive tract microbiome of dogs and found types of viruses that threaten human health, providing technical support for the prevention and control of early warning of diseases caused by environmental contaminant viruses.

MiR-590-5p promotes cisplatin resistance via targeting hMSH2 in ovarian cancer.

OBJECTIVE: The mechanisms of ovarian cancer generate chemotherapy resistance are still unclear. This study aimed to explore the role of microRNA (miR)-590-5p in regulating hMSH2 expression and cisplatin resistance in ovarian cancer. METHODS: MiR-590-5p was identified as a regulator of hMSH2 with miRDB database and Target Scan database. Then cisplatin sensitive cell line (SKOV3) and resistant cell line (SKOV3-DDP) of ovarian cancer were cultured for cell functional assay and molecular biology assay. The expression levels of MiR-590-5p and hMSH2 were compared between the two cell lines. Dual luciferase reporter assay was used to verify the targeted regulatory relationship between miR-590-5p and hMSH2. CCK-8 assay and cell apoptosis assay were utilized to assess the role of MiR-590-5p and hMSH2 in cell viability under cisplatin. RESULTS: The expression of hMSH2 was significantly decreased, and miR-590-5p was significantly up-regulated in SKOV3-DDP. Up-regulation of hMSH2 weakened the viability of SKOV3 and SKOV3-DDP cell under cisplatin. Transfection with miR­590-5p mimics reduced the expression of hMSH2 and enhanced the viability of ovarian cancer cells under cisplatin, whereas inhibition of miR­590-5p increased the expression of hMSH2, and decreased ovarian cancer cells' viability under cisplatin. Furthermore, luciferase reporter assay showed that hMSH2 was a direct target of miR-590-5p. CONCLUSION: The present study demonstrates that miR­590-5p promotes cisplatin resistance of ovarian cancer via negatively regulating hMSH2 expression. Inhibition of miR­590-5p decreases ovarian cancer cells' viability under cisplatin. Thus miR­590-5p and hMSH2 may serve as therapeutic targets for cisplatin resistant ovarian cancer.

Current genetic strategies to investigate gene functions in Trichoderma reesei.

The filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina, Ascomycota) is a well-known lignocellulolytic enzymes-producing strain in industry. To increase the fermentation titer of lignocellulolytic enzymes, random mutagenesis and rational genetic engineering in T. reesei were carried out since it was initially found in the Solomon Islands during the Second World War. Especially the continuous exploration of the underlying regulatory network during (hemi)cellulase gene expression in the post-genome era provided various strategies to develop an efficient fungal cell factory for these enzymes' production. Meanwhile, T. reesei emerges competitiveness potential as a filamentous fungal chassis to produce proteins from other species (e.g., human albumin and interferon α-2b, SARS-CoV-2 N antigen) in virtue of the excellent expression and secretion system acquired during the studies about (hemi)cellulase production. However, all the achievements in high yield of (hemi)cellulases are impossible to finish without high-efficiency genetic strategies to analyze the proper functions of those genes involved in (hemi)cellulase gene expression or secretion. Here, we in detail summarize the current strategies employed to investigate gene functions in T. reesei. These strategies are supposed to be beneficial for extending the potential of T. reesei in prospective strain engineering.

Cinnamaldehyde Targets the LytTR DNA-Binding Domain of the Response Regulator AgrA to Attenuate Biofilm Formation of Listeria monocytogenes.

