Exploring the role of detoxification genes in the resistance of Bursaphelenchus xylophilus to different exogenous nematicidal substances using transcriptomic analyses.

Bursaphelenchus xylophilus (Pine wood nematode, PWN) has become a worldwide forest disease due to its rapid infection ability, high lethality and difficulty in control. The main means of countering B. xylophilus is currently chemical control, but nematicides can present problems such as environmental pollution and drug resistance. The development of novel environmentally-friendly nematicides has thus become a focus of recent research. In this study, BxUGT3 and BxUGT34, which might be related to detoxification, were investigated by comparing transcriptomic and WGCNA approaches. Three other genes with a similar expression pattern, BxUGT13, BxUGT14, and BxUGT16, were found by gene family analysis. Further bioassays and qPCR assays confirmed that these five genes showed significant changes in transcript levels upon exposure to α-pinene and carvone, demonstrating that they respond to exogenous nematicidal substances. Finally, RNAi and bioassays showed that B. xylophilus with silenced BxUGT16 had increased mortality in the face of α-pinene and carvone stress, suggesting that BxUGT16 plays an important role in detoxification. Taken together, this study used novel molecular research methods, explored the detoxification mechanism of B. xylophilus at a transcriptomic level, and revealed a molecular target for the development of novel biopesticides.

Mutation of glucan synthase catalytic subunit in Beauveria bassiana affects fungal growth and virulence.

Beauveria bassiana is a well-known entomopathogenic fungus that parasitizes on a variety of insect species. Glucan in the cell wall of B. bassiana plays a crucial role in its structure and growth and is also involved in the activation of the host insect's immune system. Glucan biosynthesis is primarily regulated by glucan synthase, however, it is unclear if the glucan synthase catalytic subunit gene (GluS) affects the growth and virulence of B. bassiana. In this study, we constructed the mutant of the B. bassiana glucan synthase catalytic subunit (ΔGluS) by homologous recombination and observed that glucan synthase knockout affects both spore germination and cell area. Further enzyme-based assays along with gene expression analysis of glucan synthase revealed a significant downregulation in the mutant strains compared to the wild type of B. bassiana. Moreover, the virulence of ΔGluS strains against gypsy moth (Lymantria dispar) showed no significant difference compared to the wild-type strains when injected, while the spraying gypsy moths with the conidia of ΔGluS was significantly more lethal than spraying the conidia of the wild type. Altogether, our study constructed a new, highly efficient B. bassiana mutant that can be used for pest control and provides a readily transferable method for other insect-entomopathogenic interaction studies.

Comparative analysis of the immune system and expression profiling of Lymantria dispar infected by Beauveria bassiana.

Lymantria dispar is one of the most devastating forest pests worldwide. Fungal biopesticides have great potential as alternatives owing to their high lethality to pests and eco-friendly feature, which is, however, often severely compromised by the pests' innate immunity. A better understanding of the antifungal immune system in L. dispar would significantly facilitate the development of the biopesticide. Here, we investigated phylogenetic characteristics of immunity-related genes as well as the tissue expression patterns in L. dispar after the infection of an entomopathogen Beauveria bassiana using RNA-sequencing data. Results showed most immune genes remain at a low level of response after 24 h post-infection (HPI). Almost all genes in the Toll pathway were significantly up-regulated at 48 HPI, and SPH1, SPN6, Toll6, Toll12, Myd88, pelle, and Drosal were significantly down-regulated at 72 HPI. Immunoblotting analysis revealed that the protein levels of ßGRP3 and PPO1 were significantly upregulated at 24 and 48 HPI, while Myd88 was downregulated at 24 HPI, which was further confirmed by Quantitative real-time PCR experiments. Moreover, the relative content of H2O2, a potent reactive oxygen species (ROS), was significantly increased with the decrease of the total antioxidant capacity, indicating that oxidative stress system positively participates in the clearance of the pathogenic fungus. Together, our study provides detailed genetic characteristics of antifungal immunity as well as profiling of the host defense against entomopathogenic infection, and comprehensive insight into molecular interaction between L. dispar and the entomopathogen.

