A fungal CFEM-containing effector targets NPR1 regulator NIMIN2 to suppress plant immunity.

Colletotrichum fructicola causes a broad range of plant diseases worldwide and secretes many candidate proteinous effectors during infection, but it remains largely unknown regarding their effects in conquering plant immunity. Here, we characterized a novel effector CfEC12 that is required for the virulence of C. fructicola. CfEC12 contains a CFEM domain and is highly expressed during the early stage of host infection. Overexpression of CfEC12 suppressed BAX-triggered cell death, callose deposition and ROS burst in Nicotiana benthamiana. CfEC12 interacted with apple MdNIMIN2, a NIM1-interacting (NIMIN) protein that putatively modulates NPR1 activity in response to SA signal. Transient expression and transgenic analyses showed that MdNIMIN2 was required for apple resistance to C. fructicola infection and rescued the defence reduction in NbNIMIN2-silenced N. benthamiana, supporting a positive role in plant immunity. CfEC12 and MdNPR1 interacted with a common region of MdNIMIN2, indicating that CfEC12 suppresses the interaction between MdNIMIN2 and MdNPR1 by competitive target binding. In sum, we identified a fungal effector that targets the plant salicylic acid defence pathway to promote fungal infection.

A fungal effector suppresses plant immunity by manipulating DAHPS-mediated metabolic flux in chloroplasts.

Plant secondary metabolism represents an important and ancient form of defense against pathogens. Phytopathogens secrete effectors to suppress plant defenses and promote infection. However, it is largely unknown, how fungal effectors directly manipulate plant secondary metabolism. Here, we characterized a fungal defense-suppressing effector CfEC28 from Colletotrichum fructicola. Gene deletion assays showed that ∆CfEC28-mutants differentiated appressoria normally on plant surface but were almost nonpathogenic due to increased number of plant papilla accumulation at attempted penetration sites. CfEC28 interacted with a family of chloroplast-localized 3-deoxy-d-arabinose-heptulonic acid-7-phosphate synthases (DAHPSs) in apple. CfEC28 inhibited the enzymatic activity of an apple DAHPS (MdDAHPS1) and suppressed DAHPS-mediated secondary metabolite accumulation through blocking the manganese ion binding region of DAHPS. Dramatically, transgene analysis revealed that overexpression of MdDAHPS1 provided apple with a complete resistance to C. fructicola. We showed that a novel effector CfEC28 can be delivered into plant chloroplasts and contributes to the full virulence of C. fructicola by targeting the DAHPS to disrupt the pathway linking the metabolism of primary carbohydrates with the biosynthesis of aromatic defense compounds. Our study provides important insights for understanding plant-microbe interactions and a valuable gene for improving plant disease resistance.

Sufficient coumarin accumulation improves apple resistance to Cytospora mali under high-potassium status.

Cytospora canker, caused by Cytospora mali, is the most destructive disease in production of apples (Malus domestica). Adding potassium (K) to apple trees can effectively control this disease. However, the underlying mechanisms of apple resistance to C. mali under high-K (HK) status remain unknown. Here, we found that HK (9.30 g/kg) apple tissues exhibited high disease resistance. The resistance was impeded when blocking K channels, leading to susceptibility even under HK conditions. We detected a suite of resistance events in HK apple tissues, including upregulation of resistance genes, callose deposition, and formation of ligno-suberized tissues. Further multiomics revealed that the phenylpropanoid pathway was reprogrammed by increasing K content from low-K (LK, 4.30 g/kg) status, leading to increases of 18 antifungal chemicals. Among them, the physiological concentration of coumarin (1,2-benzopyrone) became sufficient to inhibit C. mali growth in HK tissues, and exogenous application could improve the C. mali resistance of LK apple branches. Transgenic apple calli overexpressing beta-glucosidase 40 (MdBGLU40), which encodes the enzyme for coumarin synthesis, contained higher levels of coumarin and exhibited high resistance to C. mali even under LK conditions. Conversely, the suppression of MdBGLU40 through RNAi reduced coumarin content and resistance in HK apple calli, supporting the importance of coumarin accumulation in vivo for apple resistance. Moreover, we found that the upregulation of transcription factor MdMYB1r1 directly activated MdBGLU40 and the binding affinity of MdMYB1r1 to the MdBGLU40 promoter increased in HK apple tissue, leading to high levels of coumarin and resistance in HK apple. Overall, we found that the accumulation of defensive metabolites strengthened resistance in apple when raising K from insufficient to optimal status, and these results highlight the optimization of K content in fertilization practices as a disease management strategy.