The Agr quorum sensing (QS) system is known to contribute to biofilm formation in Listeria monocytogenes. Cinnamaldehyde, a natural food preservative, is considered an inhibitor of Agr-mediated QS in L. monocytogenes. However, the exact mechanism by which cinnamaldehyde acts on Agr remains unclear. In this study, we assessed the effects of cinnamaldehyde on the histidine kinase AgrC and the response regulator AgrA in the Agr system. AgrC kinase activity was not influenced by cinnamaldehyde, and binding between AgrC and cinnamaldehyde was not observed when microscale thermophoresis (MST) was performed, indicating that AgrC was not the target of cinnamaldehyde. AgrA is specifically bound to the agr promoter (P2) to activate the transcription of the Agr system. However, AgrA-P2 binding was prevented by cinnamaldehyde. The interaction between cinnamaldehyde and AgrA was further confirmed with MST. Two conserved amino acids, Asn-178 and Arg-179, located in the LytTR DNA-binding domain of AgrA, were identified as the key sites for cinnamaldehyde-AgrA binding by alanine mutagenesis and MST. Coincidentally, Asn-178 was also involved in the AgrA-P2 interaction. Taken together, these results suggest that cinnamaldehyde acts as a competitive inhibitor of AgrA in AgrA-P2 binding, which leads to suppressed transcription of the Agr system and reduced biofilm formation in L. monocytogenes. IMPORTANCE Listeria monocytogenes can form biofilms on various food contact surfaces, posing a serious threat to food safety. Biofilm formation of L. monocytogenes is positively regulated by the Agr quorum sensing system. Thus, an alternative strategy for controlling L. monocytogenes biofilms is interfering with the Agr system. Cinnamaldehyde is considered an inhibitor of the L. monocytogenes Agr system; however, its exact mechanism of action is still unclear. Here, we found that AgrA (response regulator), rather than AgrC (histidine kinase), was the target of cinnamaldehyde. The conserved Asn-178 in the LytTR DNA-binding domain of AgrA was involved in cinnamaldehyde-AgrA and AgrA-P2 binding. Therefore, the occupation of Asn-178 by cinnamaldehyde suppressed transcription of the Agr system and reduced biofilm formation in L. monocytogenes. Our findings could provide a better understanding of the mechanism by which cinnamaldehyde inhibits L. monocytogenes biofilm formation.

Viromics Reveals the High Diversity of Viruses from Fishes of the Tibet Highland.

Aquaculture is important for food security and nutrition. The economy has recently been significantly threatened and the risk of zoonoses significantly increased by aquatic diseases, and the ongoing introduction of new aquatic pathogens, particularly viruses, continues to represent a hazard. Yet, our knowledge of the diversity and abundance of fish viruses is still limited. Here, we conducted a metagenomic survey of different species of healthy fishes caught in the Lhasa River, Tibet, China, and sampled intestinal contents, gills, and tissues. To be more precise, by identifying and analyzing viral genomes, we aim to determine the abundance, diversity, and evolutionary relationships of viruses in fish with other potential hosts. Our analysis identified 28 potentially novel viruses, 22 of which may be associated with vertebrates, across seven viral families. During our research, we found several new strains of viruses in fish, including papillomavirus, hepadnavirus, and hepevirus. Additionally, we discovered two viral families, Circoviridae and Parvoviridae, which were prevalent and closely related to viruses that infect mammals. These findings further expand our understanding of highland fish viruses and highlight the emerging view that fish harbor large, unknown viruses. IMPORTANCE The economy and zoonoses have recently been significantly threatened by aquatic diseases. Yet, our knowledge of the diversity and abundance of fish viruses is still limited. We identified the wide genetic diversity of viruses that these fish were harboring. Since there are currently few studies on the virome of fish living in the Tibet highland, our research adds to the body of knowledge. This discovery lays the groundwork for future studies on the virome of fish species and other highland animals, preserving the ecological equilibrium on the plateau.

Loop closure detection of visual SLAM based on variational autoencoder.

