Summary Co‐infection of apple trees with several viruses/viroids is common and decreases fruit yield and quality. Accurate and rapid detection of these viral pathogens helps to reduce losses and prevent virus spread. Current molecular detection assays used for apple viruses require specialized and expensive equipment. Here, we optimized a CRISPR/Cas12a‐based nucleic acid detection platform for the diagnosis of the most prevalent RNA viruses/viroid in apple, namely Apple necrotic mosaic virus (ApNMV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd). We detected each RNA virus/viroid directly from crude leaf extracts after simultaneous multiplex reverse transcription‐recombinase polymerase amplification (RT‐RPA) with high specificity. Positive results can be distinguished by the naked eye via oligonucleotide‐conjugated gold nanoparticles. The CRISPR/Cas12a‐RT‐RPA platform exhibited comparable sensitivity to RT‐qPCR, with limits of detection reaching 250 viral copies per reaction for ASPV and ASGV and 2500 copies for the others. However, this protocol was faster and simpler, requiring an hour or less from leaf harvest. Field tests showed 100% agreement with RT‐PCR detection for 52 samples. This novel Cas12a‐based method is ideal for rapid and reliable detection of apple viruses in the orchard without the need to send samples to a specialized laboratory.

Endophytes form symbiotic relationships with plants and constitute an important source of phytohormones and bioactive secondary metabolites for their hosts. To date, most studies of endophytes have focused on the influence of these microorganisms on plant growth and physiology and their role in plant defenses against biotic and abiotic stressors; however, to the best of our knowledge, the ability of endophytes to produce melatonin has not been reported. In the present study, we isolated and identified root-dwelling bacteria from three grapevine varieties and found that, when cultured under laboratory conditions, some of the bacteria strains secreted melatonin and tryptophan-ethyl ester. The endophytic bacterium Bacillus amyloliquefaciens SB-9 exhibited the highest level of in vitro melatonin secretion and also produced three intermediates of the melatonin biosynthesis pathway: 5-hydroxytryptophan, serotonin, and N-acetylserotonin. After B. amyloliquefaciens SB-9 colonization, the plantlets exhibited increased plant growth. Additionally, we found that, in grapevine plantlets exposed to salt or drought stress, colonization by B. amyloliquefaciens SB-9 increased the upregulation of melatonin synthesis, as well as that of its intermediates, but reduced the upregulation of grapevine tryptophan decarboxylase genes (VvTDCs) and a serotonin N-acetyltransferase gene (VvSNAT) transcription, when compared to the un-inoculated control. Colonization by B. amyloliquefaciens SB-9 was also able to counteract the adverse effects of salt- and drought-induced stress by reducing the production of malondialdehyde and reactive oxygen species (H2O2 and O2-) in roots. Therefore, our findings demonstrate the occurrence of melatonin biosynthesis in endophytic bacteria and provide evidence for a novel form of communication between beneficial endophytes and host plants via melatonin.

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To determine the value of dual-energy CT (DECT) and combined information of perfusion and angiography in diagnosing coronary artery disease (CAD), with single photon emission computed tomography (SPECT) and quantitative coronary angiography (QCA) as a reference standard. Thirty-four patients were enrolled in this study. DECT was used as a contrast-enhanced retrospectively ECG-gated scan protocol during the rest state and tubes were set at 140/100 kV. DECT angiography (DE-CTA) and DECT perfusion (DE-CTP) were calculated from two kV images. DE-CTP results were compared with SPECT and DE-CTA with QCA, respectively. The combined DE-CTP with DE-CTA data were compared to QCA in diagnosis of obstructive CAD (stenosis ≥ 50%). DECT showed diagnostic image quality in 31 patients. Using SPECT as a reference, DE-CTP had sensitivity of 68%, specificity of 93%, and sensitivity of 81%, and specificity of 92% for identifying any type of perfusion deficits on the segment- and territory-based analysis, respectively. Using QCA as a reference standard, DE-CTA showed sensitivity of 82%, specificity of 91% and accuracy of 86% for detecting ≥50% coronary stenosis on the vessel-based analysis, whereas the combination of DE-CTA and DE-CTP gave sensitivity of 90%, specificity of 86% and accuracy of 88% for detecting ≥50% coronary stenosis, respectively. Combination of DE-CTP and DE-CTA may improve diagnostic performance compared to CTA alone for the diagnosis of significant coronary stenosis.