First Report of Colletotrichum endophyticum, a Causal Agent of Leaf Spot of Bauhinia blakeana in Southwest China.

The Bauhinia blakeana is originated in South Asia and is widely planted in Chinese cities. It is distributed in Guangdong, Fujian, Hainan, Guangxi, Sichuan, and other places in China (Gu S et al. 2019). It is applied to urban greening as the street trees, garden trees, and scenic forest trees, and is an excellent landscaping tree species in South China. In August 2021, the new leaf spot disease was found in Chengdu (30°42'N, 103°51'E), and the incidence rate was about 70%. The symptoms began to appear from April to May, the worst from June to August. Firstly, the typical symptom is that round, oval, or irregular, brown and slightly concave necrotic spots begin to appear at the edge of the leaves, and the color of the spots changes from light brown to dark brown. Gradually, the number of necrotic spots increases and the spots spread from the edge of the leaf to the middle of the leaf. There is an obvious dark brown boundary between the diseased part and the healthy part, and their yellow-green halos around the spots. Finally, the leaves turn yellow and fall off. On September 1, 2021, infected tissue from samples was cut into small pieces 5 × 5 mm, surface sterilized for 30 seconds in 3% NaClO, 60seconds in 75% ethanol, rinsed three times in sterile water, placed on potato dextrose agar (PDA) amended with streptomycin sulfate (50 µg/mL), and incubated at 25°C in a dark. Finally, 10 typical isolates exhibited the morphology described as Colletotrichum endophyticum (De Silva et al. 2019). After 6 days, the colony diameter reached 63.4 to 67.7mm and had white to pale orange aerial mycelium, but was grey-green at the base. Black conidia formed after 10 days, which were round, oval, elongated spindle-shaped, with sharp ends, measuring 3.25 to 5.85 x 1.95 to 2.60µm (average: 6.18 x 2.28µm). Since the 10 isolated strains were consistent in morphology, a representative strain was selected from the 10 isolated strains to continue the next test. For molecular identification, DNA was extracted from 10 fungal colonies (the 10 fungal colonies used to isolate genomic DNA were derived from single isolates) using a plant genomic DNA extraction kit (Solarbio, Beijing). The 5.8S nuclear ribosomal genes with the two flanking internal transcribed spacer (ITS), the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), partial sequences of the actin (ACT) and beta-tubulin (TUB2) genes were amplified and sequenced using the primer pairs ITS4/ITS5 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn. 1999), GDF1/GDR1 (Guerber et al. 2003) and T1/Btub4R (O'Donnell and Cigelink. 1997; Aveskamp et al. 2009), respectively (Fang Qiu et al. 2021). Sequences were deposited in GenBank (ITS:OK560626; ACT:OK562583; GAPDH:OK562584; TUB2:OK562585). BLAST analysis showed >98% identity with several reference sequences of C. endophyticum previously deposited in GenBank. To confirm pathogenicity and fulfill Koch's postulates, the pathogenic fungal cakes were inoculated on the leaves of 5-year-old B. blakeana, and the sterile medium was used as a control. Three fungal cakes were placed on each leaf and repeated three times. Five days later, the inoculated plants showed the similar symptoms observed in diseased plants; controls remained asymptomatic. The C. endophyticum was re-isolated from the infected leaves and identified by morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results, confirming Koch's postulates. This is the first report of B. blakeana leaf spot caused by C. endophyticum in China.

Mesenchymal Stem Cell Utilization for In Vitro Donor Liver Machine Perfusion Preservation: Current Status and Future Directions.

