DYRK1A is a multifunctional host factor that regulates coronavirus replication in a kinase-independent manner.

Coronaviruses (CoVs) pose a major threat to human and animal health worldwide, which complete viral replication by hijacking host factors. Identifying host factors essential for the viral life cycle can deepen our understanding of the mechanisms of virus-host interactions. Based on our previous genome-wide CRISPR screen of α-CoV transmissible gastroenteritis virus (TGEV), we identified the host factor dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), but not DYRK1B, as a critical factor in TGEV replication. Rescue assays and kinase inhibitor experiments revealed that the effect of DYRK1A on viral replication is independent of its kinase activity. Nuclear localization signal modification experiments showed that nuclear DYRK1A facilitated virus replication. Furthermore, DYRK1A knockout significantly downregulated the expression of the TGEV receptor aminopeptidase N (ANPEP) and inhibited viral entry. Notably, we also demonstrated that DYRK1A is essential for the early stage of TGEV replication. Transmission electron microscopy results indicated that DYRK1A contributes to the formation of double-membrane vesicles in a kinase-independent manner. Finally, we validated that DYRK1A is also a proviral factor for mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. In conclusion, our work demonstrated that DYRK1A is an essential host factor for the replication of multiple viruses, providing new insights into the mechanism of virus-host interactions and facilitating the development of new broad-spectrum antiviral drugs.IMPORTANCECoronaviruses, like other positive-sense RNA viruses, can remodel the host membrane to form double-membrane vesicles (DMVs) as their replication organelles. Currently, host factors involved in DMV formation are not well defined. In this study, we used transmissible gastroenteritis virus (TGEV) as a virus model to investigate the regulatory mechanism of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) on coronavirus. Results showed that DYRK1A significantly inhibited TGEV replication in a kinase-independent manner. DYRK1A knockout (KO) can regulate the expression of receptor aminopeptidase N (ANPEP) and endocytic-related genes to inhibit virus entry. More importantly, our results revealed that DYRK1A KO notably inhibited the formation of DMV to regulate the virus replication. Further data proved that DYRK1A is also essential in the replication of mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. Taken together, our findings demonstrated that DYRK1A is a conserved factor for positive-sense RNA viruses and provided new insights into its transcriptional regulation activity, revealing its potential as a candidate target for therapeutic design.

Novel mutation N588 residue in the NS1 protein of feline parvovirus greatly augments viral replication.

Feline parvovirus (FPV) infection is highly fatal in felines. NS1, which is a key nonstructural protein of FPV, can inhibit host innate immunity and promote viral replication, which is the main reason for the severe pathogenicity of FPV. However, the mechanism by which the NS1 protein disrupts host immunity and regulates viral replication is still unclear. Here, we identified an FPV M1 strain that is regulated by the NS1 protein and has more pronounced suppression of innate immunity, resulting in robust replication. We found that the neutralization titer of the FPV M1 strain was significantly lower than that of the other strains. Moreover, FPV M1 had powerful replication ability, and the FPV M1-NS1 protein had heightened efficacy in repressing interferon-stimulated genes (ISGs) expression. Subsequently, we constructed an FPV reverse genetic system, which confirmed that the N588 residue of FPV M1-NS1 protein is a key amino acid that bolsters viral proliferation. Recombinant virus containing N588 also had stronger ability to inhibit ISGs, and lower ISGs levels promoted viral replication and reduced the neutralization titer of the positive control serum. Finally, we confirmed that the difference in viral replication was abolished in type I IFN receptor knockout cell lines. In conclusion, our results demonstrate that the N588 residue of the NS1 protein is a critical amino acid that promotes viral proliferation by increasing the inhibition of ISGs expression. These insights provide a reference for studying the relationship between parvovirus-mediated inhibition of host innate immunity and viral replication while facilitating improved FPV vaccine production.IMPORTANCEFPV infection is a viral infectious disease with the highest mortality rate in felines. A universal feature of parvovirus is its ability to inhibit host innate immunity, and its ability to suppress innate immunity is mainly accomplished by the NS1 protein. In the present study, FPV was used as a viral model to explore the mechanism by which the NS1 protein inhibits innate immunity and regulates viral replication. Studies have shown that the FPV-NS1 protein containing the N588 residue strongly inhibits the expression of host ISGs, thereby increasing the viral proliferation titer. In addition, the presence of the N588 residue can increase the proliferation titer of the strain 5- to 10-fold without affecting its virulence and immunogenicity. In conclusion, our findings provide new insights and guidance for studying the mechanisms by which parvoviruses suppress innate immunity and for developing high-yielding FPV vaccines.