Loop closure detection is an important module for simultaneous localization and mapping (SLAM). Correct detection of loops can reduce the cumulative drift in positioning. Because traditional detection methods rely on handicraft features, false positive detections can occur when the environment changes, resulting in incorrect estimates and an inability to obtain accurate maps. In this research paper, a loop closure detection method based on a variational autoencoder (VAE) is proposed. It is intended to be used as a feature extractor to extract image features through neural networks to replace the handicraft features used in traditional methods. This method extracts a low-dimensional vector as the representation of the image. At the same time, the attention mechanism is added to the network and constraints are added to improve the loss function for better image representation. In the back-end feature matching process, geometric checking is used to filter out the wrong matching for the false positive problem. Finally, through numerical experiments, the proposed method is demonstrated to have a better precision-recall curve than the traditional method of the bag-of-words model and other deep learning methods and is highly robust to environmental changes. In addition, experiments on datasets from three different scenarios also demonstrate that the method can be applied in real-world scenarios and that it has a good performance.

Phenyllactic acid application to control Listeria monocytogenes biofilms and its growth in milk and spiced beef.

Listeria monocytogenes, as a food-associated pathogen, is able to develop biofilms on different surfaces of food contact, which seriously threatens food safety. Phenyllactic acid (PLA) exhibits excellent inhibitory effects on many bacterial strains including L. monocytogenes. Our study aimed to investigate effects of PLA on L. monocytogenes biofilms and its growth in milk and on spiced beef. Biofilm biomass was measured by the microplate method and biofilm structure was observed by electron microscopy. Growth of L. monocytogenes in food samples was determined by colony counting. Results from the agar dilution method demonstrated that L. monocytogenes 10403S had a PLA minimum inhibitory concentration (MIC) value of 6 mg/ml. Sub-inhibitory concentrations of PLA could inhibit biofilm formation by reducing the secretion of exopolysaccharides and extracellular proteins in L. monocytogenes. PLA at concentrations above 1/2MIC could destroy mature biofilms of L. monocytogenes by decreasing the exopolysaccharides and extracellular proteins in the biofilm framework. Both swimming and swarming motilities of L. monocytogenes were inhibited by PLA. The hemolytic activity of L. monocytogenes was inactivated by PLA. However, the capacity to attach and invade Caco-2 cells was not affected by PLA. The results displayed that PLA had no effect on the expression of genes associated with motility, but reduced the expression level of the hly gene encoding Listeria hemolysin. When added to ultra-high temperature (UHT) whole and pasteurized milk, PLA at 3 mg/ml inhibited L. monocytogenes growth through 14 days of storage at 4 °C. PLA at concentrations ≥3 mg/ml significantly reduced L. monocytogenes counts on spiced beef samples during storage. PLA has potential as an alternative antimicrobial to control L. monocytogenes contamination and its biofilms in food industry.

One-Step Route to Fe2O3 and FeSe2 Nanoparticles Loaded on Carbon-Sheet for Lithium Storage.

Iron-based anode materials, such as Fe2O3 and FeSe2 have attracted widespread attention for lithium-ion batteries due to their high capacities. However, the capacity decays seriously because of poor conductivity and severe volume expansion. Designing nanostructures combined with carbon are effective means to improve cycling stability. In this work, ultra-small Fe2O3 nanoparticles loaded on a carbon framework were synthesized through a one-step thermal decomposition of the commercial C15H21FeO6 [Iron (III) acetylacetonate], which could be served as the source of Fe, O, and C. As an anode material, the Fe2O3@C anode delivers a specific capacity of 747.8 mAh g-1 after 200 cycles at 200 mA g-1 and 577.8 mAh g-1 after 365 cycles at 500 mA g-1. When selenium powder was introduced into the reaction system, the FeSe2 nano-rods encapsulated in the carbon shell were obtained, which also displayed a relatively good performance in lithium storage capacity (852 mAh g-1 after 150 cycles under the current density of 100 mA·g-1). This study may provide an alternative way to prepare other carbon-composited metal compounds, such as FeNx@C, FePx@C, and FeSx@C, and found their applications in the field of electrochemistry.

Andrographolide Inhibits Biofilm and Virulence in Listeria monocytogenes as a Quorum-Sensing Inhibitor.