Changes in planta K nutrient content altered the interaction pattern between Nicotiana benthamiana and Alternaria longipes.

Potassium (K) fertilisation has frequently been shown to enhance plant resistance against pathogens, though the mechanisms remain elusive. This study investigates the interaction dynamics between Nicotiana benthamiana and the pathogen Alternaria longipes under different planta K levels. On the host side, adding K activated the expressions of three NLR (nucleotide-binding domain and leucine-rich repeat-containing proteins) resistance genes, including NbRPM1, NbR1B23 and NbNBS12. Silencing these NLRs attenuated resistance in high-K (HK, 40.8 g/kg) plant, whereas their overexpression strengthened resistance in low-K (LK, 23.9 g/kg) plant. Typically, these NLRs mainly strengthened plant resistance via promoting the expression of pathogenesis-related genes (PRs), ROS burst and synthesis of antifungal metabolites in HK plant. On the pathogen side, the expression of effectors HKCSP1, HKCSP2 and LKCSP were shown to be related to planta K content. A. longipes mainly expressed effectors HKCSP1 and HKCSP2 in HK plant to interfere host resistance. HKCSP1 physically interacted with NbRPM1 to promote the degradation of NbRPM1, then attenuated related resistance in HK N. benthamiana. Meanwhile, HKCSP2 directly interacted with NbPR5 to suppress resistance in HK plant. In LK plant, A. longipes mainly deployed LKCSP that interacted with NbR1B23 to interfere reduce resistance in N. benthamiana. Overall, our research insights that both pathogen and host mobilise distinct strategies to outcompete each other during interactions in different K nutrient environments.

CfCpmd1 Regulates Pathogenicity and Sexual Development of Plus and Minus Strains in Colletotrichum fructicola Causing Glomerella Leaf Spot on Apple in China.

Colletotrichum fructicola is a devastating fungal pathogen of diverse plants. Sexually compatible plus and minus strains occur in the same ascus. However, the differentiation mechanism of plus and minus strains remains poorly understood. Here, we characterized a novel Cys2-His2-containing transcription factor CfCpmd1. The plus CfCpmd1 deletion mutant (Δ+CfCpmd1) resulted in slow hyphal growth and a fluffy cotton-like colony, and the minus deletion mutant (Δ-CfCpmd1) exhibited characters similar to the wild type (WT). Δ+CfCpmd1 led to defective perithecial formation, whereas Δ-CfCpmd1 produced more and smaller perithecia. The normal mating line was developed by pairing cultures of Δ-CfCpmd1 and plus WT, whereas a weak line was observed between Δ+CfCpmd1 and minus WT. Conidial production was completely abolished in both plus and minus mutants. When inoculated on non-wounded apple leaves with mycelial plugs, Δ-CfCpmd1 was nonpathogenic because of failure to develop conidia and appressoria, while Δ+CfCpmd1 could infect apple leaves by appressoria differentiated directly from hyphal tips, even though no conidia formed. Collectively, our results demonstrate that CfCpmd1 of C. fructicola is an important gene related to plus and minus strain differentiation, which also affects hyphal growth, sporulation, appressorium formation, and pathogenicity.

Chaetoglobosin A Contributes to the Antagonistic Action of Chaetomium globosum Strain 61239 Toward the Apple Valsa Canker Pathogen Cytospora mali.

Apple Valsa canker (AVC) weakens apple trees and significantly reduces apple production in China and other East Asian countries. Thus far, very few AVC-targeting biocontrol resources have been described. Here, we present a thorough description of a fungal isolate (Chaetomium globosum, 61239) that has strong antagonistic action toward the AVC causal agent Cytospora mali. Potato dextrose broth culture filtrate of strain 61239 completely suppressed the mycelial growth of C. mali on potato dextrose agar, and strongly constrained the development of AVC lesions in in vitro infection assays. ultra-performance liquid chromatography (UPLC) and HPLC-MS/MS investigations supported the conclusion that strain 61239 produces chaetoglobosin A, an antimicrobial metabolite that inhibits C. mali. Using genome sequencing, we discovered a gene cluster in strain 61239 that may be responsible for chaetoglobosin A production. Two of the cluster's genes-cheA, a PKS-NRPS hybrid enzyme, and cheB, an enoyl reductase-were individually silenced, which significantly decreased chaetoglobosin A accumulation as well as the strain's antagonistic activity against C. mali. Together, the findings of our investigation illustrate the potential use of Chaetomium globosum for the management of AVC disease and emphasize the significant contribution of chaetoglobosin A to the antagonistic action of strain 61239.