Drug screening identified that handelin inhibits feline calicivirus infection by inhibiting HSP70 expression in vitro.

Feline calicivirus (FCV) is considered one of the major pathogens of cats worldwide and causes upper respiratory tract disease in all cats. In some cats, infection is by a highly virulent strain of FCV (vs.-FCV), which can cause severe and fatal systemic disease symptoms. At present, few antiviral drugs are approved for clinical treatment against FCV. Therefore, there is an imminent need for effective FCV antiviral agents. Here, we used observed a cytopathic effect (CPE) assay to screen 1746 traditional Chinese medicine monomer compounds and found one that can effectively inhibit FCV replication, namely, handelin, with an effective concentration (EC50) value of approximately 2.5 µM. Further study showed that handelin inhibits FCV replication via interference with heat shock protein 70 (HSP70), which is a crucial host factor and plays a positive role in regulating viral replication. Moreover, handelin and HSP70 inhibitors have broad-spectrum antiviral activity. These findings indicate that handelin is a potential candidate for the treatment of FCV infection and that HSP70 may be an important drug target.

Phase I/II clinical trial of efficacy and safety of EGCG oxygen nebulization inhalation in the treatment of COVID-19 pneumonia patients with cancer.

BACKGROUND: The antiviral drug Nirmatrelvir was found to be a key drug in controlling the progression of pneumonia during the infectious phase of COVID-19. However, there are very few options for effective treatment for cancer patients who have viral pneumonia. Glucocorticoids is one of the effective means to control pneumonia, but there are many adverse events. EGCG is a natural low toxic compound with anti-inflammatory function. Thus, this study was designed to investigate the safety and efficacy of epigallocatechin-3-gallate (EGCG) aerosol to control COVID-19 pneumonia in cancer populations. METHODS: The study was designed as a prospective, single-arm, open-label phase I/II trial at Shandong Cancer Hospital and Institute, between January 5, 2023 to March 31,2023 with viral pneumonia on radiographic signs after confirmed novel coronavirus infection. These patients were treated with EGCG nebulization 10 ml three times daily for at least seven days. EGCG concentrations were increased from 1760-8817umol/L to 4 levels with dose escalation following a standard Phase I design of 3-6 patients per level. Any grade adverse event caused by EGCG was considered a dose-limiting toxicity (DLT). The maximum tolerated dose (MTD) is defined as the highest dose with less than one-third of patients experiencing dose limiting toxicity (DLT) due to EGCG. The primary end points were the toxicity of EGCG and CT findings, and the former was graded by Common Terminology Criteria for Adverse Events (CTCAE) v. 5.0. The secondary end point was the laboratory parameters before and after treatment. RESULT: A total of 60 patients with high risk factors for severe COVID-19 pneumonia (factors such as old age, smoking and combined complications)were included in this phase I-II study. The 54 patients in the final analysis were pathologically confirmed to have tumor burden and completed the whole course of treatment. A patient with bucking at a level of 1760 umol/L and no acute toxicity associated with EGCG has been reported at the second or third dose gradients. At dose escalation to 8817umol/L, Grade 1 adverse events of nausea and stomach discomfort occurred in two patients, which resolved spontaneously within 1 hour. After one week of treatment, CT showed that the incidence of non-progression of pneumonia was 82% (32/39), and the improvement rate of pneumonia was 56.4% (22/39). There was no significant difference in inflammation-related laboratory parameters (white blood cell count, lymphocyte count, IL-6, ferritin, C-reactive protein and lactate dehydrogenase) before and after treatment. CONCLUSION: Aerosol inhalation of EGCG is well tolerated, and preliminary investigation in cancer population suggests that EGCG may be effective in COVID-19-induced pneumonia, which can promote the improvement of patients with moderate pneumonia or prevent them from developing into severe pneumonia. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05758571. Date of registration: 8 February 2023.

The CDE region of feline Calicivirus VP1 protein is a potential candidate subunit vaccine.