Liver transplantation is the only effective treatment for end-stage liver disease. Currently, the shortage of high-quality donors has led to the exploration of the use of marginal organs. However, several factors limit the in vitro long-term preservation and long-distance transport of livers, which can also lead to ischemia-reperfusion injuries, resulting in poor prognosis. Therefore, an efficient and convenient strategy to improve this situation is urgently required. Normothermic machine perfusion (NMP) is expected to improve the liver environment in vitro and provide better evaluation indices for organ repair mechanisms. Mesenchymal stem cells (MSCs) can repair damaged hepatocytes or exert their protective effects via paracrine mechanisms, such as the release of extracellular vesicles (EVs). We hypothesized that combining the regenerative ability of MSCs and the significant advantages of NMP may improve the quality and utilization rate of organs, especially marginal organs. In this study, we review different strategies for liver preservation in vitro, as well as their strengths and weaknesses. We also introduce MSCs, derived EVs, and MSCs applications in liver preservation in vitro. Finally, we discuss the current challenges and future trends of MSCs applications for in vitro liver preservation. We envision novel bioreactor designs that employ 3D cell culturing and offer the possibility to reconstruct MSCs microenvironments to promote cell growth and biofunction expression. Large-scale MSCs production can be combined with normothermic machine perfusion to enhance in vitro liver preservation, thereby promoting donor organ function to benefit recipients in need of liver transplantation.

Sublethal concentration of emamectin benzoate inhibits the growth of gypsy moth by inducing digestive dysfunction and nutrient metabolism disorder.

BACKGROUND: Gypsy moth (Lymantria dispar) is one of the most important pests in the world. Emamectin benzoate (EMB) is widely used in the control of agricultural and forestry pests. Here, we explored the sublethal effects of EMB on gypsy moths in order to better understand the toxicological mechanism of EMB. RESULTS: The sublethal concentration of EMB exposure significantly decreased the larvae body weight. To further explore the mechanism, indicators related to digestion and nutrient metabolism were detected. The results showed that EMB exposure caused midgut damage, reduced the activities of digestive enzymes and changed the content of sugar and amino acids. Moreover, the expression of insulin/phosphoinositide-3-kinase (PI3K)/forkhead box protein O (FoxO) pathway and sugar metabolism-related genes was abnormal. The expression of insulin receptor (InR), chico, PI3K, and protein kinase B (Akt) significantly reduced, and that of phosphatase and tensin homologue (PTEN) and FoxO increased. The expression of glycogen phosphorylase (GP) was upregulation and that of glycogen synthase (GS), trehalase (TRE) and trehalose-phosphate synthase (TPS) were downregulation. All results indicated that EMB inhibits the growth of gypsy moth by inducing midgut injury, digestive dysfunction and nutrient metabolism disorder. In addition, EMB caused midgut injury may be related to apoptosis or a collateral effect of the damage in other tissues, and more extensive and deeper research is still needed to investigate the detailed mechanism. CONCLUSION: Our finding strengthens the understanding of the sublethal effect of EMB, and provides a theoretical basis for the application of EMB in the prevention and control of gypsy moth.

Transcriptome Analysis Reveals the Important Role of WRKY28 in Fusarium oxysporum Resistance.

Root rot of Populus davidiana × P. alba var. pyramidalis Louche (Pdpap) is caused by Fusarium oxysporum. We used RNA sequencing to study the molecular mechanisms and response pattern of Pdpap infected by F. oxysporum CFCC86068. We cloned the PdpapWRKY28 transcription factor gene and transformed the recombinant vector pBI121-PdpapWRKY28 into Pdpap. The resistance function of PdpapWRKY28 was verified using physiological and biochemical methods. By means of RNA sequencing, we detected 1,403 differentially expressed genes (DEGs) that are common in the different treatments by F. oxysporum. Furthermore, we found that overexpression of the PdpapWRKY28 gene may significantly improve the resistance of Pdpap plants to F. oxysporum. Our research reveals a key role for PdpapWRKY28 in the resistance response of Pdpap to F. oxysporum. Additionally, our results provide a theoretical basis for in-depth research on resistance breeding to combat root rot.

Long Noncoding RNA (lncRNA) MT1JP Suppresses Hepatocellular Carcinoma (HCC) in vitro.

