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Colletotrichum fructicola shows morphological and genetic differences in plus and minus strains. However, the mechanism of the differentiation between two types of strains is still largely unclear. Our early transcriptome analysis revealed that CfHMG expression differed in plus and minus strains. To define the functions of the CfHMG gene, we constructed gene deletion mutants by homologous recombination. We found that a CfHMG deletion mutant of the minus strain, CfHMG-M, could lead to a reduction in perithecium sizes and densities on media and sterile perithecium formation compared with the minus wild type (WT), whereas there was no effect for the plus mutant CfHMG-P. In co-cultures between CfHMG-P and minus WT, CfHMG-M and plus WT, or CfHMG-P and CfHMG-M, the quantities of perithecia were all reduced significantly. When conidial suspensions were inoculated on non-wounded apple fruit, it was found that the virulence of the minus mutant decreased significantly but not for the plus one. Further, we found that the virulence decrease in minus mutants was caused by a decrease in the conidium germination rate. Our results indicate that CfHMG of C. fructicola plays an important role in the mating line formation between the plus and minus strain for both strains and differentially regulates the perithecium size, density, fertilization, and virulence of the minus strain. The results are significant for further detecting the differentiated mechanisms between the plus and minus strains in Colletotrichum fungi.

Abstract Fusarium oxysporum f. sp. cubense (Foc) causes Fusarium wilt of banana (Musa spp.), a notorious soil-borne vascular fungal disease threatening the global banana industry. Phytopathogens secrete effectors to suppress plant immunity. However, little is known about the effectors of Foc race 4 (Foc4). In this study, we built a streamlined screening system (candidate effector prediction, RNA-seq-based expression level analysis, and cell death manipulative activity assessment based on transient expression in Nicotiana benthamiana) to identify candidate virulence-related effectors. In total, 80 candidate effector genes (CEGs) differentially expressed during plant colonization were predicted; 12 out of 15 characterized CEGs, including FoSSP17, could suppress BAX-triggered programmed cell death (PCD) in N. benthamiana and were induced during the infection of plants. FoSSP17 encodes a novel protein conserved in the Fusarium genus. FoSSP17 gene deletion mutants were not affected in vegetative growth and conidiation but showed reduced virulence. Furthermore, the deletion mutants triggered higher expression levels of host defense-related genes including PR1, PR3, PR5, and PR10. Signal peptide activity assay and subcellular localization assay suggested that FoSSP17 is a conventional secretory protein that exerts cell-death-suppressive activity inside plant cells. In addition, FoSSP17 suppressed pattern-triggered immunity in plants by inhibiting reactive oxygen species (ROS) accumulation, reducing callose deposition, and suppressing the expression of NbLOX and NbERF1 genes related to jasmonic acid (JA)-pathway and ethylene (ET)-pathway, respectively. Overall, a systemic screening of Foc4 candidate effectors reveals that FoSSP17 contributes to the virulence of Foc4 and suppresses pattern-triggered immunity in plants.

The prolonged disregard for the Gross Ecosystem Product (GEP) has limited the effectiveness of regional ecological conservation. However, identifying and safeguarding areas with high GEP, and optimizing their distribution and spatial structure, are of great immense for preserving ecosystem services (ESs). Our goal is to refine the spatial configuration of ecological network (EN), and subsequently augment and sustain the stability of ESs. We have specifically chosen Hechi City, situated in the Karst region of southwest China, as our case study. Firstly, we devised a GEP evaluation system to pinpoint crucial areas as ecological sources. Subsequently, we utilized the circuit theory and minimum cumulative resistance model to construct the preliminary EN. Through the circuit theory model identified the key nodes in the EN and optimized the EN. Finally, by combining complex network analysis methods, we evaluated the robustness and topological characteristics of the EN. Our results showed that the area of ecological sources increased from 71 to 161 after optimization. The number of corridors increased from 161 to 423, resulting in an overall increment of 8.8% in the source area. The connectivity and stability of the optimized EN can be significantly enhanced. The addition of new nodes increases the overall importance of the network, making the importance of each node more balanced. Additionally, the complex network analysis method provided a detailed understanding about the spatial topology of the overall and local elements of the EN. The study results can provide a valuable reference for optimizing, restoring and evaluating ENs in karst areas.

Abstract Glomerella leaf spot (GLS) is a devastating fungal disease that damages the leaves and fruits and reduces tree vigor of apple (Malus domestica). The pathogen infection mechanism, however, remains elusive. Histone-modifying enzymes, which regulate eukaryotic chromatin conformation and gene expression, are key epigenetic factors controlling fungal development, virulence, and secondary metabolism. To dissect the epigenetic regulation of GLS pathogenesis, we characterized a histone deacetylase gene Cfhos2 in Colletotrichum fructicola, the causing agent of GLS. Cfhos2 deletion mutants were mildly reduced in vegetative growth rate, but almost lost pathogenicity on apple leaves. Cfhos2 deletion mutants induced strong plant defense responses manifested by epidermal cell browning, granulation, and distortion of pathogen invasive hyphae. The mutants also showed defect in appressorial development on cellophane, but not on parafilm or on apple leaf surface, suggesting that the defect in appressorial development is surface-dependent. RNA-seq based transcriptome analysis highlighted that Cfhos2 regulates secondary metabolism-related virulence genes during infection. Moreover, the expression of an apple defense-related F-box protein was strongly induced by infection with Cfhos2 deletion mutants. Taken together, we demonstrate that Cfhos2 is a key epigenetic factor regulating appressorium development, virulence gene expression, and GLS pathogenesis in C. fructicola. The results provide important information for understanding the virulence mechanisms of C. fructicola.