BACKGROUND: Feline calicivirus (FCV) infection causes severe upper respiratory disease in cats, but there are no effective vaccines available for preventing FCV infection. Subunit vaccines have the advantages of safety, low cost and excellent immunogenicity, but no FCV subunit vaccine is currently available. The CDE protein is the dominant neutralizing epitope region of the main antigenic structural protein of FCV, VP1. Therefore, this study evaluated the effectiveness of the CDE region as a truncated FCV VP1 protein in preventing FCV infection to provide a strategy for developing potential FCV subunit vaccines. RESULTS: Through the prediction of FCV VP1 epitopes, we found that the E region is the dominant neutralizing epitope region. By analysing the spatial structure of VP1 protein, 13 amino acid sites in the CD and E regions were found to form hydrogen bonding interactions. The results show the presence of these interaction forces supports the E region, helping improve the stability and expression level of the soluble E protein. Therefore, we selected the CDE protein as the immunogen for the immunization of felines. After immunization with the CDE protein, we found significant stimulation of IgG, IgA and neutralizing antibody production in serum and swab samples, and the cytokine TNF-α levels and the numbers of CD4+ T lymphocytes were increased. Moreover, a viral challenge trial indicated that the protection generated by the CDE subunit vaccine significantly reduced the incidence of disease in animals. CONCLUSIONS: For the first time, we studied the efficacy of the CDE protein, which is the dominant neutralizing epitope region of the FCV VP1 protein, in preventing FCV infection. We revealed that the CDE protein can significantly activate humoral, mucosal and cellular immunity, and the resulting protective effect can significantly reduce the incidence of animal disease. The CDE region of the FCV capsid is easy to produce and has high stability and excellent immunogenicity, which makes it a candidate for low-cost vaccines.

Head-to-head comparison of perfluorobutane contrast-enhanced US and multiparametric MRI for breast cancer: a prospective, multicenter study.

BACKGROUND: Multiparametric magnetic resonance imaging (MP-MRI) has high sensitivity for diagnosing breast cancers but cannot always be used as a routine diagnostic tool. The present study aimed to evaluate whether the diagnostic performance of perfluorobutane (PFB) contrast-enhanced ultrasound (CEUS) is similar to that of MP-MRI in breast cancer and whether combining the two methods would enhance diagnostic efficiency. PATIENTS AND METHODS: This was a head-to-head, prospective, multicenter study. Patients with breast lesions diagnosed by US as Breast Imaging Reporting and Data System (BI-RADS) categories 3, 4, and 5 underwent both PFB-CEUS and MP-MRI scans. On-site operators and three reviewers categorized the BI-RADS of all lesions on two images. Logistic-bootstrap 1000-sample analysis and cross-validation were used to construct PFB-CEUS, MP-MRI, and hybrid (PFB-CEUS + MP-MRI) models to distinguish breast lesions. RESULTS: In total, 179 women with 186 breast lesions were evaluated from 17 centers in China. The area under the receiver operating characteristic curve (AUC) for the PFB-CEUS model to diagnose breast cancer (0.89; 95% confidence interval [CI] 0.74, 0.97) was similar to that of the MP-MRI model (0.89; 95% CI 0.73, 0.97) (P = 0.85). The AUC of the hybrid model (0.92, 95% CI 0.77, 0.98) did not show a statistical advantage over the PFB-CEUS and MP-MRI models (P = 0.29 and 0.40, respectively). However, 90.3% false-positive and 66.7% false-negative results of PFB-CEUS radiologists and 90.5% false-positive and 42.8% false-negative results of MP-MRI radiologists could be corrected by the hybrid model. Three dynamic nomograms of PFB-CEUS, MP-MRI and hybrid models to diagnose breast cancer are freely available online. CONCLUSIONS: PFB-CEUS can be used in the differential diagnosis of breast cancer with comparable performance to MP-MRI and with less time consumption. Using PFB-CEUS and MP-MRI as joint diagnostics could further strengthen the diagnostic ability. Trial registration Clinicaltrials.gov; NCT04657328. Registered 26 September 2020. IRB number 2020-300 was approved in Chinese PLA General Hospital. Every patient signed a written informed consent form in each center.

Adaptive Mutation in the Main Protease Cleavage Site of Feline Coronavirus Renders the Virus More Resistant to Main Protease Inhibitors.