INTRODUCTION: The purpose of this study was to evaluate the effects and mechanisms of the long noncoding RNA (lncRNA) MT1JP on hepatocellular carcinoma (HCC) in vitro. PATIENTS AND METHODS: Thirty pairs of tumor and adjacent normal tissues were collected from HCC patients. Tissue pathology and MT1JP expression were evaluated by hematoxylin and eosin staining and in situ hybridization (ISH), respectively. The correlation between MT1JP and HCC prognosis was investigated. MTT assays, cloning, flow cytometry, transwell assays, and wound-healing assays were used to evaluate the effects of MT1JP on HCC cell lines. RT-qPCR and Western blot were used to measure the relative mRNA and protein expression levels. RESULTS: The expression of MT1JP was downregulated in HCC tumor tissues compared with that in adjacent normal tissues, while the percent survival was significantly greater in the high MT1JP expression group than in the low MT1JP expression group (P=0.0238). In vitro, overexpression of MT1JP suppressed the proliferation, invasion, and migration, reduced colony cell number, increased cell apoptosis, and induced G1-phase cell cycle arrest in Bel-7402 and Huh-7 cells. Meanwhile, the mRNA and protein expression levels of RUNX3 and P21 were significantly upregulated, whereas those of MMP2 and MMP9 were significantly downregulated, in Bel-7402 and Huh-7 cells overexpressing MT1JP (all P<0.001). CONCLUSION: LncRNA MT1JP may function as a tumor suppressor in HCC. Overexpression of MT1JP suppressed HCC cell biological activities through the regulation of RUNX3.

Alveolar macrophage dysfunction and cytokine storm in the pathogenesis of two severe COVID-19 patients.

BACKGROUND: The novel coronavirus pneumonia COVID-19 caused by SARS-CoV-2 infection could lead to a series of clinical symptoms and severe illnesses, including acute respiratory distress syndrome (ARDS) and fatal organ failure. We report the fundamental pathological investigation in the lungs and other organs of fatal cases for the mechanistic understanding of severe COVID-19 and the development of specific therapy in these cases. METHODS: The autopsy and pathological investigations of specimens were performed on bodies of two deceased cases with COVID-19. Gross anatomy and histological investigation by Hematoxylin and eosin (HE) stained were reviewed on each patient. Alcian blue/periodic acid-Schiff (AB-PAS) staining and Masson staining were performed for the examinations of mucus, fibrin and collagen fiber in lung tissues. Immunohistochemical staining was performed on the slides of lung tissues from two patients. Real-time PCR was performed to detect the infection of SARS-CoV-2. Flow cytometry analyses were performed to detect the direct binding of S protein and the expression of ACE2 on the cell surface of macrophages. FINDINGS: The main pathological features in lungs included extensive impairment of type I alveolar epithelial cells and atypical hyperplasia of type II alveolar cells, with formation of hyaline membrane, focal hemorrhage, exudation and pulmonary edema, and pulmonary consolidation. The mucous plug with fibrinous exudate in the alveoli and the dysfunction of alveolar macrophages were characteristic abnormalities. The type II alveolar epithelial cells and macrophages in alveoli and pulmonary hilum lymphoid tissue were infected by SARS-CoV-2. S protein of SARS-CoV-2 directly bound to the macrophage via the S-protein-ACE2 interaction. INTERPRETATION: Infection of alveolar macrophage by SARS-CoV-2 might be drivers of the "cytokine storm", which might result in damages in pulmonary tissues, heart and lung, and lead to the failure of multiple organs . FUNDING: Shanghai Guangci Translational Medical Research Development Foundation, Shanghai, China.

Titanium and zirconium complexes bearing new tridentate [OSO] bisphenolato-based ligands: synthesis, characterization and catalytic properties for alkene polymerization.