Polysaccharides are the most important effective components of Lycii fructus, which has a variety of biological activities and broad application prospects in the fields of medicine and food. In this study, we reported a novel arabinogalactan LFP-80-W1 with potential immunostimulatory activity. LFP-80-W1 was a continuous symmetrical single-peak with an average molecular weight of 4.58 × 104 Da and was mainly composed of arabinose and galactose. Oligosaccharide sequencing analyses and NMR data showed that the LFP-80-W1 domain consists of a repeated 1,6-linked β-Galp main chain with branches arabinoglycan and arabinogalactan at position C-3. Importantly, we found that LFP-80-W1 could activate the MAPK pathway and promote the release of NO, IL-6, and TNF-α cytokines in vitro. Therefore, our findings suggest that the homogeneous arabinogalactan from Lycii fructus, can be used as a natural immunomodulator.

Identifying and protecting key sites of ecological assets and improving spatial connectivity and accessibility are important measures taken to protect ecological diversity. This study takes Guangxi as the research area. Based on the gross ecosystem product (GEP), the ecological source is identified, and the initial ecological network (EN) is constructed by identifying the ecological corridor with the minimum cumulative resistance model. The internal defects of the initial ecological network are extracted using the circuit theory, the priority areas for restoration and protection with clear spatial positions are determined according to the complex network analysis, and the network’s performance before and after optimization is comprehensively evaluated. The results show that 456 initial ecological sources and 1219 ecological corridors have been identified, forming the initial ecological network of Guangxi. Based on the circuit theory, 168 ecological barriers, 83 ecological pinch points, and 71 ecological stepping stones were extracted for network optimization. After optimizing the ecological network, there are 778 ecological sources with a total area of 73,950.56 km2 and 2078 ecological corridors with a total length of 23,922.07 km. The GEP of the optimized structure is 13.33% higher than that of the non-optimized structure. The priority areas for protection are distributed in a large area, and the attached GEP reaches USD 118 billion, accounting for 72% of the total GEP attached to the optimized ecological source area. The priority areas for restoration are scattered in small patches, with a GEP of USD 19.27 billion. The robustness and connectivity of the optimized ecological network have been improved obviously. This study attempts to identify key sites of ecological assets and the priority regions for restoration and conservation using genuine geographical location and reference materials for regional ecological network optimization and implementation.

Pueraria lobata (Willd) (Pueraria montana var. lobata (Willd.) Maesen & S. M. Almeida ex Sanjappa & Predeep) is an important herbal medicine used in many countries. In P. lobata plants showing symptoms of mosaic, yellow spots, and mottling, mixed infection of new viruses provisionally named Pueraria lobata-associated emaravirus (PloAEV, genus Emaravirus), Pueraria lobata-associated crinivirus (PloACV, genus Crinivirus), and isolate CQ of the previously reported kudzu mosaic virus (KuMV-CQ, genus Begomovirus) was confirmed through high-throughput sequencing. PloAEV has five RNA segments, encoding a putative RNA-dependent RNA polymerase, glycoprotein precursor, nucleocapsid protein, movement protein, and P5, respectively. PloACV has two RNA segments, encoding 11 putative proteins. Only PloAEV could be mechanically transmitted from mixed infected symptomatic kudzu to Nicotiana benthamiana plants. All three viruses were detected in 35 symptomatic samples collected from five different growing areas, whereas no viruses were detected in 21 non-symptomatic plants, suggesting a high association between these three viruses. Thus, this study provides new knowledge on the diversity and molecular characteristics of viruses in P. lobata plants affected by the viral disease.

Abstract Hepatocellular carcinoma (HCC) is a common and high-mortality cancer worldwide. Numerous microRNAs have crucial roles in the progression of different cancers. However, identifying the important microRNAs and the target biological function of the microRNA in HCC progression is difficult. In this study, we selected highly expressed microRNAs with different read counts as candidate microRNAs and then tested whether the microRNAs were differentially expressed in HCC tumour tissues, and we found that their expression was related to the HCC prognosis. Then, we investigated the effects of microRNAs on the cell growth and mobility of HCC using a real-time cell analyser (RTCA), colony formation assay and subcutaneous xenograft models. We further used deep-sequencing technology and bioinformatic analyses to evaluate the main functions of the microRNAs. We found that miR-103a was one of the most highly expressed microRNAs in HCC tissues and that it was upregulated in HCC tissue compared with the controls. In addition, high miR-103a expression was associated with poor patient prognosis, and its overexpression promoted HCC cell growth and mobility. A functional enrichment analysis showed that miR-103a mainly promoted glucose metabolism and inhibited cell death. We validated this analysis, and the data showed that miR-103a promoted glucose metabolism-likely function and directly inhibited cell death via ATP11A and EIF5. Therefore, our study revealed that miR-103a may act as a key mediator in HCC progression.

Abstract 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

Abstract 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 Graphic abstract

Fusarium wilt of banana (Musa spp.), a typical vascular wilt disease caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense race 4 (Foc4), seriously threatens banana production worldwide. Pathogens, including vascular wilt fungi, secrete small cysteine-rich proteins during colonization. Some of these proteins are required for pathogenicity. In this study, 106 small secretory proteins that contain a classic N-terminal signal peptide were identified using bioinformatic methods in Foc4. Among them, 11 proteins were selected to show transient expressions in tobacco. Interestingly, transient expression of FoSsp1 in tobacco, an uncharacterized protein (of 145 aa), induced necrotic cell death reactive oxygen burst, and callous deposition. Furthermore, the expression of FoSSP1 in Foc4 wild type (WT) was up-regulated during the stage of banana roots colonization. A split-marker approach was used to knock out FoSSP1 in the Foc4 WT strain. Compared with the WT, the deletion mutant Fossp1 was normal in growth rate but increased in conidiation and virulence. RT-qPCR analysis showed that the expression of four conidiation regulator genes in the Fossp1 deletion mutant was significantly decreased compared to the WT strain. In addition, the expression of four pathogenesis-related genes of bananas infected with Fossp1 deletion mutant was down-regulated in comparison with that of the WT. In summary, these results suggested that FoSSP1 is a putative elicitor that negatively regulates conidiation and pathogenicity in Foc4.

