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Abstract Background Cell wall integrity (CWI) is crucial for fungal growth, pathogenesis, and adaptation to extracellular environments. Calcofluor white (CFW) is a cell wall perturbant that inhibits fungal growth, yet little is known about how phytopathogenic fungi respond to the CFW-induced stress. Results In this study, we unveiled a significant discovery that CFW triggered the translocation of the transcription factor CgCrzA from the cytoplasm to the nucleus in Colletotrichum gloeosporioides. This translocation was regulated by an interacting protein, CgMkk1, a mitogen-activated protein kinase involved in the CWI pathway. Further analysis revealed that CgMkk1 facilitated nuclear translocation by phosphorylating CgCrzA at the Ser280 residue. Using chromatin immunoprecipitation sequencing, we identified two downstream targets of CgCrzA, namely CgCHS5 and CgCHS6, which are critical for growth, cell wall integrity, and pathogenicity as chitin synthase genes. Conclusions These findings provide a novel insight into the regulatory mechanism of CgMkk1-CgCrzA-CgChs5/6, which enables response of the cell wall inhibitor CFW and facilitates infectious growth for C. gloeosporioides.

Pinus, a conifer, dominates the world’s forest ecosystems. But it is seriously infected with pine wood nematode (PWN). Transcription factors (TFs) are key regulators in regulating plant resistance. However, the molecular mechanism of TFs remains thus far unresolved in P. thunbergii inoculated with Bursaphelenchus xylophilus. Here, we used RNA-seq technology to identify differentially expressed TFs in resistant and susceptible pines. The results show that a total of 186 differentially expressed transcription factors (DETFs), including 99 upregulated and 87 downregulated genes were identified. Gene ontology (GO) enrichment showed that the highly enriched differentially expressed TFs were responsible for secondary biosynthetic processes. According to KEGG pathway analysis, the differentially expressed TFs were related to chaperones and folding catalysts, phenylpropanoid biosynthesis, and protein processing in the endoplasmic reticulum. Many TFs such as NAC, LBD, MYB, bHLH, and WRKY were determined to be quite abundant in the DETFs. Moreover, the NAC transcription factor PtNAC9 was upregulated in PWN-resistant and susceptible P. thunbergii and especially distinctly upregulated in resistant pines. By purifying recombinant PtNAC9 protein in vitro, we found that overexpression of PtNAC9 at the early stage of B. xylophilus infection could reduce the degree of disease. We also demonstrated the content of salicylic acid (SA) and the related genes were increased in the PtNAC9 protein-treated plants. These results could be helpful in enhancing our understanding of the resistance mechanism underlying different resistant pine.

Pinus is an important economic tree species, but pine wilt disease (PWD) seriously threatens the survival of pine trees. PWD caused by Bursaphelenchus xylophilus is a major quarantine disease worldwide that causes significant economic losses. However, more information about its molecular pathogenesis is needed, resulting in a lack of effective prevention and treatment measures. In recent years, effectors have become a hot topic in exploring the molecular pathogenic mechanism of pathogens. Here, we identified a specific effector, BxNMP1, from B. xylophilus. In situ hybridization experiments revealed that BxNMP1 was specifically expressed in dorsal gland cells and intestinal cells, and RT–qPCR experiments revealed that BxNMP1 was upregulated in the early stage of infection. The sequence of BxNMP1 was different in the avirulent strain, and when BxNMP1-silenced B. xylophilus was inoculated into P. thunbergii seedlings, the disease severity significantly decreased. We demonstrated that BxNMP1 interacted with the thaumatin-like protein PtTLP-L2 in P. thunbergii. Additionally, we found that the β-1,3-glucanase PtGLU interacted with PtTLP-L2. Therefore, we hypothesized that BxNMP1 might indirectly interact with PtGLU through PtTLP-L2 as an intermediate mediator. Both targets can respond to infection, and PtTLP-L2 can enhance the resistance of pine trees. Moreover, we detected increased salicylic acid contents in P. thunbergii seedlings inoculated with B. xylophilus when BxNMP1 was silenced or when the PtTLP-L2 recombinant protein was added. In summary, we identified a key virulence effector of PWNs, BxNMP1. It positively regulates the pathogenicity of B. xylophilus and interacts directly with PtTLP-L2 and indirectly with PtGLU. It also inhibits the expression of two targets and the host salicylic acid pathway. This study provides theoretical guidance and a practical basis for controlling PWD and breeding for disease resistance.