Listeria monocytogenes is a major foodborne pathogen that can cause listeriosis in humans and animals. Andrographolide is known as a natural antibiotic and exhibits good antibacterial activity. We aimed to investigate the effect of andrographolide on two quorum-sensing (QS) systems, LuxS/AI-2 and Agr/AIP of L. monocytogenes, as well as QS-controlled phenotypes in this study. Our results showed that neither luxS expression nor AI-2 production was affected by andrographolide. Nevertheless, andrographolide significantly reduced the expression levels of the agr genes and the activity of the agr promoter P2. Results from the crystal violet staining method, confocal laser scanning microscopy (CLSM), and field emission scanning electron microscopy (FE-SEM) demonstrated that andrographolide remarkably inhibited the biofilm-forming ability of L. monocytogenes 10403S. The preformed biofilms were eradicated when exposed to andrographolide, and reduced surviving cells were also observed in treated biofilms. L. monocytogenes treated with andrographolide exhibited decreased ability to secrete LLO and adhere to and invade Caco-2 cells. Therefore, andrographolide is a potential QS inhibitor by targeting the Agr QS system to reduce biofilm formation and virulence of L. monocytogenes.

Advances and Challenges in Heavy-Metal-Free InP Quantum Dot Light-Emitting Diodes.

Light-emitting diodes based on colloidal quantum dots (QLEDs) show a good prospect in commercial application due to their narrow spectral linewidths, wide color range, excellent luminance efficiency, and long operating lifetime. However, the toxicity of heavy-metal elements, such as Cd-based QLEDs or Pb-based perovskite QLEDs, with excellent performance, will inevitably pose a serious threat to people's health and the environment. Among heavy-metal-free materials, InP quantum dots (QDs) have been paid special attention, because of their wide emission, which can, in principle, be tuned throughout the whole visible and near-infrared range by changing their size, and InP QDs are generally regarded as one of the most promising materials for heavy-metal-free QLEDs for the next generation displays and solid-state lighting. In this review, the great progress of QLEDs, based on the fundamental structure and photophysical properties of InP QDs, is illustrated systematically. In addition, the remarkable achievements of QLEDs, based on their modification of materials, such as ligands exchange of InP QDs, and the optimization of the charge transport layer, are summarized. Finally, an outlook is shown about the challenge faced by QLED, as well as possible pathway to enhancing the device performance. This review provides an overview of the recent developments of InP QLED applications and outlines the challenges for achieving the high-performance devices.

Benzalkonium Chloride Adaptation Increases Expression of the Agr System, Biofilm Formation, and Virulence in Listeria monocytogenes.

Benzalkonium chloride (BC) is widely used for disinfection in food industry. However, prolonged exposure to BC may lead to the emergence of BC adapted strains of Listeria monocytogenes, an important foodborne pathogen. Until now, two communication systems, the LuxS/AI-2 system and the Agr system, have been identified in L. monocytogenes. This study aimed to investigate the role of communication systems in BC adaptation and the effect of BC adaptation on two communication systems and the communication-controlled behaviors in L. monocytogenes. Results demonstrated that the Agr system rather than the LuxS system plays an important role in BC adaptation of L. monocytogenes. Neither luxS expression nor AI-2 production was affected by BC adaptation. On the other hand, the expression of the agr operon and the activity of the agr promoter were significantly increased after BC adaptation. BC adaptation enhanced biofilm formation of L. monocytogenes. However, swarming motility was reduced by BC adaptation. Data from qRT-PCR showed that flagella-mediated motility-related genes (flaA, motA, and motB) were downregulated in BC adapted strains. BC adaptation increased the ability of L. monocytogenes to adhere to and invade Caco-2 cells but did not affect the hemolytic activity. Compared with the wild-type strains, the expression levels of virulence genes prfA, plcA, mpl, actA, and plcB increased more than 2-fold in BC adapted strains; however, lower than 2-fold changes in the expression of hemolysis-associated gene hly were observed. Our study suggests that BC adaptation could increase the expression of the Agr system and enhance biofilm formation, invasion, and virulence of L. monocytogenes, which brings about threats to food safety and public health. Therefore, effective measures should be taken to avoid the emergence of BC adapted strains of L. monocytogenes.