Abstract Background This research was to develop a special method for enriching Circulating tumor cells (CTCs) of Hepatocellular carcinoma (HCC) by Glypican-3 immunoliposomes (GPC3-IML), and to analyze the correlation between the CTCs count and tumor malignancy, as well as to investigate the mutation characteristics of CTC-derived NGS. Results In this study characterization of physical parameters was performed with the preparation of GPC3-IML. CTCs in peripheral blood of HCC patients were further separated and identified. Immunofluorescence was used to identify CTCs for further counting. By this means, the correlation between CTCs count and clinicopathological features was analyzed, and the genetic mutation characteristics of NGS derived from CTCs were investigated and compared with that of tissue NGS. Results showed that compared with EpCAM and vimentin, GPC-3 had a stronger CTCs separation ability. There was a correlation between "positive" count of CTCs (≥ 5 PV-CTC per 7.5 ml blood) and BCLC stage (P = 0.055). The result of CTC-NGS was consistent with that of tissue-NGS in 60% cases, revealing that KMT2C was a common highly-frequent mutated gene. Conclusion The combination of immunomagnetic separation of CTCs and anti-tumor marker identification technology can be regarded as a new technology of CTCs detection in peripheral blood of patients with HCC. Trial registration EHBHKY2020-k-024. Registered 17 August 2020—Retrospectively registered

Abstract 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.

Abstract Background It is very important to develop a highly efficient cerebrospinal fluid (CSF) detection system with diagnosis and prediction function, for which the detection of circulating tumor cells (CTCs) in CSF is a good choice. In contrast to the past use of epithelial EpCAM as CTCs separation target, a cytoplasm protein of GFAP antibody was first selected to construct highly-sensitive immunomagnetic liposome beads (IMLs). The validation and efficiency of this system in capturing CTCs for brain tumors were measured both in vitro and in vivo. The associations between the numbers of CTCs in patients with their clinical characteristics were further analyzed. Results Our data show that CTCs can be successfully isolated from CSF and blood samples from 32 children with brain tumors. The numbers of CTCs in CSF were significantly higher than those in blood. The level of CTCs in CSF was related to the type and location of the tumor rather than its stage. The higher the CTCs number is, the more possibly the patient will suffer from poor prognosis. Genetic testing in GFAP CTC-DNA by sanger sequencing, q-PCR and NGS methods indicated that the isolated CTCs (GFAP+/EGFR+) are the related tumor cell. For example, the high expression of NPR3 gene in CSF CTCs was consistent with that of tumor tissue. Conclusions The results indicated that GFAP-IML CTCs isolation system, combined with an EGFR immunofluorescence assay of antitumor marker, can serve as a brand-new method for the identification of CTCs for brain tumors. Via lumbar puncture, a minimally invasive procedure, this technique may play a significant role in the clinical diagnosis and drug evaluation of brain tumors.

An amendment to this paper has been published and can be accessed via the original article.

Colletotrichum fructicola is a plant-pathogenic fungus with a broad host range. It causes significant losses to important crops, including apple, pear, strawberry, and other Rosaceae and non-Rosaceae species. To date, two short read–based C. fructicola genomes are publicly available, but both are fragmented. In this study, we re-sequenced the genome of C. fructicola using nanopore long-read technology and refined the assembly with Hi-C map data. The resulting high-quality assembly is an important resource for further comparative and experimental studies with C. fructicola.

Abstract Background The wide variation in the size of fungal genomes is well known, but the reasons for this size variation are less certain. Here, we present a chromosome-scale assembly of ectophytic Peltaster fructicola, a surface-dwelling extremophile, based on long-read DNA sequencing technology, to assess possible mechanisms associated with genome compaction. Results At 18.99 million bases (Mb), P. fructicola possesses one of the smallest known genomes sequence among filamentous fungi. The genome is highly compact relative to other fungi, with substantial reductions in repeat content, ribosomal DNA copies, tRNA gene quantity, and intron sizes, as well as intergenic lengths and the size of gene families. Transposons take up just 0.05% of the entire genome, and no full-length transposon was found. We concluded that reduced genome sizes in filamentous fungi such as P. fructicola, Taphrina deformans and Pneumocystis jirovecii occurred through reduction in ribosomal DNA copy number and reduced intron sizes. These dual mechanisms contrast with genome reduction in the yeast fungus Saccharomyces cerevisiae, whose small and compact genome is associated solely with intron loss. Conclusions Our results reveal a unique genomic compaction architecture of filamentous fungi inhabiting plant surfaces, and broaden the understanding of the mechanisms associated with compaction of fungal genomes.

Abstract Gene therapy is emerging as a valid method for the treatment of ovarian cancer, including small interfering RNA (siRNA). Although it is so powerful, few targeting efficient gene delivery systems seriously hindered the development of gene therapy. In this study, we synthesized a novel gene vector PEG-GO-PEI-FA by functionalized graphene oxide (GO), in which folic acid (FA) can specifically bind to the folate receptor (FR), which is overexpressed in ovarian cancer. Characterizations of the nanocomplexes were evaluated by dynamic light scattering (DLS), atomic force microscopy (AFM), and Fourier transform infrared spectroscopy (FTIR). The siRNA condensation ability and stability were assessed by agarose gel electrophoresis. Cellular uptake efficiency and lysosomal escape ability in ovarian cancer cells were investigated by confocal laser scanning microscopy. Furthermore, cellular biosafety of the system and inhibitory of the siRNA tolerability were evaluated by CCK-8 assay. The size of the PEG-GO-PEI-FA nanocomplexes was 216.1 ± 2.457 nm, exhibiting mild cytotoxicity in ovarian cancer cells. With high uptake efficiency, PEG-GO-PEI-FA can escape from the lysosome rapidly and release the gene. Moreover, PEG-GO-PEI-FA/siRNA can effectively inhibit the growth of ovarian cancer cells. By and large, the PEG-GO-PEI-FA/siRNA may offer a promising strategy for siRNA delivery in the treatment of FR-positive ovarian carcinoma or similar tumors.