The rapid global emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused serious health problems, highlighting the urgent need for antiviral drugs. The viral main protease (Mpro) plays an important role in viral replication and thus remains the target of choice for the prevention or treatment of several viral diseases due to high sequence and structural conservation. Prolonged use of viral protease inhibitors can lead to the development of mutants resistant to those inhibitors and to many of the available antiviral drugs. Here, we used feline infectious peritonitis virus (FIPV) as a model to investigate its development of resistance under pressure from the Mpro inhibitor GC376. Passage of wild-type (WT) FIPV in the presence of GC376 selected for a mutation in the nsp12 region where Mpro cleaves the substrate between nsp12 and nsp13. This mutation confers up to 3-fold resistance to GC376 and nirmatrelvir, as determined by EC50 assay. In vitro biochemical and cellular experiments confirmed that FIPV adapts to the stress of GC376 by mutating the nsp12 and nsp13 hydrolysis site to facilitate cleavage by Mpro and release to mediate replication and transcription. Finally, we demonstrate that GC376 cannot treat FIP-resistant mutants that cause FIP in animals. Taken together, these results suggest that Mpro affects the replication of coronaviruses (CoVs) and the drug resistance to GC376 by regulating the amount of RdRp from a distant site. These findings provide further support for the use of an antiviral drug combination as a broad-spectrum therapy to protect against contemporary and emerging CoVs. IMPORTANCE CoVs cause serious human infections, and antiviral drugs are currently approved to treat these infections. The development of protease-targeting therapeutics for CoV infection is hindered by resistance mutations. Therefore, we should pay attention to its resistance to antiviral drugs. Here, we identified possible mutations that lead to relapse after clinical treatment of FIP. One amino acid substitution in the nsp12 polymerase at the Mpro cleavage site provided low-level resistance to GC376 after selection exposure to the GC376 parental nucleoside. Resistance mutations enhanced FIPV viral fitness in vitro and attenuated the therapeutic effect of GC376 in an animal model of FIPV infection. Our research explains the evolutionary characteristics of coronaviruses under antiviral drugs, which is helpful for a more comprehensive understanding of the molecular basis of virus resistance and provides important basic data for the effective prevention and control of CoVs.

Effects of plaque characteristics and artery hemodynamics on the residual stenosis after carotid artery stenting.

OBJECTIVE: Carotid artery stenting (CAS) has become an alternative strategy to carotid endarterectomy for carotid artery stenosis. Residual stenosis was an independent risk factor for restenosis, with the latter affecting the long-term outcomes of CAS. This multicenter study aimed to evaluate the echogenicity of plaques and hemodynamic alteration by color duplex ultrasound (CDU) examination and investigate their effects on the residual stenosis after CAS. METHODS: From June 2018 to June 2020, 454 patients (386 males and 68 females) with a mean age of 67.2 ± 7.9 years, who underwent CAS from 11 advanced stroke centers in China were enrolled. One week before recanalization, CDU was used to evaluate the responsible plaques, including the morphology (regular or irregular), echogenicity of the plaques (iso-, hypo-, or hyperechoic) and calcification characteristics (without calcification, superficial calcification, inner calcification, and basal calcification). One week after CAS, the alteration of diameter and hemodynamic parameters were evaluated by CDU, and the occurrence and degree of residual stenosis were determined. In addition, magnetic resonance imaging was performed before and during the 30-day postprocedural period to identify new ischemic cerebral lesions. RESULTS: The rate of composite complications, including cerebral hemorrhage, symptomatic new ischemic cerebral lesions, and death after CAS, was 1.54% (7/454 cases). The rate of residual stenosis after CAS was 16.3% (74/454 cases). After CAS, both the diameter and peak systolic velocity (PSV) improved in the preprocedural 50% to 69% and 70% to 99% stenosis groups (P < .05). Compared with the groups without residual stenosis and with <50% residual stenosis, the PSV of all three segments of stent in the 50% to 69% residual stenosis group were the highest, and the difference in the midsegment of stent PSV was the largest (P < .05). Logistic regression analysis showed that preprocedural severe (70% to 99%) stenosis (odds ratio [OR], 9.421; P = .032), hyperechoic plaques (OR, 3.060; P = .006) and plaques with basal calcification (OR, 1.885; P = .049) were independent risk factors for residual stenosis after CAS. CONCLUSIONS: Patients with hyperechoic and calcified plaques of the carotid stenosis are at a high risk of residual stenosis after CAS. CDU is an optimal, simple and noninvasive imaging method to evaluate plaque echogenicity and hemodynamic alterations during the perioperative period of CAS, which can help surgeons to select the optimal strategies and prevent the occurrence of residual stenosis.

Diagnostic power of vertebral hydroxyapatite concentration measurements in spectral CT for osteoporosis-associated fractures and impact of intravenous contrast administration.