Phase-shift-mediated sensitive detection of propagating ultra-confined graphene plasmons.

The ultra-confined plasmon field supported by graphene provides an ideal platform for enhanced light-matter interactions and studies of fundamental physical phenomena. On the other hand, the intrinsic ultra-short plasmon wavelength obstructs in-plane detectability of plasmon behaviors, like wavelength variations induced by biomolecule or dragging current. The detection of plasmon wavefront and its spatial shift relies on scattering-type scanning near-field microscopy with a spatial resolution of 20 nm. Here we propose a configuration which can efficiently separate ultra-confined plasmon region from detection region, guaranteeing both field confinement and in-plane sensitive detection of wavelength variations. As an example, the application in detecting Fizeau drag effect is demonstrated. Our study can be applied for detecting strong light-matter interactions, including fundamental physical studies and biosensing applications.

Urolithin B, a Gut Microbiota Metabolite, Reduced Susceptibility to Myocardial Arrhythmic Predisposition after Hypoxia.

Cardiomyocyte apoptosis, neural remodeling, and gap junction channel change play critical roles in ventricular arrhythmia (VA) after acute myocardial infarction (AMI). Urolithin B (UB), one of the gut metabolites of ellagitannins, a class of antioxidant polyphenols, has various biological activities, but its direct role in cardiomyocyte apoptosis, neural remodeling, and gap junction channel change after AMI remains elusive. We investigated whether urolithin B reduced susceptibility of myocardial arrhythmic after myocardial infarction (MI). In vitro, the cardiomyocytes were subjected to hypoxia (94% N2/5% CO2/1% O2) for 3 hours. Cardiomyocyte apoptosis was assessed by TUNEL staining and western blotting. Urolithin B was found to decrease the number of apoptotic cells after hypoxia. Moreover, there was a substantial decrease in the expression of neural remodeling markers in the urolithin B treatment group. Urolithin B significantly increased the expression level of gap junction channel protein. Mechanistically, urolithin B inhibited cardiomyocyte apoptosis by activating Akt/the mammalian target of rapamycin (mTOR) pathway, and the protection of urolithin B against cardiomyocyte apoptosis was compromised with Akt gene silencing. Furthermore, urolithin B suppressed nuclear translocation of nuclear factor-kB (NF-κB) to facilitate nerve remodeling. Taken together, our findings suggested that UB reduced the occurrence of myocardial arrhythmias after hypoxia via regulation of the Akt/mTOR pathway and NF-κB nuclear translocation, which highlights the potential of UB as a novel therapy for ischemic heart disease.

Self-Assembly of a 3D Hollow BiOBr@Bi-MOF Heterostructure with Enhanced Photocatalytic Degradation of Dyes.

Considering the flexibility, adjustable pore structure, and abundant active sites of metal-organic frameworks (MOFs), rational design and fine control of the MOF-based hetero-nanocrystals is a highly important and challenging subject. In this work, self-assembly of a 3D hollow BiOBr@Bi-MOF microsphere was fabricated through precisely controlled dissociation kinetics of the self-sacrificial template (BiOBr) for the first time, where the residual quantity of BiOBr and the formation of Bi-MOF were carefully regulated by changing the reaction time and the capability of coordination. Meanwhile, the hollow microstructure was formed in BiOBr@Bi-MOF through the Oswald ripening mechanism to separate photogenerated electron-hole pairs and increase the adsorption capacity of Bi-MOF for dyes, which significantly enhanced the photocatalytic degradation efficiency of RhB from 56.4% for BiOBr to 99.4% for the optimal BiOBr@Bi-MOF microsphere. This research broadens the selectivity of semiconductor/MOF hetero-nanocrystals with reasonable design and flexible synthesis.