Abstract Tumor cells with heterogeneity and diversity can express different markers. At present, positive separation of circulating tumor cells (CTC) taking EpCAM as the marker was used in most cases which could be one-sided, while this study successfully prepared four antibody-modified magnetic immunoliposomes (MIL) by using the self-assembled liposome with antibody derivatives. This study aims to explore the separation efficiency and clinical detection feasibility of single or combined use of MIL with multi-tumor markers on different tumors. Captured CTC were stained with CK-FITC, CD45-PE and DAPI, and fluorescence microscope was used for the observation, analysis and calculation. The result indicated that the CTC number positive rate in blood samples of four different magnetic balls on the same patient could be up to 87.5% in 32 patients with 14 different kinds tumors. While the effect of directly mixed separation by four kinds of magnetic balls was not satisfying. It suggested that the MIL of multi-tumor markers could be a powerful tool for CTC separation in application of tumor screening and prognosis.

Lipopolysaccharide (LPS) is a peculiar component of the outer membrane (OM) of many Gram-negative bacteria that renders these bacteria highly impermeable to many toxic molecules, including antibiotics. LPS is assembled at the OM by a dedicated intermembrane transport system, the Lpt (LPS transport) machinery, composed of seven essential proteins located in the inner membrane (IM) (LptB2CFG), periplasm (LptA), and OM (LptDE). Defects in LPS transport compromise LPS insertion and assembly at the OM and result in an overall modification of the cell envelope and its permeability barrier properties. LptA is a key component of the Lpt machine. It connects the IM and OM sub-complexes by interacting with the IM protein LptC and the OM protein LptD, thus enabling the LPS transport across the periplasm. Defects in Lpt system assembly result in LptA degradation whose stability can be considered a marker of an improperly assembled Lpt system. Indeed, LptA recruitment by its IM and OM docking sites requires correct maturation of the LptB2CFG and LptDE sub-complexes, respectively. These quality control checkpoints are crucial to avoid LPS mistargeting. To further dissect the requirements for the complete Lpt transenvelope bridge assembly, we explored the importance of LPS presence by blocking its synthesis using an inhibitor compound. Here, we found that the interruption of LPS synthesis results in the degradation of both LptA and LptD, suggesting that, in the absence of the LPS substrate, the stability of the Lpt complex is compromised. Under these conditions, DegP, a major chaperone–protease in Escherichia coli, is responsible for LptD but not LptA degradation. Importantly, LptD and LptA stability is not affected by stressors disturbing the integrity of LPS or peptidoglycan layers, further supporting the notion that the LPS substrate is fundamental to keeping the Lpt transenvelope complex assembled and that LptA and LptD play a major role in the stability of the Lpt system.

To explore the effect of nano-attapulgite clay (NAC) on the durability of concrete, two kinds of NAC (calcined raw ore and calcined high viscosity ore: calcination at 650 °C for 2 h) were used to study their effects on the durability of concrete, mainly discussing the changes of chloride ion permeability and the resistivity of concrete with NAC. The effect of NAC on the strength of concrete was analyzed by testing the compressive strength of concrete. The two-electrode method, four-electrode method, and concrete resistivity tester were used to analyze the relationship between the testing method and concrete resistivity, and the effect of NAC on concrete resistivity was analyzed. The influence of NAC on the chloride corrosion resistance of concrete was analyzed by measuring the chloride diffusion coefficient, and the relationship between the chloride diffusion coefficient and resistivity was established. The diffusion process of chloride ions in concrete was analyzed by theoretical derivation and numerical simulation. The results show that: calcining raw ore NAC can improve the compressive strength of concrete, while calcining high-viscosity ore reduces the compressive strength of concrete. At the age of 28 days, the strength of concrete mixed with calcined raw ore is about 7.10% higher than that of ordinary concrete, while the compressive strength of concrete mixed with calcined high-viscosity ore is about 4.32% lower than that of common concrete. The resistivity of concrete mixed with calcined raw ore increases the fastest, and the 56 days age is about 15.8% and 29.6% higher than that of ordinary concrete and calcined high-viscosity ore. There is a good negative correlation between concrete resistivity and chloride diffusion coefficient. At 28 days, the incorporation of calcined raw ore concrete decreased by about 19.9% and 49.4% compared with ordinary concrete and calcined high-viscosity ore, respectively. After 10 years of decline, the chloride ion content is 11.1% and 23.2% lower than that of ordinary concrete and concrete mixed with calcined high viscosity ore.