Apple (Malus domestica Borkh.), an economically important tree fruit worldwide, frequently suffers from temperature stress during growth and development, which strongly affects the yield and quality. Heat shock protein 20 (HSP20) genes play crucial roles in protecting plants against abiotic stresses. However, they have not been systematically investigated in apple. In this study, we identified 41 HSP20 genes in the apple 'Golden Delicious' genome. These genes were unequally distributed on 15 different chromosomes and were classified into 10 subfamilies based on phylogenetic analysis and predicted subcellular localization. Chromosome mapping and synteny analysis indicated that three pairs of apple HSP20 genes were tandemly duplicated. Sequence analysis revealed that all apple HSP20 proteins reflected high structure conservation and most apple HSP20 genes (92.6%) possessed no introns, or only one intron. Numerous apple HSP20 gene promoter sequences contained stress and hormone response cis-elements. Transcriptome analysis revealed that 35 of 41 apple HSP20 genes were nearly unchanged or downregulated under normal temperature and cold stress, whereas these genes exhibited high-expression levels under heat stress. Subsequent qRT-PCR results showed that 12 of 29 selected apple HSP20 genes were extremely up-regulated (more than 1,000-fold) after 4 h of heat stress. However, the heat-upregulated genes were barely expressed or downregulated in response to cold stress, which indicated their potential function in mediating the response of apple to heat stress. Taken together, these findings lay the foundation to functionally characterize HSP20 genes to unravel their exact role in heat defense response in apple.

Endophytes have been verified to synthesize melatonin in vitro and promote abiotic stress-induced production of endogenous melatonin in grape (Vitis vinifera L.) roots. This study aimed to further characterize the biotransformation of tryptophan to melatonin in the endophytic bacterium Pseudomonas fluorescens RG11 and to investigate its capacity for enhancing endogenous melatonin levels in the roots of different grape cultivars. Using ultra performance liquid chromatography-tandem mass spectrometry combined with 15N double-labeled L-tryptophan as the precursor for melatonin, we detected isotope-labeled 5-hydroxytryptophan, serotonin, N-acetylserotonin, and melatonin, but tryptamine was not detected during the in vitro incubation of P. fluorescens RG11. Furthermore, the production capacity of these four compounds peaked during the exponential growth phase. RG11 colonization increased the endogenous levels of 5-hydroxytryptophan, N-acetylserotonin, and melatonin, but reduced those of tryptamine and serotonin, in the roots of the Red Globe grape cultivar under salt stress conditions. Quantitative real-time PCR revealed that RG11 reduced the transcription of grapevine tryptophan decarboxylase and serotonin N-acetyltransferase genes when compared to the un-inoculated control. These results correlated with decreased reactive oxygen species bursts and cell damage, which were alleviated by RG11 colonization under salt stress conditions. Additionally, RG11 promoted plant growth and enhanced the levels of endogenous melatonin in different grape cultivars. Intraspecific variation in the levels of melatonin precursors was found among four grape cultivars, and the associated root crude extracts appeared to significantly induce RG11 melatonin biosynthesis in vitro. Overall, this study provides useful information that enhances the existing knowledge of a potential melatonin synthesis pathway in rhizobacteria, and it reveals plant–rhizobacterium interactions that affect melatonin biosynthesis in plants subjected to abiotic stress conditions.

PurposeChronic rhinosinusitis with nasal polyps (CRSwNP), a mainly Th2 cytokine-mediated disease, often involves mucus secretion. Recent evidence suggests that transmembrane protein 16A (TMEM16A), a calcium-activated Cl- channel (CaCC), can regulate mucus secretion from airway epithelium by transepithelial electrolyte transport and hydration. However, the role of TMEM16A in mucin production/secretion in the airway epithelium is not clear. This study was conducted to determine the role of TMEM16A in mediating mucin secretion in human nasal polyp epithelial cells (HNPECs) induced by IL-13.MethodsHuman sinonasal mucosa tissue and dissociated sinonasal epithelium from control subjects and patients with CRSwNP were assessed for the expression of TMEM16A and the secretion of human mucin 5AC (MUC5AC) by immunohistochemistry, Western blot analysis, and enzyme-linked immuno-sorbent assay (ELISA). A model of the Th2 inflammatory environment was created by exposure of primary air-liquid interface (ALI)-cultured HNPECs to interleukin-13 (IL-13) for 14 days, with subsequent assessment of TMEM16A expression in cell lysates by Western blotting and MUC5AC secretion in apical washings of cells by ELISA.ResultsThe expressions of TMEM16A and MUC5AC were increased in human nasal polyp tissue and dissociated nasal polyp epithelium. TMEM16A was detected in IL-13-treated HNPECs, specifically in MUC5AC-positive cells but not in ciliated cells. IL-13 treatment increased percentages of TMEM16A-positive cells, MUC5AC-positive cells, and cells coexpressing TMEM16A/MUC5AC, the expression of TMEM16A protein, and the secretion of MUC5AC. T16Ainh-A01, a TMEM16A inhibitor, attenuated these IL-13-induced effects.ConclusionsThe expression of TMEM16A and MUC5AC are increased in CRSwNP, which might be a direct effect of Th2 cytokines present in the sinonasal mucosa in CRSwNP. Down-regulation of TMEM16A expression and MUC5AC secretion in HNPECs by T16Ainh-A01 indicates that TMEM16A might play an important role in mucin secretion in upper airway inflammatory diseases.

The demonstration that IFN-γ and IL-13 both significantly reduce ciliated cell differentiation and CBF in CRSwNP patients, and IL-13 additionally induces significant goblet cell hyperplasia and MUC5AC mucin expression, as well as IL-17 significantly increases MUC5B mucin expression, suggests that these inflammatory cytokines may be potential therapeutic targets in the management of CRSwNP.