OBJECTIVES: To evaluate the diagnostic power of using vertebral hydroxyapatite concentration measurements in unenhanced and contrast-enhanced spectral CT for detecting and predicting the risk of osteoporosis-associated fractures. METHODS: L1 of 210 patients (105 men, 105 women; mean age, 64 years, range, 19-103 years) who had undergone spectral CT examinations from January 1, 2018, to March 1, 2019, were retrospectively analyzed. Patient data for 3 years after spectral CT were retrieved from electronic medical record information systems to obtain the incidence of osteoporotic fractures. Baseline vertebral cancellous hydroxyapatite concentration from unenhanced and contrast-enhanced late-arterial-phase images was measured. The receiver operating characteristic curves were used to evaluate the diagnostic power for detecting and predicting the 3-year risk of osteoporosis-associated fractures using hydroxyapatite concentrations in both phases. RESULTS: The hydroxyapatite concentrations in both phases had good diagnostic power to detect fractures at baseline. The sensitivity and specificity for predicting one or more osteoporosis-associated fractures within 3 years after spectral CT were 76.80% and 93.10%, respectively, using the cutoff of 74.79 mg/cm3 in vertebral hydroxyapatite concentration in the unenhanced CT phase, and 82.87% and 82.76%, respectively, using the cutoff of 84.65 mg/cm3 in the late-arterial phase. Furthermore, there was no significant difference in the diagnosis between unenhanced and enhanced CT-derived hydroxyapatite concentrations (p = 0.360). CONCLUSIONS: Both unenhanced and enhanced spectral CT-derived hydroxyapatite concentrations can accurately detect and predict future risk of osteoporosis-associated fractures. The hydroxyapatite concentration assessed in the late-arterial phase may have a similar diagnostic efficacy to that in the unenhanced phase. KEY POINTS: • A cutoff of 74.79 mg/cm3 of vertebral hydroxyapatite concentration in the unenhanced CT scans had 76.80% sensitivity and 93.10% specificity to predict one or more osteoporosis-associated fractures within 3 years after spectral CT examinations. • A cutoff of 84.65 mg/cm3 of vertebral hydroxyapatite concentration in the late-arterial-enhanced CT scans had 82.87% sensitivity and 82.76% specificity to predict one or more osteoporosis-associated fractures within 3 years after spectral CT examinations. • The hydroxyapatite concentration assessed in the late-arterial phase may have a similar diagnostic efficacy to that in the unenhanced phase.

Effects of Reductive Soil Disinfestation Combined with Liquid-Readily Decomposable Compounds and Solid Plant Residues on the Bacterial Community and Functional Composition.

Reductive soil disinfestation (RSD) incorporated with sole plant residues or liquid-readily decomposable compounds is an effective management strategy to improve soil health. However, the synthetic effects of RSD incorporated with liquid-readily decomposable compounds and solid plant residues on soil ecosystem services remain unclear. Field experiments were carried out to investigate the effects of untreated soil (CK), RSD incorporated with sawdust (SA), molasses (MO), and their combinations (SA + MO) on the bacterial community and functional composition. The results showed that RSD treatments significantly altered soil bacterial community structure compared to CK treatment. The bacterial community structure and composition in MO and SA + MO treatments were clustered compared to SA treatment. This was mainly attributed to the readily decomposable carbon sources in molasses having a stronger driving force to reshape the soil microbial community during the RSD process. Furthermore, the functional compositions, such as the disinfestation efficiency of F. oxysporum (96.4 - 99.1%), abundances of nitrogen functional genes, soil metabolic activity, and functional diversity, were significantly increased in all of the RSD treatments. The highest disinfestation efficiency and abundances of denitrification (nirS and nrfA) and nitrogen fixation (nifH) genes were observed in SA + MO treatment. Specifically, SA + MO treatment enriched more abundant beneficial genera, e.g., Oxobacter, Paenibacillus, Cohnella, Rummeliibacillus, and Streptomyces, which were significantly and positively linked to disinfestation efficiency, soil metabolic activity, and denitrification processes. Our results indicated that combining RSD practices with liquid-readily decomposable compounds and solid plant residues could effectively improve soil microbial community and functional composition.

Nitrogen-doped graphene for tetracycline removal via enhancing adsorption and non-radical persulfate activation.

Nitrogen-doped graphene (NG) was synthesized via direct thermal annealing treatment. The obtained NG showed outstanding removal ability for tetracycline (TC) ascribed to enhanced adsorption and persulfate activation. The maximum TC adsorption capacity calculated from the Langmuir model of NG was 227.3 mg/g, which was 1.66 times larger than nitrogen-free graphene. The coexistence of NG and persulfate (PS) exhibited complete degradation of TC within 120 min attributed to the successful modification of nitrogen. Further analysis demonstrated that non-radical electron transfer was the dominant degradation pathway, which was different from the widely acknowledgeable radical mechanism. An electron donor-mediator-acceptor system was introduced, in which TC, NG, and PS performed as electron donor, mediator, and acceptor, respectively. The potential intermediates in the TC degradation process were detected and toxicity assessment was also performed. In addition, more than 75.8% of total organic carbon was removed, and excellent reusability was manifested in multiple adsorption and degradation experiments.