Drug nanocrystals, one of most common drug delivery systems, enable the delivery of poorly water-soluble drugs with high drug loading and enhanced dissolution. The rapid clearance and uncontrolled drug release of drug nanocrystals limit their delivery efficiency and clinical application. Herein, an amphiphilic co-polymer, poly oligo(ethylene glycol) methacrylate-b-poly(styrene–co-4-formylphenyl methacrylate) (POEGMA-b-P (St-co-FPMA), PPP), characterized by a hydrophilic part with bottlebrush-like oligo(ethylene glycol) methacrylate (OEGMA) side chains, was synthesized as stabilizers to fabricate a high-drug-loading nanocrystal micelle (053-PPP NC micelle) using the chronic myeloid leukemia (CML) drug candidate N-(2-methyl-5-(3-(trifluoromethyl)benzamido)phenyl)-4-(methylamino)pyrimidine-5-carboxamide (CHMFL-ABL-053 or 053) as a model drug. The 053-PPP NC micelle was characterized and subjected to in vitro and in vivo studies. It featured a worm-like shape of small size, high drug loading (~50%), high colloidal stability, and controlled release in vitro. The presence of the 053-PPP NC micelle resulted in a long-circulation property and a much higher AUC. The 053-PPP NC micelle induced higher accumulation in the tumor tissues under multiple continuous administration. For in vivo efficacy, the 053-PPP NC micelle with a longer dosing interval (96 h), beneficial for improving patient adherence, demonstrated superiority to the 053-F127 NC. The proposed stabilizer PPP and the 053-PPP NC micelle with high drug loading enables drug delivery with long circulation and controlled release of drugs. It is also promising for the development of more efficient nanocrystal-based intravenous injection formulations for poorly water-soluble drugs. It might also offer new possibilities for potential clinical application of the CML candidate drug 053.

Bitter rot and Glomerella leaf spot (GLS) of apples, caused by Colletotrichum species, are major diseases of apples around the world. A total of 98 isolates were obtained from apple fruits with bitter rot, and 53 isolates were obtained from leaves with leaf spot in the primary apple production regions in China. These isolates were characterized morphologically, and five gene regions (ITS, ACT, GAPDH, CHS-1 and TUB2) were sequenced for each isolate. A phylogenetic analysis, combined with a comparison of the morphological, cultural and pathogenic characters, sorted bitter rot isolates into six species: C. alienum, C. fructicola, C. gloeosporioides sensu stricto, C. nymphaeae, C. siamense and one new species, C. orientalis Dandan Fu & G.Y. Sun. Among these, C. siamense was the predominant pathogen associated with bitter rot. Isolates from leaf spot were identified as two species, C. aenigma and C. fructicola. This is the first report of C. orientalis as an apple bitter rot pathogen worldwide, and the results provide important insights into the diversity of Colletotrichum species in China.

Circulating Tumor Cells (CTCs) are already present in the peripheral blood of patients with early tumors and even precancerous lesions. The objective of this study was to determine the count of CTCs in peripheral blood from high-risk population(HRP), healthy subjects and patients with Pan-cancer. The CTCs in the peripheral blood from HRP and cancer patients were enriched and identified based on the positive sorting method by epithelial cell adhesion molecular (EpCAM) liposome magnetic bead (Ep-LMB) and Vimentin liposome magnetic bead (Vi-LMB). Simultaneously, further analysis was carried out focusing on the clinical characteristics of patients by collecting the peripheral blood samples from healthy subjects as the parallel control. According to the results, the prepared LMBs had high specificity and stability, resulting in an average (Av) proliferation rate of over 90% for each cell line, and the average capture rate of higher than 80%. In terms of CTCs count detection in clinical blood samples, the average count was 0.9 (Ep: Av=0.6, Vi: Av=0.3), 2.4 (Ep: Av=1.4, Vi: Av=0.8) and 7.3 (Ep: Av=4.0, Vi: Av=3.3) in healthy subjects, HRP and total cancer patients, respectively. Besides, there was no obvious difference in the average count of CTCs among patients with different cancer types. While count of CTCs in the aforementioned cancer patients was statistically different from that in healthy subjects and patients with HRP. The survival time of cancer patients whose number of CTCs is greater than the average is significantly increased. Collectively, the study confirmed that CTCs can achieve early tumor detection and auxiliary diagnosis, and its number is related to the occurrence and development of tumors, and CTCs can be detected in HRP and sub-health population.

Abstract Ring rot, one of the most destructive diseases of apple worldwide, is caused primarily by Botryosphaeria dothidea and 8. kuwatsukai. Here, we sequenced the genomes of 8. dothidea strain PG45 (44.3 Mb with 5.12 % repeat rate) and 8. kuwatsukai epitype strain PG2 (48.0 Mb with 13.02 % repeat rate), and conducted a comparative analysis of these two genomes, as well as other sequenced fungal genomes, in order to understand speciation and distinctive patterns of evolution of pathogenicity-related genes. Pair-wise genome alignments revealed that the two species are highly syntenic (96.74 % average sequence identity). Both species encode a significant number of pathogenicity-related genes, e.g.carbohydrateactive enzymes (CAZYs), plant cell wall degrading enzymes (PCWDEs), secondary metabolites (SMs) biosynthetic enzymes, cytochrome P450 enzymes (CYPs), and secreted peptidases, in comparison to all additional sequenced fungal species involved in various life-styles. The number of pathogenicity-related genes in 8. dothidea and 8. kuwatsukai is higher than other genomes of Botryosphaeriaceae pathogens (Macrophomina phaseolina and Neofusicoccum parvum), suggesting a secondary round of Botryosphaeria-lineage expansion in the family. There were, however, also significant differences in the genomes of the two Botryosphaeria species. Botryosphaeria kuwatsukai, which infects only apple and pear, apparently lost a set of SMs genes, CAZYs and PCWDEs, possibly as a result of host specialization. Botryosphaeria kuwatsukai contained significantly more transposable elements and higher value of repeat induced point (RIP) index than B. dothidea. Our results will be instrumental in understanding how both phytopathogens interact with their plant hosts and in designing efficient strategies for disease control and molecular breeding to help ensure global apple production and food security.