Pine wilt disease, caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus), is a major quarantine forest disease that poses a threat to various pine species, including Pinus massoniana (masson pine), worldwide. Breeding of PWN-resistant pine trees is an important approach to prevent the disease. To expedite the production of PWN-resistant P. massoniana accessions, we investigated the effects of maturation medium treatments on somatic embryo development, germination, survival, and rooting. Furthermore, we evaluated the mycorrhization and nematode resistance of regenerated plantlets. Abscisic acid was identified as the main factor affecting maturation, germination, and rooting of somatic embryos in P. massoniana, resulting in a maximum of 34.9 ± 9.4 somatic embryos per ml, 87.3 ± 9.1% germination rate, and 55.2 ± 29.3% rooting rate. Polyethylene glycol was identified as the main factor affecting the survival rate of somatic embryo plantlets, with a survival rate of up to 59.6 ± 6.8%, followed by abscisic acid. Ectomycorrhizal fungi inoculation with Pisolithus orientalis enhanced the shoot height of plantlets regenerated from embryogenic cell line (ECL) 20-1-7. Ectomycorrhizal fungi inoculation also improved the survival rate of plantlets during the acclimatization stage, with 85% of mycorrhized plantlets surviving four months after acclimatization in the greenhouse, compared with 37% non-mycorrhized plantlets. Following PWN inoculation, the wilting rate and the number of nematodes recovered from ECL 20-1-7 were lower than those recovered from ECL 20-1-4 and 20-1-16. The wilting ratios of mycorrhizal plantlets from all cell lines were significantly lower than those of non-mycorrhizal regenerated plantlets. This plantlet regeneration system and mycorrhization method could be used in the large-scale production of nematode-resistance plantlets and to study the interaction between nematode, pines, and mycorrhizal fungi.

The migratory plant-parasitic nematode Bursaphelenchus xylophilus is the pathogen of the pine wilt disease (PWD), causing serious damage to pine forests in China. During the process of plant resistance to multiple pathogens, plant immunity plays a key role. In this current study, the pathogen-associated molecular pattern (PAMP) BxCDP1 in B. xylophilus has been identified, but the host target protein of BxCDP1 and its key amino acid region inducing the plant immunity have yet to be elucidated. We found that BxCDP1 could trigger superoxide production, H2O2 production, and callose deposits. A RING-H2 finger protein 1 (RHF1) of Pinus thunbergii was screened and characterized as a target protein of BxCDP1 by yeast two-hybrid and co-immunoprecipitation (Co-IP). Moreover, two peptides (namely M9 and M16) proved to be key regions of BxCDP1 to induce PAMP-triggered immunity (PTI) in Nicotiana benthamiana, which also induced the expression of pathogenesis-related (PR) genes (PtPR-3, PtPR-4, and PtPR-5) in P. thunbergii and enhanced the resistance of the host to B. xylophilus. These results indicate that BxCDP1 plays a critical role in the interaction between B. xylophilus and P. thunbergii, and both peptides M9 and M16 have the potential to be developed and utilized as immune inducers of pines against B. xylophilus in future.