Influence of Sleeve Gastrectomy on Skeletal Muscular Fat Infiltration Measured by MRI in Patients with Metabolic Syndrome: Preliminary Results.

INTRODUCTION: Several researchers have focused on the role of skeletal muscle in metabolic problems in recent years. We aimed to evaluate influence of sleeve gastrectomy on skeletal muscular fat infiltration determined by magnetic resonance imaging in patients with metabolic syndrome (MetS). METHODS: Sixty five MetS patients (male/female, 20/45; mean age, 35.5 years ± 6.6 [standard deviation]; age range, 22-59 years) enrolled in our study. Prior to and 1 year after sleeve gastrectomy, patients underwent routine measurement of skeletal muscular fat concentration (denoted by proton density fat fraction, PDFF) and chemical indexes. The associations of skeletal muscular fat concentration with other variables were determined using multiple linear regression analysis. RESULTS: Difference between skeletal muscular PDFF at baseline (4.46 ± 2.01%) and PDFF 1-year after sleeve gastrectomy (3.00 ± 1.47%) was significant. Multivariable predictors of baseline skeletal muscular PDFF by descending order of standardized coefficient were fasting serum glucose (0.459; p = 0.001), age (0.395; p < 0.001), systolic pressure (0.319; p = 0.029), insulin (0.030; p = 0.026), white cell count (0.302; p = 0.007), diastolic pressure (-0.301; p = 0.046), and total alkaline phosphatase (-0.474; p < 0.001) all at baseline. Furthermore, multivariable predictors of change in PDFF were serum total cholesterol (3.510; p < 0.001), total alkaline phosphatase (0.535; p < 0.001), estrogen (0.457; p < 0.001), diastolic pressure (0.352; p < 0.001), systolic pressure (-0.409; p < 0.001), high-density lipoprotein cholesterol (-0.719; p < 0.001), insulin (-0.774; p < 0.001), C-reactive protein (-0.900; p < 0.001), triglyceride (-1.756; p < 0.001), and low-density lipoprotein cholesterol (-2.854; p < 0.001) all at baseline. CONCLUSION: Sleeve gastrectomy could alleviate myosteatosis in MetS patients during 1-year follow-up. The extent of remission on skeletal muscular fat infiltration after sleeve gastrectomy was influenced by baseline metabolic problems related to serum glucose, serum lipid, and blood pressure level.

Tissue-specific expression of GhnsLTPs identified via GWAS sophisticatedly coordinates disease and insect resistance by regulating metabolic flux redirection in cotton.

Cotton (Gossypium hirsutum) is constantly attacked by pathogens and insects. The most efficient control strategy is to develop resistant varieties using broad-spectrum gene resources. Several resistance loci harboured by superior varieties have been identified through genome-wide association studies. However, the key genes and/or loci have not been functionally identified. In this study, we identified a locus significantly associated with Verticillium wilt (VW) resistance, and within a 145.5-kb linkage disequilibrium, two non-specific lipid transfer protein genes (named GhnsLTPsA10) were highly expressed under Verticillium pathogen stress. The expression of GhnsLTPsA10 significantly increased in roots upon Verticillium dahliae stress but significantly decreased in leaves under insect attack. Furthermore, GhnsLTPsA10 played antagonistic roles in positively regulating VW and Fusarium wilt resistance and negatively mediating aphid and bollworm resistance in transgenic Arabidopsis and silenced cotton. By combining transcriptomic, histological and physiological analyses, we determined that GhnsLTPsA10-mediated phenylpropanoid metabolism further affected the balance of the downstream metabolic flux of flavonoid and lignin biosynthesis. The divergent expression of GhnsLTPsA10 in roots and leaves coordinated resistance of cotton against fungal pathogens and insects via the redirection of metabolic flux. In addition, GhnsLTPsA10 contributed to reactive oxygen species accumulation. Therefore, in this study, we elucidated the novel function of GhnsLTP and the molecular association between disease resistance and insect resistance, balanced by GhnsLTPsA10. This broadens our knowledge of the biological function of GhnsLTPsA10 in crops and provides a useful locus for genetic improvement of cotton.

Evolution, expression and functional analysis of cultivated allotetraploid cotton DIR genes.