The performance of reinforced concrete bridge deck pavement will be gradually reduced with the increase of operation time, so the reasonable maintenance measures should be taken to ensure the performance and safety of the bridge structures. The objectives and their hierarchical structures from both state position and enterprise position of the decision makers in bridge engineering decision-making problems were discussed. By the existing deterioration model, this paper studies the bridge operation safety maintenance strategy, establishes the mathematical model of the influence of different degrees of maintenance measures on the structural performance, combines with the cost-benefit theory, establishes the maintenance risk decision criteria for the decision makers with different objectives, and makes the decision on the selection of the bridge deck pavement scheme based on the enterprise position through an example.

Autophagy is a cellular mechanism responsible for delivering protein aggregates or damaged organelles to lysosomes for degradation. It is also simultaneously a precise regulatory process, which is crucial for dealing with hunger, oxidative stress, and pathogen defense. Neutrophil Extracellular Traps (NETs), which form a part of a newly described bactericidal process, are reticular structures composed of a DNA backbone and multiple functional proteins, formed via a process known as NETosis. NETs exert their anti-infection activity by capturing pathogenic microorganisms, inhibiting their spread and inactivating virulence factors. However, NETs may also activate an immune response in non-infectious diseases, leading to tissue damage. Although the mechanism underlying this phenomenon is unclear, a large number of studies have suggested that autophagy may be involved. Autophagy-mediated NETs not only induce inflammation and tissue damage, but can also lead to cell senescence, malignant transformation, and cell death. Autophagy-dependent NETs also play a beneficial role in the hostwith respect to pathogen clearance and immune defense. Through careful review of the literature, we have found that the distinct roles of autophagy in NETosis may be dependent on the extent of autophagy and the specific manner in which it was induced. This article summarizes numerous recent studies, and reviews the role of autophagy-driven NETosis in various diseases, in the hope that this will lead to the development of more effective treatments.

Abstract Background Colletotrichum is a fungal genus in Ascomycota that contain many plant pathogens. Among all Colletotrichum genomes that have been sequenced, C. fructicola contains the largest number of candidate virulence factors, such as plant cell wall degrading enzymes, secondary metabolite (SM) biosynthetic enzymes, secreted proteinases, and small secreted proteins. Systematic analysis of the expressional patterns of these factors would be an important step toward identifying key virulence determinants. Results In this study, we obtained and compared the global transcriptome profiles of four types of infection-related structures: conidia, appressoria, infected apple leaves, and cellophane infectious hyphae (bulbous hyphae spreading inside cellophane) of C. fructicola. We also compared the expression changes of candidate virulence factors among these structures in a systematic manner. A total of 3189 genes were differentially expressed in at least one pairwise comparison. Genes showing in planta-specific expressional upregulations were enriched with small secreted proteins (SSPs), cytochrome P450s, carbohydrate-active enzymes (CAZYs) and secondary metabolite (SM) synthetases, and included homologs of several known candidate effectors and one SM gene cluster specific to the Colletotrichum genus. In conidia, tens of genes functioning in triacylglycerol biosynthesis showed coordinately expressional upregulation, supporting the viewpoint that C. fructicola builds up lipid droplets as energy reserves. Several phosphate starvation responsive genes were coordinately up-regulated during early plant colonization, indicating a phosphate-limited in planta environment immediately faced by biotrophic infectious hyphae. Conclusion This study systematically analyzes the expression patterns of candidate virulence genes, and reveals biological activities related to the development of several infection-related structures of C. fructicola. Our findings lay a foundation for further dissecting infection mechanisms in Colletotrichum and identifying disease control targets.

Abstract Background Ectophytic fungi occupy the waxy plant surface, an extreme environment characterized by prolonged desiccation, nutrient limitation, and exposure to solar radiation. The nature of mechanisms that facilitate adaptation to this environment remains unclear. In this study, we sequenced the complete genome of an ectophytic fungus, Ramichloridium luteum, which colonizes the surface of apple fruit, and carried out comparative genomic and transcriptome analysis. Results The R. luteum genome was 28.18 Mb and encoded 9466 genes containing 1.85% repetitive elements. Compared with cell-penetrating pathogens, genes encoding plant cell wall degrading enzymes (PCWDEs), PTH11-like G protein-coupled receptors (GPCRs) and effectors were drastically reduced. In contrast, genes encoding cutinases and secretory lipases were strikingly expanded, and four of nine secretory lipases were probably acquired by horizontal gene transfer from Basidiomycota. Transcriptomic analysis revealed elevated expression of genes involved in cuticle degradation (cutinase, secretory lipase) and stress responses (melanin biosynthesis, aquaporins, lysozymes and HOG pathway). Conclusions Taken together, our results highlight genomic features associated with evolution of surface niche adaptation by the ectophytic fungus R. luteum, namely the contraction of PCWDEs, PTH11-like GPCRs and effectors, and the expansion of cuticle degradation and stress tolerance.

The Ascomycetes fungus Colletotrichum fructicola causes severe diseases on a wide range of crops, fruits, and vegetables. Its pathogenic mechanisms, however, remain poorly understood. Mitogen-activated protein kinases (MAPKs) are conserved regulators of fungal development and pathogenesis. In this study, a Fus3/Kss1-related MAPK from C. fructicola was functionally characterized via gene deletion. On potato dextrose agar (PDA) and oatmeal agar media, the CfPMK1 gene deletion mutants (ΔCfPMK1) were slightly reduced in radial growth rate, severely limited in aerial hyphal differentiation and hyphal melanization, and formed deformed perithecia that were smaller in size and more compactly organized relative to wild type. When artificially inoculated on plants, conidia of these mutants failed to differentiate appressoria or penetrate cuticle, and their pathogenicity defect could not be rescued by wounding plant tissue prior to inoculation. On PDA, ΔCfPMK1 mutants were hypersensitive to osmotic stresses, but were more tolerant to membrane and cell wall stresses. Genetic complementation rescued all phenotypic changes associated with CfPMK1 gene deletion. Based on GFP fusion expression, CfPMK1 protein accumulation was detected at all life stages, and the accumulation level was higher in nascent appressoria relative to conidia. Overall, this study identified CfPMK1 as a key regulator of appressorium and sexual development, pathogenesis, and stress tolerance in C. fructicola.