Two Putative Pheromone Receptors, but Not Their Cognate Pheromones, Regulate Female Fertility in the Atypical Mating Fungus Colletotrichum fructicola.

Colletotrichum fungi are a group of damaging phytopathogens with atypical mating type loci (harboring only MAT1-2-1 but not MAT1-1-1) and complex sexual behaviors. Sex pheromones and their cognate G-protein-coupled receptors are conserved regulators of fungal mating. These genes, however, lose function frequently among Colletotrichum species, indicating a possibility that pheromone signaling is dispensable for Colletotrichum sexual reproduction. We have identified two putative pheromone-receptor pairs (PPG1:PRE2, PPG2:PRE1) in C. fructicola, a species that exhibits plus-to-minus mating type switching and plus-minus-mediated mating line development. Here, we report the generation and characterization of gene-deletion mutants for all four genes in both plus and minus strain backgrounds. Single-gene deletion of pre1 or pre2 had no effect on sexual development, whereas their double deletion caused self-sterility in both the plus and minus strains. Moreover, double deletion of pre1 and pre2 caused female sterility in plus-minus outcrossing. Double deletion of pre1 and pre2, however, did not inhibit perithecial differentiation or plus-minus-mediated enhancement of perithecial differentiation. Contrary to the results with pre1 and pre2, double deletion of ppg1 and ppg2 had no effect on sexual compatibility, development, or fecundity. We concluded that pre1 and pre2 coordinately regulate C. fructicola mating by recognizing novel signal molecule(s) distinct from canonical Ascomycota pheromones. The contrasting importance between pheromone receptors and their cognate pheromones highlights the complicated nature of sex regulation in Colletotrichum fungi.

[Structure-activity relationship of Lycium barbarum polysaccharides].

As a traditional Chinese herb and functional food, the fruits of Lycium barbarum has been widely used for thousands of years in China. L. barbarum polysaccharides(LBPs) are predominant active components, which have immunomodulatory, antioxidant, hypoglycemic, neuroprotective, anti-tumor, and prebiotic activities. The molecular weight, monosaccharide composition, glycosidic bond, branching degree, protein content, chemical modification, and spatial structure of LBPs are closely related to their biological activity. Based on the previous studies of this research team, this paper systematically combed and integrated the research progress of structure, function, and structure-activity relationship of LBPs. At the same time, some problems restricting the clarification of the structure-activity relationship of LBPs were considered and prospected, hoping to provide references for the high value utilization of LBPs and in-depth exploration of their health value.

Adaptive Responses of Pseudomonas aeruginosa to Treatment with Antibiotics.

Pseudomonas aeruginosa is among the highest priority pathogens for drug development because of its resistance to antibiotics, extraordinary adaptability, and persistence. Antipseudomonal research is strongly encouraged to address the acute scarcity of innovative antimicrobial lead structures. In an effort to understand the physiological response of P. aeruginosa to clinically relevant antibiotics, we investigated the proteome after exposure to ciprofloxacin, levofloxacin, rifampicin, gentamicin, tobramycin, azithromycin, tigecycline, polymyxin B, colistin, ceftazidime, meropenem, and piperacillin-tazobactam. We further investigated the response to CHIR-090, which represents a promising class of lipopolysaccharide biosynthesis inhibitors currently under evaluation. Radioactive pulse-labeling of newly synthesized proteins followed by two-dimensional polyacrylamide gel electrophoresis was used to monitor the acute response of P. aeruginosa to antibiotic treatment. The proteomic profiles provide insights into the cellular defense strategies for each antibiotic. A mathematical comparison of these response profiles based on upregulated marker proteins revealed similarities of responses to antibiotics acting on the same target area. This study provides insights into the effects of commonly used antibiotics on P. aeruginosa and lays the foundation for the comparative analysis of the impact of novel compounds with precedented and unprecedented modes of action.

A novel hexa-segmented dsRNA mycovirus confers hypovirulence in the phytopathogenic fungus Diaporthe pseudophoenicicola.