Abstract Background The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a devastating pathogen of many Pinus species in China. The aim of this study was to understand the interactive molecular mechanism of PWN and its host by comparing differentially expressed genes and candidate effectors from three transcriptomes of B. xylophilus at different infection stages. Results In total, 62, 69 and 46 candidate effectors were identified in three transcriptomes (2.5 h postinfection, 6, 12 and 24 h postinoculation and 6 and 15 d postinfection, respectively). In addition to uncharacterized pioneers, other candidate effectors were involved in the degradation of host tissues, suppression of host defenses, targeting plant signaling pathways, feeding and detoxification, which helped B. xylophilus survive successfully in the host. Seven candidate effectors were identified in both our study and the B. xylophilus transcriptome at 2.5 h postinfection, and one candidate effector was identified in all three transcriptomes. These common candidate effectors were upregulated at infection stages, and one of them suppressed pathogen-associated molecular pattern (PAMP) PsXEG1-triggered cell death in Nicotiana benthamiana. Conclusions The results indicated that B. xylophilus secreted various candidate effectors, and some of them continued to function throughout all infection stages. These various candidate effectors were important to B. xylophilus infection and survival, and they functioned in different ways (such as breaking down host cell walls, suppressing host defenses, promoting feeding efficiency, promoting detoxification and playing virulence functions). The present results provide valuable resources for in-depth research on the pathogenesis of B. xylophilus from the perspective of effectors.

Selecting suitable promoters to drive gene overexpression can provide significant insight into the development of engineered bacteria. In this study, we analyzed the transcriptome data of Burkholderia pyrrocinia JK-SH007 and identified 54 highly expressed genes. The promoter sequences were located using genome-wide data and scored using the prokaryotic promoter prediction software BPROM to further screen out 18 promoter sequences. We also developed a promoter trap system based on two reporter proteins adapted for promoter optimization in B. pyrrocinia JK-SH007: firefly luciferase encoded by the luciferase gene set (Luc) and trimethoprim (TP)-resistant dihydrofolate reductase (TPr). Ultimately, eight constitutive promoters were successfully inserted into the probe vector and transformed into B. pyrrocinia JK-SH007. The transformants were successfully grown on Tp antibiotic plates, and firefly luciferase expression was determined by measuring the relative light unit (RLU). Five of the promoters (P4, P9, P10, P14, and P19) showed 1.01–2.51-fold higher activity than the control promoter λ phage transcriptional promoter (PRPL). The promoter activity was further validated via qPCR analysis, indicating that promoters P14 and P19 showed stable high transcription levels at all time points. Then, GFP and RFP proteins were overexpressed in JK-SH007. In addition, promoters P14 and P19 were successfully used to drive gene expression in Burkholderia multivorans WS-FJ9 and Escherichia coli S17-1. The two constitutive promoters can be used not only in B. pyrrocinia JK-SH007 itself to gene overexpression but also to expand the scope of application.

IntroductionFor patients with epidermal growth factor receptor (EGFR)-mutated lung cancer who undergo surgery, adjuvant tyrosine kinase inhibitor (TKI) therapy other than osimertinib is an alternative option. We aimed to discuss the long-term safety and efficacy of TKI treatment in real-world data.MethodsFrom January 2011 to May 2020, patients with stage II–III EGFR-mutated adenocarcinoma who underwent cancer resection surgery at a single center were enrolled. The primary endpoint was disease relapse, and the secondary endpoint was overall survival. In total, 30 patients were included in the study. In our study, all patients underwent complete resection using video-assisted thoracoscopic surgery. The patients were divided into a dose interruption (prolonged interval use) group and non-dose adjustment group.ResultsThe patients' pathological stages were II–III. The initial EGFR TKIs were mostly gefitinib (n = 25, 83%), and others were erlotinib (n = 3, 10%) and afatinib (n = 2, 6%). The mean disease-free survival (DFS) was 53.3 months. The 2- and 5-year DFS rate was 90.0 and 73.3%, respectively. The median TKI treatment duration in this study was 44.5 months (range, 6–133 months), which was the longest in the literature review. Of these patients, nine had dose interruption. We compared the two groups and found no treatment differences between them. There were no significant side effect potentials between both groups.ConclusionTo our knowledge, this study provides the longest experience of TKI in patients with resected EGFR mutations and also provided a dose reduction strategy (prolonged medication interval) for patients who had intolerable side effects.