A number of new sulfur-bridged tridentate [OSO] bisphenolato-based ligand precursors S(2-CH2-4-tBu-6-R-C6H2OH)2 [R = CMe3 (H2L1), CMe2Ph (H2L2), CMePh2 (H2L3), CPh3 (H2L4), and C(p-Tol)3 (H2L5)] were synthesized by reactions of Na2S·9H2O with 2 eq. of the corresponding 2-(bromomethyl)-4-(tert-butyl)-6-R-phenol. Their neutral titanium complexes [S(2-CH2-4-tBu-6-R-C6H2O)2]TiCl2 [R = CMe3 (1), CMe2Ph (2), CMePh2 (3), CPh3 (4), and C(p-Tol)3 (5)] were synthesized in high yields by direct HCl-elimination reactions of TiCl4 with the corresponding ligand precursors in toluene. Ionic titanium complexes [NHEt3][S(2-CH2-4-tBu-6-R-C6H2O)2TiCl3] [R = CMe3 (6), CMePh2 (7)] and [NH2Et2][S(2-CH2-4-tBu-6-R-C6H2O)2TiCl3] [R = CMe3 (8) and CMePh2 (9)] were obtained in high yields from the reactions of TiCl4 with the corresponding ligand precursors in the presence of 2 eq. of triethylamine or diethylamine. Neutral zirconium complexes [S(2-CH2-4-tBu-6-R-C6H2O)2]ZrCl2(THF) [R = CMe2Ph (10·THF), and CMePh2 (11·THF)] were synthesized by reactions of ZrCl4 with 1 eq. of the dilithium salt of the corresponding ligand precursors Li2L in THF. The new titanium and zirconium complexes were characterized by 1H and 13C NMR, IR and elemental analyses. The molecular structures of complexes 4, 6 and 10·THF were determined by single-crystal X-ray diffraction analysis. The X-ray crystallography analysis reveals that titanium complex 4 has a five-coordinating environment surrounding the central metal atom, while the titanium complex 6 and the THF-solvated zirconium complex 10·THF possess a six-coordinating pseudo-octahedral environment around the central metal atom. Upon activation with MAO or AliBu3/Ph3CB(C6F5)4, all these titanium and zirconium complexes exhibit moderate to high catalytic activities for ethylene polymerization and ethylene/1-hexene copolymerization with moderate to high comonomer incorporation, and the ionic titanium complexes 6, 7, 8 and 9 show lower catalytic activity than their corresponding neutral complexes under similar conditions.

In situ growing of CoO nanoparticles on g-C3N4 composites with highly improved photocatalytic activity for hydrogen evolution.

CoO/g-C3N4 hybrid catalyst is facilely prepared for application to photocatalytic H2 evolution from water splitting by the vacuum rotation-evaporation and in situ thermal method. The physical and chemical properties of CoO/g-C3N4 are determined by a series of characterization methods. The g-C3N4 with 0.6 wt% Co loading exhibits superior photocatalytic hydrogen evolution activity with an H2 evolution amount of 23.25 mmol g-1 after 5 h. The obtained 0.6 wt% CoO/g-C3N4 can split water to generate 0.39 mmol g-1 H2 without sacrificial agent and noble metal, while the pure g-C3N4 is inactive under the same reaction conditions. The remarkable enhancement of photocatalytic H2 evolution activity of CoO/g-C3N4 composites is mainly ascribed to the effective separation of electron-hole pairs and charge transfer. The work creates new opportunities for the design of low-cost g-C3N4-based photocatalysts with high photocatalytic H2 evolution activity from overall water splitting.

Combination of acoustic radiation force impulse imaging, serological indexes and contrast-enhanced ultrasound for diagnosis of liver lesions.