A novel hexa-segmented double-stranded RNA (dsRNA) mycovirus was isolated and characterized from the filamentous phytopathogenic fungus Diaporthe pseudophoenicicola and was named Diaporthe pseudophoenicicola chrysovirus 1 (DpCV1). The full-length cDNAs of dsRNA1-6 were 3335, 3030, 3039, 2980, 963, and 780 bp, respectively. Sequence analysis indicated the presence of nine open reading frames (ORFs) in the DpCV1 genome. ORF1 in dsRNA1 putatively encoded the RNA-dependent RNA polymerase (RdRp) and ORF3 in dsRNA2 encoded a capsid protein (CP). The seven remaining ORFs, ORF2 in dsRNA2, ORF4 in dsRNA3, ORF6, seven in dsRNA4, ORF8 in dsRNA5, and ORF9 in dsRNA6, encoded proteins with unknown functions. Phylogenetic analysis revealed that DpCV1 is closely related to members of the cluster I group within the family Chrysoviridae but formed a separate clade. Importantly, all the six segments of DpCV1 were cured successfully through single spore isolation to obtain the isogenic virus-free strains. DpCV1 can confer hypovirulence to the fungal host of Diaporthe pseudophoenicicola. Compared with the virus-free strain, WC02 harbouring the DpCV1 is more sensitive to fungicide prochloraz. Furthermore, the cell wall of DpCV1 infected strain was loose and enlarged. This is the first report of a hexa-segmented tentative chrysovirus in D. pseudophoenicicola.

Dual effect of ultraviolet B on cholesterol efflux and regulated by ultraviolet radiation resistance-associated gene-mediated autophagy.

OBJECTIVE: In addition to diet and metabolism, the occurrence of foam cells and atherosclerosis are also related to environmental factors. Individual studies have shown that ultraviolet B (UVB) can regulate the progression of atherosclerosis, but with different results. Whether or not UVB has a dual effect on atherosclerosis and what mechanism is involved has not been reported. METHODS: After THP-1-derived foam cells were treated with UVB in different ways, the effects of UVB on foam cells were investigated by western blotting, cholesterol efflux experiment, oil red O staining and other methods. RESULTS: UVB plays a dual role on foam cell formation, and this effect is related to cholesterol efflux. UVB of 50 mJ/cm2 can promote cholesterol efflux in foam cells, while UVB of 200 mJ/cm2 can inhibit cholesterol efflux. UVB induces cholesterol efflux from foam cells in an autophagy-dependent manner, as the beneficial effect of UVB at 50 mJ/cm2 can be reversed by the autophagy inhibitor 3-Methyladenine (3-MA). In addition, silencing the expression of ultraviolet radiation resistance-associated gene (UVRAG) can inhibit autophagy and reduce cholesterol efflux, and overexpressing UVRAG yields the opposite result. CONCLUSION: In conclusion, our research proves that UVB exhibits a dual role in foam cell formation by regulating cholesterol efflux. Further more, we also reveal that UVRAG-mediated autophagy is the underlying mechanism of UVB-induced cholesterol efflux.

1,8-Dihydroxynaphthalene Melanin Biosynthesis in Colletotrichum fructicola Is Developmentally Regulated and Requires the Cooperative Function of Two Putative Zinc Finger Transcription Factors.

In ascomycetes, 1,8-dihydroxynaphthalene (DHN) melanin plays important protective functions and its production is usually coupled with development and environmental stress responses. The regulation of melanin biosynthesis, however, remains obscure. Colletotrichum fructicola is a phytopathogen with a broad host range that produces melanized appressoria and perithecia. In this study, we annotated melanin genes in a high-quality C. fructicola genome and characterized two zinc finger transcription factors (TFs) (cmr1 and cmr2) that form a loosely organized gene cluster with several melanin biosynthesis genes. Deleting either TF abolished melanization in both mycelia and perithecia but did not affect appressoria. The deletion mutants also showed perithecial development defects. Overexpressing cmr1 in Δcmr2 strongly activated the expression of melanin biosynthesis genes including pks1, scd1, t4hr1, and thr1 and caused hyper-accumulation of charcoal to black pigment(s). On the other hand, overexpressing cmr2 in Δcmr1 activated pks1, t4hr1, and thr1, but not scd1. We conclude that proper DHN melanin accumulation in C. fructicola requires the cooperative function of two in-cluster TFs that also regulate perithecial development. The study clarifies DHN melanin regulations in C. fructicola and expands the function of melanin in-cluster TFs to sex regulation.