The conformational dynamics of a compound has a large effect on ligand/receptor binding. Here, the authors employ NMR spectroscopy to study ligand binding to the enzyme LpxC, discovering an inhibitor envelope that was not identifiable by crystallography and subsequently developing a highly potent inhibitor.

Here, we report a novel (+) ssRNA mycovirus, Phoma matteucciicola ourmia-like virus 1 (PmOLV1), isolated from Phoma matteucciicola strain LG915-1. The genome of PmOLV1 was 2603 nucleotides long and contained a single open reading frame (ORF), which could be translated into a product of RNA-dependent RNA polymerase (RdRp) by both standard and mitochondrial genetic codons. Cellular fractionation assay indicated that PmOLV1 RNAs are likely more enriched in mitochondria than in cytoplasm. Phylogenetic analysis indicated that PmOLV1 is a new member of the genus Penoulivirus (recently proposed) within the family Botourmiaviridae.

Lipid A is an essential basal component of lipopolysaccharide of most Gram-negative bacteria. Inhibitors targeting LpxC, a conserved enzyme in lipid A biosynthesis, are antibiotic candidates against Gram-negative pathogens. Here we report the characterization of the role of lipid A in Coxiella burnetii growth in axenic media, monkey kidney cells (BGMK and Vero), and macrophage-like THP-1 cells by using a potent LpxC inhibitor -LPC-011. We first determined the susceptibility of C. burnetii LpxC to LPC-011 in a surrogate E. coli model. In E. coli, the minimum inhibitory concentration (MIC) of LPC-011 against C. burnetii LpxC is < 0.05 μg/mL, a value lower than the inhibitor's MIC against E. coli LpxC. Considering the inhibitor's problematic pharmacokinetic properties in vivo and Coxiella's culturing time up to 7 days, the stability of LPC-011 in cell cultures was assessed. We found that regularly changing inhibitor-containing media was required for sustained inhibition of C. burnetii LpxC in cells. Under inhibitor treatment, Coxiella has reduced growth yields in axenic media and during replication in non-phagocytic cells, and has a reduced number of productive vacuoles in such cells. Inhibiting lipid A biosynthesis in C. burnetii by the inhibitor was shown in a phase II strain transformed with chlamydial kdtA. This exogenous KdtA enzyme modifies Coxiella lipid A with an α-Kdo-(2 → 8)-α-Kdo epitope that can be detected by anti-chlamydia genus antibodies. In inhibitor-treated THP-1 cells, Coxiella shows severe growth defects characterized by poor vacuole formation and low growth yields. Coxiella progenies prepared from inhibitor-treated cells retain the capability of normally infecting all tested cells in the absence of the inhibitor, which suggests a dispensable role of lipid A for infection and early vacuole development. In conclusion, our data suggest that lipid A has significance for optimal development of Coxiella-containing vacuoles, and for robust multiplication of C. burnetii in macrophage-like THP-1 cells. Unlike many bacteria, C. burnetii replication in axenic media and non-phagocytic cells was less dependent on normal lipid A biosynthesis.

The fungal genus Colletotrichum contains hemibiotrophic phytopathogens being highly variable in host and tissue specificities. We sequenced a C. fructicola genome (1104-7) derived from an isolate of apple in China and compared it with the reference genome (Nara_gc5) derived from an isolate of strawberry in Japan. Mauve alignment and BlastN search identified 0.62 Mb lineage-specific (LS) genomic regions in 1104-7 with a length criterion of 10 kb. Genes located within LS regions evolved more dynamically, and a strongly elevated proportion of genes were closely related to non-Colletotrichum sequences. Two LS regions, containing nine genes in total, showed features of fungus-to-fungus horizontal transfer supported by both gene order collinearity and gene phylogeny patterns. We further compared the gene content variations among 13 Colletotrichum and 11 non-Colletotrichum genomes by gene function annotation, OrthoMCL grouping and CAFE analysis. The results provided a global evolutionary picture of Colletotrichum gene families, and identified a number of strong duplication/loss events at key phylogenetic nodes, such as the contraction of the detoxification-related RTA1 family in the monocot-specializing graminicola complex and the expansions of several ammonia production-related families in the fruit-infecting gloeosporioides complex. We have also identified the acquirement of a RbsD/FucU fucose transporter from bacterium by the Colletotrichum ancestor. In sum, this study summarized the pathogenic evolutionary features of Colletotrichum fungi at multiple taxonomic levels and highlights the concept that the pathogenic successes of Colletotrichum fungi require shared as well as lineage-specific virulence factors.

Characterization of newly isolated mycoviruses may contribute to understanding of the evolution and diversity of viruses. Here, a deep sequencing approach was used to analyze the double-stranded RNA (dsRNA) mycoviruses isolated from field-collected P. striiformis samples in China. Database searches showed the presence of at least four totivirus-like sequences, termed Puccinia striiformis virus 1 to 4 (PsV1 to 4). All of these identified sequences contained two overlapping open reading frames (ORFs) which encode a putative coat protein (CP) and an RNA-dependent RNA polymerase (RdRp) showing similar structures to members of the genus Totivirus. Each PsV contained a -1 ribosomal frameshifting region with a slippery site and a pseudoknot structure in the overlapped regions of these ORFs, indicating that the RdRp is translated as a CP-RdRp fusion. Phylogenetic analyses based on RdRp and CP suggested that these novel viruses belong to the genus Totivirus in the family Totiviridae. The presences of these PsVs were further validated by transmission electron microscope (TEM) and RT-PCR. Taken together, our results demonstrate the presence of diverse, novel totiviruses in the P. striiformis field populations.