BACKGROUND: Dirigent (DIR) proteins mediate regioselectivity and stereoselectivity during lignan biosynthesis and are also involved in lignin, gossypol and pterocarpan biosynthesis. This gene family plays a vital role in enhancing stress resistance and in secondary cell-wall development, but systematical understanding is lacking in cotton. RESULTS: In this study, 107 GbDIRs and 107 GhDIRs were identified in Gossypium barbadense and Gossypium hirsutum, respectively. Most of these genes have a classical gene structure without intron and encode proteins containing a signal peptide. Phylogenetic analysis showed that cotton DIR genes were classified into four distinct subfamilies (a, b/d, e, and f). Of these groups, DIR-a and DIR-e were evolutionarily conserved, and segmental and tandem duplications contributed equally to their formation. In contrast, DIR-b/d mainly expanded by recent tandem duplications, accompanying with a number of gene clusters. With the rapid evolution, DIR-b/d-III was a Gossypium-specific clade involved in atropselective synthesis of gossypol. RNA-seq data highlighted GhDIRs in response to Verticillium dahliae infection and suggested that DIR gene family could confer Verticillium wilt resistance. We also identified candidate DIR genes related to fiber development in G. barbadense and G. hirsutum and revealed their differential expression. To further determine the involvement of DIR genes in fiber development, we overexpressed a fiber length-related gene GbDIR78 in Arabidopsis and validated its function in trichomes and hypocotyls. CONCLUSIONS: These findings contribute novel insights towards the evolution of DIR gene family and provide valuable information for further understanding the roles of DIR genes in cotton fiber development as well as in stress responses.

Immune-Related Long Non-coding RNA Constructs a Prognostic Signature of Ovarian Cancer.

BACKGROUND: Since ovarian cancer leads to the poor prognosis in women all over the world, we aim to construct an immune-related lncRNAs signature to improve the survival of ovarian cancer patients. METHODS: Normal and cancer patient samples and corresponding clinical data of ovarian were obtained from The Genotype-Tissue Expression (GTEx) portal and The Cancer Genome Atlas (TCGA) database. The predictive signature was constructed by the lasso penalty Cox proportional hazard regression model. The division of different risk groups was accounting for the optimal critical value of the time-dependent Receiver Operating Characteristic (ROC) curve. Finally, we validated and evaluated the application of this prognostic signature based on the clinical factors, chemo-sensitivity and immune status of different risk groups. RESULTS: The signature was established from 145 DEirlncRNAs and can be shown as an independent prognostic risk factor with accurate prediction on overall survival in ovarian cancer patients. Further analysis on the application of the prognostic signature showed that patients with low-risk had a better sensitivity to chemotherapy and a higher immunogenicity. CONCLUSION: We constructed and verified an effective signature based on DEirlncRNA pairs, which could predict the prognosis, drug sensitivity and immune status of ovarian cancer patients and promote the prognostic estimation and individualized treatment.

Photodynamic inactivation of planktonic Staphylococcus aureus by sodium magnesium chlorophyllin and its effect on the storage quality of lettuce.

Photodynamic inactivation (PDI) is a fast and effective non-heat sterilization technology. This study established an efficient blue light-emitting diode (LED) PDI with the photosensitizer sodium magnesium chlorophyllin (SMC) to eradicate Staphylococcus aureus in food. The antibacterial mechanisms were determined by evaluating DNA integrity, protein changes, morphological alteration, and the potency of PDI to eradicate S. aureus on lettuce was evaluated. Results showed that planktonic S. aureus could not be clearly observed on the medium after treatment with 5.0 µmol/L SMC for 10 min (1.14 J/cm2). Bacterial cell DNA and protein were susceptible to SMC-mediated PDI, and cell membranes were found to be disrupted. Moreover, SMC-mediated PDI effectively reduced 8.31 log CFU/mL of S. aureus on lettuce under 6.84 J/cm2 radiant exposure (30 min) with 100 µmol/L SMC, and PDI displayed a potent ability to restrain the weight loss as well as retard the changes of color difference of the lettuce during 7 day storage. The study will enrich our understanding of the inactivation of S. aureus by PDI, allowing for the development of improved strategies to eliminate bacteria in the food industry.

The fungal community outperforms the bacterial community in predicting plant health status.