The CfMcm1 Regulates Pathogenicity, Conidium Germination, and Sexual Development in Colletotrichum fructicola.

Glomerella leaf spot (GLS), caused by Colletotrichum fructicola, is a severe disease worldwide on apple, causing defoliation, leaf and fruit spot, and substantial yield loss. However, little is known about its molecular mechanisms of pathogenesis. Previous transcriptome analysis revealed that a transcription factor, CfMcm1, was induced during leaf infection. In the present work, expression pattern analysis verified that the CfMcm1 gene was strongly expressed in conidia and early infection. Phenotypic analysis revealed that the gene deletion mutant ΔCfMcm1 lost pathogenicity to apple leaves by inhibiting conidial germination and appressorium formation. In addition to appressorium-mediated pathogenicity, ΔCfMcm1 colonization and hyphal extension in wounded apple fruit was also reduced, and conidial germination mode and conidial color were altered. ΔCfMcm1 displayed impairment of cell wall integrity and response to stress caused by oxidation, osmosis, and an acid environment. Furthermore, the deletion mutant produced fewer and smaller perithecia and no ascospores. In contrast, melanin deposition in mycelia of ΔCfMcm1 was strengthened. Further comparative transcriptome and quantitative PCR analysis revealed that CfMcm1 modulated expression of genes related to conidial development (CfERG5A, CfERG5B, CfHik5, and CfAbaA), appressorium formation (CfCBP1 and CfCHS7), pectin degradation (CfPelA and CfPelB), sexual development (CfMYB, CfFork, CfHMG, and CfMAT1-2-1), and melanin biosynthesis (CfCmr1, CfPKS1, CfT4HR1, CfTHR1, and CfSCD1). Our results demonstrated that CfMcm1 is a pivotal regulator possessing multiple functions in pathogenicity, asexual and sexual reproduction, and melanin biosynthesis.

Sustainable Apple Disease Management in China: Challenges and Future Directions for a Transforming Industry.

Apple trees are grown worldwide, and consuming fresh apple fruit is associated with many health benefits. China produces about half of the world's apple supply. However, apple growing in China differs sharply from that in western countries in terms of the prevalent diseases and corresponding management strategies. For instance, family-owned small-scale orchards dominate China's apple industry, and manual bagging of fruit has been a long-standing practice for controlling fruit diseases. In recent years, rural labor shortages have been increasingly challenging the traditional production system, and China's apple industry is experiencing a rapid transition to much larger-scale enterprises featuring high-density orchards with advanced automation and mechanization. Associated with this transition are new challenges and grower demands that are changing the face of apple disease management. This Feature Article summarizes the ongoing transformation of China's apple industry in the context of sustainable disease management.

Comparison of mitochondrial genomes provides insights into intron dynamics and evolution in Botryosphaeria dothidea and B. kuwatsukai.

Botryosphaeria dothidea is one of the most common fungal pathogens on a large number of hosts worldwide. Botryosphaeria dothidea and B. kuwatsukai are also the main causal agents of apple ring rot. In this study, we sequenced, assembled and annotated the circular mitogenomes of 12 diverse B. dothidea isolates (105.7-114.8 kb) infecting various plants including apple, and five diverse B. kuwatsukai isolates (118.0-124.6 kb) from apple. B. dothidea mitogenomes harboured a set of 29-31 introns and 48-52 ORFs. In contrast, B. kuwatsukai mitogenomes harboured more introns (32-34) and ORFs (51-54). The variation in mitogenome sizes was associated mainly with different numbers of introns and insertions of mobile genetic elements. Interestingly, B. dothidea and B. kuwatsukai displayed distinct intron distribution patterns, with three intron loci showing presence/absence dynamics in each species. Large numbers of introns (57% in B. dothidea and 49% in B. kuwatsukai) were most likely obtained through horizontal transfer from non-Dothideomycetes. The mitochondrial gene phylogeny supported the differentiation of the two species. Overall, this study sheds light into the mitochondrial evolution of the plant pathogens B. dothidea and B. kuwatsukai, and intron distribution patterns could be useful markers for studies on population diversity.

Molecular Dissection of Perithecial Mating Line Development in Colletotrichum fructicola, a Species with a Nontypical Mating System Featuring Plus-to-Minus Switch and Plus-Minus-Mediated Sexual Enhancement.