ABSTRACT The infectious diseases caused by multidrug-resistant bacteria pose serious threats to humankind. It has been suggested that an antibiotic targeting LpxC of the lipid A biosynthetic pathway in Gram-negative bacteria is a promising strategy for curing Gram-negative bacterial infections. However, experimental proof of this concept is lacking. Here, we describe our discovery and characterization of a biphenylacetylene-based inhibitor of LpxC, an essential enzyme in the biosynthesis of the lipid A component of the outer membrane of Gram-negative bacteria. The compound LPC-069 has no known adverse effects in mice and is effective in vitro against a broad panel of Gram-negative clinical isolates, including several multiresistant and extremely drug-resistant strains involved in nosocomial infections. Furthermore, LPC-069 is curative in a murine model of one of the most severe human diseases, bubonic plague, which is caused by the Gram-negative bacterium Yersinia pestis. Our results demonstrate the safety and efficacy of LpxC inhibitors as a new class of antibiotic against fatal infections caused by extremely virulent pathogens. The present findings also highlight the potential of LpxC inhibitors for clinical development as therapeutics for infections caused by multidrug-resistant bacteria. IMPORTANCE The rapid spread of antimicrobial resistance among Gram-negative bacilli highlights the urgent need for new antibiotics. Here, we describe a new class of antibiotics lacking cross-resistance with conventional antibiotics. The compounds inhibit LpxC, a key enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria, and are active in vitro against a broad panel of clinical isolates of Gram-negative bacilli involved in nosocomial and community infections. The present study also constitutes the first demonstration of the curative treatment of bubonic plague by a novel, broad-spectrum antibiotic targeting LpxC. Hence, the data highlight the therapeutic potential of LpxC inhibitors against a wide variety of Gram-negative bacterial infections, including the most severe ones caused by Y. pestis and by multidrug-resistant and extensively drug-resistant carbapenemase-producing strains.

In this paper, an atomic force microscope (AFM) was used to study the nano-morphology characteristics of styrene-butadiene-styrene block copolymer (SBS) modified bitumen. The effects of SBS content and short-term aging on the nano-morphology parameters of bitumen were studied. The correlation between nano-morphology parameters and main pavement performances of SBS modified bitumen was analyzed. The results show that with the increase of SBS content, the average area of bee-like structure and the proportions of bee-like structure area of SBS modified bitumen increases gradually, the roughness of the whole image and the roughness of the lightweight component region decreases gradually, but the increasing or decreasing trend is no longer obvious when the content of SBS is more than 4%. The maximum height difference of SBS modified bitumen is linear with the content of SBS. The larger the content of SBS, the smaller the influence of short-term aging on the nano-morphology of SBS modified bitumen. The penetration degree will decrease and the softening point will increase with the increase of the proportion of bee-like structure area of SBS modified bitumen. The ductility will decrease with the increase of the roughness of the lightweight component region. The ductility and the Brookfield viscosity will increase with the increase of the maximum height difference. The research results provided a reference for defining quantifiable nano-parameters of SBS modified bitumen and establishing the relationship between nano-parameters and pavement performances.

Hyperammonemia is frequently seen in tumor microenvironments as well as in liver diseases where it can lead to severe brain damage or death. Ammonia induces autophagy, a mechanism that tumor cells may use to protect themselves from external stresses. However, how cells sense ammonia has been unclear. Here we show that culture medium alone containing Glutamine can generate milimolar of ammonia at 37 degrees in the absence of cells. In addition, we reveal that ammonia acts through the G protein-coupled receptor DRD3 (Dopamine receptor D3) to induce autophagy. At the same time, ammonia induces DRD3 degradation, which involves PIK3C3/VPS34-dependent pathways. Ammonia inhibits MTOR (mechanistic target of Rapamycin) activity and localization in cells, which is mediated by DRD3. Therefore, ammonia has dual roles in autophagy: one to induce autophagy through DRD3 and MTOR, the other to increase autophagosomal pH to inhibit autophagic flux. Our study not only adds a new sensing and output pathway for DRD3 that bridges ammonia sensing and autophagy induction, but also provides potential mechanisms for the clinical consequences of hyperammonemia in brain damage, neurodegenerative diseases and tumors.

Transcripts encoding Sclerotinia sclerotiorum γ-glutamyl transpeptidase (Ss-Ggt1) were found to accumulate specifically during sclerotium, apothecium, and compound appressorium development in S. sclerotiorum. To determine the requirement of this protein in these developmental processes, gene deletion mutants of Ss-ggt1 were generated and five independent homokaryotic ΔSs-ggt1 mutants were characterized. All deletion mutants overproduced sclerotial initials that were arrested in further development or eventually produced sclerotia with aberrant rind layers. During incubation for carpogenic germination, these sclerotia decayed and failed to produce apothecia. Total glutathione accumulation was approximately 10-fold higher and H2O2 hyperaccumulated in ΔSs-ggt1 sclerotia compared with the wild type. Production of compound appressoria was also negatively affected. On host plants, these mutants exhibited a defect in infection efficiency and a delay in initial symptom development unless the host tissue was wounded prior to inoculation. These results suggest that Ss-Ggt1 is the primary enzyme involved in glutathione recycling during these key developmental stages of the S. sclerotiorum life cycle but Ss-Ggt1 is not required for host colonization and symptom development. The accumulation of oxidized glutathione is hypothesized to negatively impact these developmental processes by disrupting the dynamic redox environment associated with multicellular development.