Soil salinity is one of the main factors limiting agricultural development worldwide and has an adverse effect on plant growth and yield. To date, plant growth-promoting rhizobacteria (PGPR) are considered to be one of the most promising eco-friendly strategies for improving saline soils. The bacterium Bacillus megaterium ZS-3 is an excellent PGPR strain that induces growth promotion as well as biotic stress resistance and tolerance to abiotic stress in a broad range of host plants. In this study, the potential mechanisms of protection against salinity stress by B. megaterium ZS-3 in Arabidopsis thaliana were explored. Regulation by ZS-3 improved growth in A. thaliana under severe saline conditions. The results showed that ZS-3 treatment significantly increased the biomass, chlorophyll content and carotenoid content of A. thaliana. Compared to the control, the leaf area and total fresh weight of plants inoculated with ZS-3 increased by 245% and 271%, respectively; the chlorophyll a, chlorophyll b, and carotenoid contents increased by 335%, 146%, and 372%, respectively, under salt stress. Physiological and biochemical tests showed that ZS-3 regulated the content of osmotic substances in plants under salt stress. Compared to the control, the soluble sugar content of the ZS-3-treated group was significantly increased by 288%, while the proline content was significantly reduced by 41.43%. Quantification of Na+ and K+ contents showed that ZS-3 treatment significantly reduced Na+ accumulation and increased the K+/Na+ ratio in plants. ZS-3 also isolated Na+ in vesicles by upregulating NHX1 and AVP1 expression while limiting Na+ uptake by downregulating HKT1, which protected against Na+ toxicity. Higher levels of peroxidase and catalase activity and reduced glutathione were detected in plants inoculated with ZS-3 compared to those in uninoculated plants. In addition, it was revealed that ZS-3 activates salicylic acid (NPR1 and PR1) and jasmonic acid/ethylene (AOS, LOX2, PDF1.2, and ERF1) signaling pathways to induce systemic tolerance, thereby inducing salt tolerance in plants. In conclusion, the results of this study indicate that ZS-3 has the potential to act as an environmentally friendly salt tolerance inducer that can promote plant growth in salt-stressed environments.

Abstract Background Insomnia seriously affects people’s normal lives and work. However, effective treatment strategies are scarce. The purpose of this study is to explore the efficacy and safety of Jiao-tai-wan (JTW) for ameliorating insomnia symptoms caused by disharmony of the heart and kidney. Design This is a randomized, double-blind, placebo-controlled pilot clinical trial. A total of 124 participants suffering from insomnia symptoms will be randomly assigned to the JTW or placebo group in an equal ratio. The participants will be asked to take JTW or placebo granules twice a day for 1 week. All data will be gathered at baseline and at the end of the drug intervention. The primary outcome measures will be the mean change in the Pittsburgh Sleep Quality Index (PSQI) from baseline to the end of the drug intervention. Secondary outcome measures will include the altered sleep parameters in polysomnography, 1H-magnetic resonance spectroscopy (1H-MRS) evaluation, the Disharmony of Heart and Kidney Scoring System score, and blood tests, including the levels of serum adenosine and melatonin. A laboratory test will be taken before and after treatment to assess the safety of JTW. Discussion The outcomes of this study will confirm the efficacy of JTW for the treatment of insomnia symptoms and will also be used to monitor the safety of JTW. Trial registration Chinese Clinical Trial Registry, ChiCTR1 800019239 . Registered on 1st November 2018.

Objective To compare the effectiveness of different approaches of neuraxial anesthesia in parturient women with obesity and pregnancy-induced hypertension (PIH) who undergo cesarean section (CS). Methods We retrospectively analyzed data from 108 parturient women with obesity and PIH who underwent CS. All women were divided into the following three groups according to the neuraxial anesthesia approach: spinal anesthesia (SA), epidural anesthesia (EA), and combined spinal–epidural anesthesia (CSE). Clinical variables were compared. Results The mean age of the patients was 27.3 ± 2.2 years. Women in the CSE group had a longer duration from puncture to surgery, smaller intraoperative change in mean arterial pressure, higher Apgar scores at 1 and 5 minutes, shorter surgery time, lower rates of nausea and vomiting, and lower rate of intraoperative hypotension compared with those in the SA and EA groups. Conclusion CSE takes longer to administer in parturient women with obesity and PIH who undergo CS compared with those who have SA or EA. However, CSE has several advantages over SA or EA, including a shorter surgery time, more stable intraoperative mean arterial pressure, lower rates of nausea, vomiting, and intraoperative hypotension, and better Apgar scores at 1 and 5 minutes.