The genetic regulation of Colletotrichum (Glomerella) sexual reproduction does not strictly adhere to the Ascomycota paradigm and remains poorly understood. Morphologically different but sexually compatible strain types, termed plus and minus, have been recognized, but the biological and molecular distinctions between these strain types remain elusive. In this study, we characterized the sexual behaviors of a pair of plus and minus strains of C. fructicola with the aid of live-cell nucleus-localized fluorescent protein labeling, gene expression, and gene mutation analyses. We confirmed a genetically stable plus-to-minus switching phenomenon and demonstrated the presence of both cross-fertilized and self-fertilized perithecia within the mating line (perithecia cluster at the line of colony contact) between plus and minus strains. We demonstrated that pheromone signaling genes (a-factor-like and α-factor-like pheromones and their corresponding GPCR receptors) were differently expressed between vegetative hyphae of the two strains. Moreover, deletion of pmk1 (a FUS/KSS1 mitogen-activate protein kinase) in the minus strain severely limited mating line formation, whereas deletion of a GPCR (FGSG_05239 homolog) and two histone modification factors (hos2, snt2) in the minus strain did not affect mating line development but altered the ratio between cross-fertilization and self-fertilization within the mating line. We propose a model in which mating line formation in C. fructicola involves enhanced protoperithecium differentiation and enhanced perithecium maturation of the minus strain mediated by both cross-fertilization and diffusive effectors. This study provides insights into mechanisms underlying the mysterious phenomenon of plus-minus-mediated sexual enhancement being unique to Colletotrichum fungi. IMPORTANCE Plus-minus regulation of Colletotrichum sexual differentiation was reported in the early 1900s. Both plus and minus strains produce fertile perithecia in a homothallic but inefficient manner. However, when the two strain types encounter each other, efficient differentiation of fertile perithecia is triggered. The plus strain, by itself, can also generate minus ascospore progeny at high frequency. This nontypical mating system facilitates sexual reproduction and is Colletotrichum specific; the underlying molecular mechanisms, however, remain elusive. The current study revisits this longstanding mystery using C. fructicola as an experimental system. The presence of both cross-fertilized and self-fertilized perithecia within the mating line was directly evidenced by live-cell imaging with fluorescent markers. Based on further gene expression and gene mutation analysis, a model explaining mating line development (plus-minus-mediated sexual enhancement) is proposed. Data reported here have the potential to allow us to better understand Colletotrichum mating and filamentous ascomycete sexual regulation.

Complete genome sequence of a novel mycovirus from Phoma matteucciicola.

The complete genome sequence of a novel mycovirus, Phoma matteucciicola RNA virus 1 (PmRV1), derived from Phoma matteucciicola strain LG-01, was sequenced and analyzed. The complete cDNA sequence of PmRV1 is 3432 bp in length with a GC content of 57.17%. The genome of PmRV1 contains two putative open reading frames (ORFs): ORF1 and ORF2. ORF1 encodes a hypothetical protein with significant similarity to a protein encoded by Periconia macrospinosa ambiguivirus 1 (PmAV1). ORF2 encodes a protein of 491 amino acids with a conserved RNA-dependent RNA polymerase (RdRp) domain. Additionally, the triad within domain III has an asparagine (GDN) instead of the nearly universally conserved aspartic acid (GDD). RdRp phylogeny showed that PmRV1 grouped together with PmAV1 as a sister branch of a new member of the recently proposed family of mycotombus-like viruses. This is first report of the complete sequence of a novel mycovirus, PmRV1, infecting Phoma matteucciicola strain LG-01, the causal agent of leaf blight of Curcuma wenyujin.

Protein liposomes-mediated targeted acetylcholinesterase gene delivery for effective liver cancer therapy.

BACKGROUND: Effective methods to deliver therapeutic genes to solid tumors and improve their bioavailability are the main challenges of current medical research on gene therapy. The development of efficient non-viral gene vector with tumor-targeting has very important application value in the field of cancer therapy. Proteolipid integrated with tumor-targeting potential of functional protein and excellent gene delivery performance has shown potential for targeted gene therapy. RESULTS: Herein, we prepared transferrin-modified liposomes (Tf-PL) for the targeted delivery of acetylcholinesterase (AChE) therapeutic gene to liver cancer. We found that the derived Tf-PL/AChE liposomes exhibited much higher transfection efficiency than the commercial product Lipo 2000 and shown premium targeting efficacy to liver cancer SMMC-7721 cells in vitro. In vivo, the Tf-PL/AChE could effectively target liver cancer, and significantly inhibit the growth of liver cancer xenografts grafted in nude mice by subcutaneous administration. CONCLUSIONS: This study proposed a transferrin-modified proteolipid-mediated gene delivery strategy for targeted liver cancer treatment, which has a promising potential for precise personalized cancer therapy.