Characterizing the relationship between soil biotic and abiotic properties and plant health status is crucial to understanding the pathogenesis of soil-borne diseases. Here, we compared these properties in the soils of lisianthus with different disease incidence plots and report the cause-effect relationship between soil properties and plant health status using heat treatment coupled with microbiota self/across re-inoculations. The relative importance of soil bacterial and fungal communities in predicting plant health was also analyzed. Results showed that the soils with low and high disease incidences (LDS and HDS) harbored differential microbial communities and physicochemical properties. The LDS soil had relatively low Fusarium oxysporum abundance, electrical conductivity (EC), and NO3--N content. Soil microbial community was the direct determinant of plant health. The disease-suppressive activity of the microbiome in the LDS soil could be transferred to the HDS soil. Also, the relative importance of the fungal community in predicting plant health status was greater than that of the bacterial community, as reflected by (1) the fungal community could drive more complex networks related to healthy plants and (2) the diversity and core taxa of the fungal community had higher mean predictor importance values for plant health. The relative abundances of core genera Acremonium, Mycothermus, and Chryseolinea were significantly and negatively correlated with the disease incidence and the abundances of pathogens, identifying these genera as potential disease-suppressive agents. Taken together, our results reveal a direct relationship between soil properties and plant health status, in which the fungal community composition is most important for predicting plant health status. KEY POINTS: • Soil with differing pathological groups harbors distinct microbial communities. • Soil microbial communities directly determine the plant's health status. • Fungal community is a better predictor of plant health than the bacterial community.

Establishment of an efficient seed fluorescence reporter-assisted CRISPR/Cas9 gene editing in maize.

Genome editing by clustered regularly interspaced short palindromic sequences (CRISPR)/CRISPR-associated protein 9 (Cas9) has revolutionized functional gene analysis and genetic improvement. While reporter-assisted CRISPR/Cas systems can greatly facilitate the selection of genome-edited plants produced via stable transformation, this approach has not been well established in seed crops. Here, we established the seed fluorescence reporter (SFR)-assisted CRISPR/Cas9 systems in maize (Zea mays L.), using the red fluorescent DsRED protein expressed in the endosperm (En-SFR/Cas9), embryos (Em-SFR/Cas9), or both tissues (Em/En-SFR/Cas9). All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out. We describe several case studies of the implementation of En-SFR/Cas9, Em-SFR/Cas9, and Em/En- SFR/Cas9 to identify plants not harboring the genome-editing cassette but carrying the desired mutations at target genes in single genes or in small-scale mutant libraries, and report on the successful generation of single-target mutants and/or mutant libraries with En-SFR/Cas9, Em-SFR/Cas9, and Em/En-SFR/Cas9. SFR-assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.

Effect of Polyvinyl Chloride Microplastics on Bacterial Community and Nutrient Status in Two Agricultural Soils.

Knowledge of the influence of microplastics on soil microbiome and nutrients is important for understanding the ecological consequences of microplastic pollution in terrestrial ecosystems. In this study, we investigated whether polyvinyl chloride (PVC) microplastic pollution at environmentally relevant concentrations would affect soil bacterial community and available nitrogen/phosphorus content. The results showed that although PVC microplastics at 0.1% and 1% levels did not have a significant effect on overall bacterial community diversity and composition in soil over the course of 35 days, a number of bacterial genera were significantly reduced or enriched by the presence of microplastics. Potentially due to their effect on certain functional groups, microplastics caused a significant change in soil available P content. It is noteworthy that, depending on soil type, pollution level and plasticizer presence, contrasting effects of microplastics may be observed. Further research is definitely warranted to gain a clearer picture of the threats posed by microplastic pollution in soil environments.

LncRNA HOTTIP facilitates the stemness of breast cancer via regulation of miR-148a-3p/WNT1 pathway.

Emerging evidence suggests that dysregulation of long non-coding RNA (lncRNA) plays a key role in tumorigenesis. The lncRNA, HOXA transcript at the distal tip (HOTTIP), has been reported to be up-regulated in multiple cancers, including breast cancer, and is involved in various biological processes, including the maintenance of stemness. However, the biological function and underlying modulatory mechanism of HOTTIP in breast cancer stem cells (BCSCs) remains unknown. In this study, we found that HOTTIP was markedly up-regulated in BCSCs and had a positive correlation with breast cancer progression. Functional studies revealed that overexpression of HOTTIP markedly promoted cell clonogenicity, increased the expression of the stem cell markers, OCT4 and SOX2, and decreased the expression of the differentiation markers, CK14 and CK18, in breast cancer cells. Knockdown of HOTTIP inhibited the CSC-like properties of BCSCs. Consistently, depletion of HOTTIP suppressed tumour growth in a humanized model of breast cancer. Mechanistic studies demonstrated that HOTTIP directly binds to miR-148a-3p and inhibits the mediation of WNT1, which leads to inactivation of the Wnt/ß-catenin signalling pathway. Our study is the first to report that HOTTIP regulates the CSC-like properties of BCSCs by as a molecular sponge for miR-148a-3p to increase WNT1 expression, offering a new target for breast cancer therapy.