The pinewood nematode, Bursaphelenchus xylophilus, has been determined as one of the world’s top ten plant-parasitic nematodes. It causes pine wilt, a progressive disease that affects the economy and ecologically sustainable development in East Asia. B. xylophilus secretes pathogenic proteins into host plant tissues to promote infection. However, little is known about the interaction between B. xylophilus and pines. Previous studies reported transthyretin proteins in some species and their strong correlation with immune evasion, which has also been poorly studied in B. xylophilus. In this study, we cloned and functionally validated the B. xylophilus pathogenic protein BxTTR-52, containing a transthyretin domain. An in situ hybridization assay demonstrated that BxTTR-52 was expressed mainly in the esophageal glands of B. xylophilus. Confocal microscopy revealed that BxTTR-52-RFP localized to the nucleus, cytoplasm, and plasma membrane. BxTTR-52 recombinant proteins produced by Escherichia coli could be suppressed by hydrogen peroxide and antioxidant enzymes in pines. Moreover, silencing BxTTR-52 significantly attenuated the morbidity of Pinus thunbergii infected with B. xylophilus. It also suppressed the expression of pathogenesis-related genes in P. thunbergii. These results suggest that BxTTR-52 suppresses the plant immune response in the host pines and might contribute to the pathogenicity of B. xylophilus in the early infection stages.