AIM: To assess the value of combined acoustic radiation force impulse (ARFI) imaging, serological indexes and contrast-enhanced ultrasound (CEUS) in distinguishing between benign and malignant liver lesions. METHODS: Patients with liver lesions treated at our hospital were included in this study. The lesions were divided into either a malignant tumor group or a benign tumor group according to pathological or radiological findings. ARFI quantitative detection, serological testing and CEUS quantitative detection were performed and compared. A comparative analysis of the measured indexes was performed between these groups. Receiver operating characteristic (ROC) curves were constructed to compare the diagnostic accuracy of ARFI imaging, serological indexes and CEUS, alone or in different combinations, in identifying benign and malignant liver lesions. RESULTS: A total of 112 liver lesions in 43 patients were included, of which 78 were malignant and 34 were benign. Shear wave velocity (SWV) value, serum alpha-fetoprotein (AFP) content and enhancement rate were significantly higher in the malignant tumor group than in the benign tumor group (2.39 ± 1.20 m/s vs 1.50 ± 0.49 m/s, 18.02 ± 5.01 ng/mL vs 15.96 ± 4.33 ng/mL, 2.14 ± 0.21 dB/s vs 2.01 ± 0.31 dB/s; P < 0.05). The ROC curve analysis revealed that the areas under the curves (AUCs) of SWV value alone, AFP content alone, enhancement rate alone, SWV value + AFP content, SWV value + enhancement rate, AFP content + enhancement rate and SWV value + AFP content + enhancement rate were 85.1%, 72.1%, 74.5%, 88.3%, 90.4%, 82.0% and 92.3%, respectively. The AUC of SWV value + AFP content + enhancement rate was higher than those of SWV value + AFP content and SWV value + enhancement rate, and significantly higher than those of any single parameter or the combination of any two of parameters. CONCLUSION: The combination of SWV, AFP and enhancement rate had better diagnostic performance in distinguishing between benign and malignant liver lesions than the use of any single parameter or the combination of any two of parameters. It is expected that this would provide a tool for the differential diagnosis of benign and malignant liver lesions.

Liver reserve function assessment by acoustic radiation force impulse imaging.

AIM: To evaluate the utility of liver reserve function by acoustic radiation force impulse (ARFI) imaging in patients with liver tumors. METHODS: Seventy-six patients with liver tumors were enrolled in this study. Serum biochemical indexes, such as aminotransferase (ALT), aspartate aminotransferase (AST), serum albumin (ALB), total bilirubin (T-Bil), and other indicators were observed. Liver stiffness (LS) was measured by ARFI imaging, measurements were repeated 10 times, and the average value of the results was taken as the final LS value. Indocyanine green (ICG) retention was performed, and ICG-K and ICG-R15 were recorded. Child-Pugh (CP) scores were carried out based on patient's preoperative biochemical tests and physical condition. Correlations among CP scores, ICG-R15, ICG-K and LS values were observed and analyzed using either the Pearson correlation coefficient or the Spearman rank correlation coefficient. Kruskal-Wallis test was used to compare LS values of CP scores, and the receiver-operator characteristic (ROC) curve was used to analyze liver reserve function assessment accuracy. RESULTS: LS in the ICG-R15 10%-20% group was significantly higher than in the ICG-R15 < 10% group; and the difference was statistically significant (2.19 ± 0.27 vs 1.59 ± 0.32, P < 0.01). LS in the ICG-R15 > 20% group was significantly higher than in the ICG-R15 < 10% group; and the difference was statistically significant (2.92 ± 0.29 vs 1.59 ± 0.32, P < 0.01). The LS value in patients with CP class A was lower than in patients with CP class B (1.57 ± 0.34 vs 1.86 ± 0.27, P < 0.05), while the LS value in patients with CP class B was lower than in patients with CP class C (1.86 ± 0.27 vs 2.47 ± 0.33, P < 0.01). LS was positively correlated with ICG-R15 (r = 0.617, P < 0.01) and CP score (r = 0.772, P < 0.01). Meanwhile, LS was negatively correlated with ICG-K (r = -0.673, P < 0.01). AST, ALT and T-Bil were positively correlated with LS, while ALB was negatively correlated with LS (P < 0.05). The ROC curve revealed that the when the LS value was 2.34 m/s, the Youden index was at its highest point, sensitivity was 69.2% and specificity was 92.1%. CONCLUSION: For patients with liver tumors, ARFI imaging is a useful tool for assessing liver reserve function.