Optimization of GPC3-specific chimeric antigen receptor structure and its effect on killing hepatocellular carcinoma cells.

To investigate the effect of optimized GPC3-specific chimeric antigen receptor (GPC3-CAR) structure on killing hepatocellular carcinoma (HCC) cells. We constructed three lentiviral expression vectors with different CAR structures by genetic engineering and molecular cloning techniques. These three CAR structures shared the same intracellular signaling region consisting of 4-1BB and CD3ζ, but had different hinge and transmembrane regions. Specifically, GPC3-O4-CAR contained an optimized CD8α hinge region and a 4-1BB transmembrane domain; GPC3-CD8-CAR contained an optimized CD8α hinge region and a CD8α transmembrane domain; and GPC3-ori-CAR contained an original CD8α hinge region and a 4-1BB transmembrane domain. With similar transfection efficiency, it was observed by fluorescence microscopy that GPC3-O4-CAR expression on the surface of 293 T cells was much higher than those of the other two. Cytotoxicity experiments showed that T or NK cells with GPC3-O4-CAR structure were more lethal and could secrete more IFN-γ than the other two. In conclusion, GPC3-O4-CAR can be efficiently and stably expressed on the cell surface. Moreover, both the killing effect of transduced T and NK cells on GPC3-positive HCC cells and release of IFN-γ are increased.

Exosomal DLX6-AS1 from hepatocellular carcinoma cells induces M2 macrophage polarization to promote migration and invasion in hepatocellular carcinoma through microRNA-15a-5p/CXCL17 axis.

BACKGROUND: Hepatocellular carcinoma (HCC) cells-secreted exosomes (exo) could stimulate M2 macrophage polarization and promote HCC progression, but the related mechanism of long non-coding RNA distal-less homeobox 6 antisense 1 (DLX6-AS1) with HCC-exo-mediated M2 macrophage polarization is largely ambiguous. Thereafter, this research was started to unearth the role of DLX6-AS1 in HCC-exo in HCC through M2 macrophage polarization and microRNA (miR)-15a-5p/C-X-C motif chemokine ligand 17 (CXCL17) axis. METHODS: DLX6-AS1, miR-15a-5p and CXCL17 expression in HCC tissues and cells were tested. Exosomes were isolated from HCC cells with overexpressed DLX6-AS1 and co-cultured with M2 macrophages. MiR-15a-5p/CXCL17 down-regulation assays were performed in macrophages. The treated M2 macrophages were co-cultured with HCC cells, after which cell migration, invasion and epithelial mesenchymal transition were examined. The targeting relationships between DLX6-AS1 and miR-15a-5p, and between miR-15a-5p and CXCL17 were explored. In vivo experiment was conducted to detect the effect of exosomal DLX6-AS1-induced M2 macrophage polarization on HCC metastasis. RESULTS: Promoted DLX6-AS1 and CXCL17 and reduced miR-15a-5p exhibited in HCC. HCC-exo induced M2 macrophage polarization to accelerate migration, invasion and epithelial mesenchymal transition in HCC, which was further enhanced by up-regulated DLX6-AS1 but impaired by silenced DLX6-AS1. Inhibition of miR-15a-5p promoted M2 macrophage polarization to stimulate the invasion and metastasis of HCC while that of CXCL17 had the opposite effects. DLX6-AS1 mediated miR-15a-5p to target CXCL17. DLX6-AS1 from HCC-exo promoted metastasis in the lung by inducing M2 macrophage polarization in vivo. CONCLUSION: DLX6-AS1 from HCC-exo regulates CXCL17 by competitively binding to miR-15a-5p to induce M2 macrophage polarization, thus promoting HCC migration, invasion and EMT.