Clinical verification of vimentin/EpCAM immunolipid magnetic sorting system in monitoring CTCs in arterial and venous blood of advanced tumor.

BACKGROUND: Circulating tumor cells (CTCs) are the dominant factor leading to tumor metastasis. This study aims to investigate the effect of disparate sources of CTCs on the treatment and prognosis of patients with advanced tumors by analyzing the number and gene mutations change of CTCs in arterial and venous blood in patients with advanced tumors. RESULTS: A CTCs sorting system was constructed based on Vimentin-immunolipid magnetic balls (Vi-IMB) and EpCAM immunolipid magnetic balls (Ep-IMB). Results showed that the prepared Ep-IMB and Vi-IMB had lower cytotoxicity, better specificity and sensitivity. The number of arterial CTCs was higher than that of venous CTCs, with a statistically significant difference (P < 0.05). Moreover, the prognosis of the low positive group of total CTCs in arterial blood and venous blood was higher than that of the high positive group, with a statistical significance (P < 0.05). The genetic testing results showed that the targeted drug gene mutations in tissues, arterial CTCs and venous CTCs showed a complementary trend, indicating that there was heterogeneity among different tumor samples. CONCLUSIONS: CTCs in blood can be efficiently captured by the CTCs sorting system based on Vi-LMB/Ep-LMB, and CTCs detection in arterial blood can be utilized to more accurately evaluate the prognosis and predict postoperative progress. It is further confirmed that tumor samples from disparate sources are heterogeneous, providing a reference basis for gene mutation detection before clinical targeted drug treatment, and the detection of CTCs in arterial blood has more potential clinical application value. TRIAL REGISTRATION: The Ethics Committee of Putuo Hospital, PTEC-A-2019-18-1. Registered 24 September 2019.

Expression and mechanism of exosome-mediated A FOXM1 related long noncoding RNA in gastric cancer.

BACKGROUND: Forkhead box protein M1 (FOXM1) is an oncogene regulating tumor growth and metastasis. Exosome was suggested to mediate cell communication by delivering active molecules in cancers. However, the existence of FOXM1 in circulating exosomes and the role of exosome FOXM1 in gastric cancer (GC) were not clear. This study aims to investigate the potential role of FOXM1 related long noncoding RNA (FRLnc1) in exosomes in GC. RESULTS: The prepared CD63 immunomagnetic beads (CD63-IMB) had the characteristics of good dispersity and high magnetic response. The isolated exosomes were presented with elliptical membranous particles under a transmission electron microscope (TEM), with the particle size of 89.78 ± 4.8 nm. Western blot (WB) results showed that the exosomes were rich in CD9 and CD81. The Dil-labeled exosomes were distributed around cytoplasm and nucleus of cells by imaging flow cytometry (IFC) analysis. The results of quantitative real-time PCR (qRT-PCR) revealed that the FRLnc1 expressions were up-regulated in GC cells, tumor tissues, and serum of GC patients. An obviously up-regulated FRLnc1 expression was found in serum exosomes of GC patients. Up-regulation of FRLnc1 expression was closely correlated to lymph node metastasis (LNM) and TNM stage with the combination of relevant clinicopathological parameter analysis. The in vitro functional analyses demonstrated that FRLnc1 knockdown by RNA interference suppressed cell proliferation and migration in HGC-27 cells, whereas FRLnc1 overexpression promoted cell proliferation and migration in MKN45 cells. After exosome treatment, the FRLnc1 expression was significantly increased in MKN45 cells, and the MKN45 cells showed increased ability of proliferation and migration. CONCLUSION: GC cells-derived exosomes played roles in promoting the growth and metastasis of GC by transporting FRLnc1, suggesting that FRLnc1 in the exosomes may be a potential biomarker for the diagnosis and treatment of GC. The delivery of FRLnc1 by the exosomes may provide a new way for the treatment of GC. Trial registration 2020-KYSB-094. Registered 23 March 2020-Retrospectively registered.