Abstract Objectives We aimed to test our expectation that additional administration of Traditional Chinese medicine (TCM), maxingshigan-weijing decoction, is more effective in the management of COVID-19 patients compared to those treated with routine supportive care alone. Trial design This is a multicenter, open-label 2-arm (1:1 ratio) randomized controlled trial. Participants Patients will be recruited from 3 hospitals in Wenzhou China: the First Affiliated Hospital of Wenzhou Medical University, the Second Affiliated Hospital of Wenzhou Medical University and Wenzhou Center Hospital. The inclusion and exclusion criteria are as follows: Inclusion criteria 1. Participants are 18-85 years of age, either male or female. 2. Diagnosed as positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3. Symptomatic. Mild (mild clinical symptoms without signs of pneumonia in chest X-ray) and Moderate (fever or respiratory symptom with signs of pneumonia in chest X-ray) . 1. Signed the informed consent before treatment. 2. Agreed not to enroll in any other clinical trials. 3. Inpatients Exclusion criteria 1. < 18 or > 85 years old. 2. Pregnancy and lactation. 3. Serious heart, liver, kidney and hematopoietic system diseases, abnormal liver or kidney function. 4. Suffering from other known virus pneumonia. 5. Allergic to Chinese herbal medicine or suffering from allergies. 6. Critical patients (respiratory failure treated by mechanical ventilation or shock or multiple organ failure). Intervention and comparator Patients in the control group will receive routine supportive clinically care including the therapies of anti-viral, anti-bacterial and ameliorating the related symptoms, while patients in TCM group will be asked to take maxingshigan-weijing decoction (composed of 14 Chinese herbal medicines), orally 200 mL 2 times daily, for 14 consecutive days in addition to routine supportive care as mentioned above. Maxingshigan-weijing decoction consists of 10 g of Herba Ephedra (Mahuang), 10 g of Amygdalus Communis Vas (Xingren), 45 g of Gypsum Fibrosum (Shigao), 30 g of Rhizoma phragmitis (Lugen), 20 g of Peach kernel (Taoren), 20 g of Winter Melon kernel (Dongguaren), 30 g of Trichosanthes Kirilowii Maxim (Gualou), 12 g of Pericarpium Citri Reticulatae (Chenpi), 12 g of Rhizoma Pinelliae (Jiangbanxia), 12 g of caulis bambusae in taeniis (Zhuru), 30 g of semen lepidii (Tingliz), 15 g of semen lepidii (Shichangpu), 10 g of curcuma zedoary (ezhu) and 5 g of Radix Glycyrrhizae (Gancao). Main outcomes The primary outcome will be the number of days until the clinical symptom of fever improves in the first 14 days of treatment following randomisation. Fever will be defined as an improvement when the temperature is less than 37°C. Secondary outcomes will be TCM Syndrome Scores, the time it takes until individuals have negative test results for SARS-CoV-2 nucleic acid, the proportion of cases with chest X-ray improvements and the rate of symptom (fever, cough, malaise, shortness of breath) recovery. TCM Syndrome Scoring System is a checklist covering 4 main, 7 secondary and 13 accompanying items. The 4 main items consisting of fever, cough, malaise and shortness of breath, use a four-point scale (0, 2, 4 and 6) depending on the severity; the 7 secondary items including dysphoria, diarrhea, pharyngalgia, expectoration, muscular soreness, nasal obstruction and rhinorrhoea use 0-3-point scale; the 13 accompanying items contain chest pain, headache, aversion to cold, dizziness, nausea and vomiting, anorexia, abdominal distension, dry mouth, anxiety, spontaneous sweating, insomnia, wheezing and blood tinged sputum, and each item is rated on 0-1 scale ( 0 stands for asymptomatic, 1 stands for symptomatic ). The total scores sum up to a range from 0 to 58, with higher scores indicating more severe levels of disease. Randomization Minimization method will be used, balancing the two arms for pneumonia severity. Patients are randomized (1:1 ratio) to each group. Clinical researchers will get a random sequence number which is automatically generated by a random number generator (IBM Corp., Armonk, NY, USA), and sequentially number them in an opaque envelope. Researchers will open random allocation envelopes and assign participants accordingly. Eligible patients will be randomly divided into a routine supportive care group and a routine supportive care plus oral administration of traditional Chinese medicine group, with 70 patients in each group. Blinding (masking) This is an open-label study. The statistical analysis will be carried out by the Professor of Statistics at Wenzhou Medical University, who is blinded to patient allocation. Numbers to be randomised (sample size) The previous study reported the efficacy of TCM for COVID-19 and H1N1 influenza patients, the median survival time in the TCM group is estimated as 3 days; this time will be 1.5 times longer in the control group. Accordingly, Kaplan-Meier method and log-rank test will be used. And assuming a statistical power of 70% (one-sided type-1 error of α = 5%, β = 30%) and a rate of withdrawal and loss to follow-up of 10%, we plan to include 140 participants in both groups ( TCM group = 70, control group = 70). Trial Status The trial protocol is Version 2.0, October 14, 2020. Recruitment began March, 2020, and is anticipated to be completed by December 31, 2020. Trial registration Chinese Clinical Trial Registry, ChiCTR2000030759 . Registered on 13 March 2020. Full protocol The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Chordae tendineae, referred to as heart tendinous cords, act as tendons connecting the papillary muscles to the valves in the heart. Their role is analogous to tendons in the musculoskeletal system. Despite being exposed to millions of cyclic tensile stretches over a human's lifetime, chordae tendineae rarely suffer from overuse injuries. On the other hand, musculoskeletal tendinopathy is very common and remains challenging in clinical treatment. The objective of this study was to investigate the mechanism behind the remarkable durability and resistance to overuse injuries of chordae tendineae, as well as to explore their effects on flexor tenocyte biology. The messenger RNA expression profiles of chordae tendineae were analyzed using RNA sequencing and verified by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. Interestingly, we found that periostin (Postn) and fibroblast growth factor 7 (FGF7) were expressed at significantly higher levels in chordae tendineae, compared to flexor tendons. We further treated flexor tenocytes in vitro with periostin and FGF7 to examine their effects on the proliferation, migration, apoptosis, and tendon‐related gene expression of flexor tenocytes. The results displayed enhanced cell proliferation ability at an early stage and an antiapoptotic effect on tenocytes, while treated with periostin and/or FGF7 proteins. Furthermore, there was a trend of promoted tenocyte migration capability. These findings indicated that Postn and FGF7 may represent novel cytokines to target flexor tendon healing. Clinical significance: The preliminary discovery leads to a novel idea for treating tendinopathy in the musculoskeletal system using specific molecules identified from chordae tendineae. [ABSTRACT FROM AUTHOR]