Combining thermotherapy with shoot tip culture or cryotherapy for improved virus eradication from in vitro Actinidia macrosperma.

In recent years, kiwifruit viral diseases have become increasingly prevalent in kiwifruit producing regions of China, significantly impacting both the yield and quality of kiwifruit. This has emerged as a significant constraint on the healthy and sustainable development of the kiwifruit industry. The use of virus-free propagation materials has been proven to be an effective strategy for controlling plant viral diseases. In the present study, shoot tip culture (STC), shoot tip cryotherapy (Cryo), and their combinations with thermotherapy were established to eradicate AcVA, AcVB and AcCRaV from Actinidia macrosperma. Additionally, the impact of shoot tip size on virus eradication was evaluated. Among the three confirmed viruses, AcVB was the easiest to eradicate, followed by AcVA and AcCRaV. Combining thermotherapy with shoot tip culture or cryotherapy resulted in higher virus elimination rates than shoot tip culture or cryotherapy alone. Notably, the combination of thermotherapy and 0.5-1 mm shoot tip cryotherapy was shown to be the most effective protocol which produced 50% of regenerated shoots free from all the tested viruses. To the best of our knowledge, this is the first report on virus elimination from kiwifruit infected with multiple viruses based on conventional shoot tip culture and shoot tip cryotherapy.

Milletdb: a multi-omics database to accelerate the research of functional genomics and molecular breeding of millets.

Millets are a class of nutrient-rich coarse cereals with high resistance to abiotic stress; thus, they guarantee food security for people living in areas with extreme climatic conditions and provide stress-related genetic resources for other crops. However, no platform is available to provide a comprehensive and systematic multi-omics analysis for millets, which seriously hinders the mining of stress-related genes and the molecular breeding of millets. Here, a free, web-accessible, user-friendly millets multi-omics database platform (Milletdb, http://milletdb.novogene.com) has been developed. The Milletdb contains six millets and their one related species genomes, graph-based pan-genomics of pearl millet, and stress-related multi-omics data, which enable Milletdb to be the most complete millets multi-omics database available. We stored GWAS (genome-wide association study) results of 20 yield-related trait data obtained under three environmental conditions [field (no stress), early drought and late drought] for 2 years in the database, allowing users to identify stress-related genes that support yield improvement. Milletdb can simplify the functional genomics analysis of millets by providing users with 20 different tools (e.g., 'Gene mapping', 'Co-expression', 'KEGG/GO Enrichment' analysis, etc.). On the Milletdb platform, a gene PMA1G03779.1 was identified through 'GWAS', which has the potential to modulate yield and respond to different environmental stresses. Using the tools provided by Milletdb, we found that the stress-related PLATZs TFs (transcription factors) family expands in 87.5% of millet accessions and contributes to vegetative growth and abiotic stress responses. Milletdb can effectively serve researchers in the mining of key genes, genome editing and molecular breeding of millets.

Rapid and Visual Detection of Actinidia Chlorotic Ringspot-Associated Virus Using One-Step Reverse-Transcription Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay.

Actinidia chlorotic ringspot-associated virus (AcCRaV) occurs widely in major kiwifruit producing areas of China and is often accompanied by coinfecting viruses, affecting the growth, yield, and quality of kiwifruit. Therefore, a rapid and sensitive detection method is crucial for diagnosing and developing effective AcCRaV management strategies. In this study, a one-step reverse-transcription recombinase polymerase amplification combined with a lateral flow dipstick (RT-RPA-LFD) assay was developed for rapid detection of AcCRaV. Specific primers and a probe were designed based on the conserved region of the coat protein gene sequence of AcCRaV. The one-step RT-RPA reaction can be performed at 35 and 40°C within 10 to 30 min, and the amplification results can be read directly on the LFD within 5 min. The detection limit of the one-step RT-RPA-LFD assay was 10-8 ng (about 20 viral copies), which was equal with one-step RT-qPCR and 100 times more sensitive than one-step RT-PCR. Moreover, the one-step RT-RPA-LFD assay was successfully applied to detect AcCRaV from crude extracts, and the entire detection process can be completed within 40 min. These results indicate that the RT-RPA-LFD assay is a simple, rapid, and sensitive strategy that can be used for accurate diagnosis of AcCRaV-infected kiwifruit plants in the field. To our knowledge, this is the first study applying the one-step RT-RPA-LFD assay to detect a kiwifruit virus.

Micrografting: An Old Dog Plays New Tricks in Obligate Plant Pathogens.

Micrografting, which was developed almost 50 years ago, has long been used for virus eradication, micropropagation, regeneration, rejuvenation, and graft compatibility. Recently, micrografting has been used for studies of long-distance trafficking and signaling of molecules between scions and rootstocks. The graft transmissiveness of obligate plant pathogens, such as viruses, viroids, and phytoplasmas, facilitated the use of micrografting to study biological indexing and pathogen transmission, pathogen-induced graft incompatibility, and screening for the pathogen resistance during the past 20 years. The present study provides comprehensive information on the latter subjects. Finally, prospects are proposed to direct further studies.

Cryopreservation of shoot tips, evaluations of vegetative growth, and assessments of genetic and epigenetic changes in cryo-derived plants of Actinidia spp.

A droplet-vitrification protocol was described for cryopreservation of shoot tips of kiwifruit 'Yuxiang' (Actinidia chinensis var. deliciosa). No significant differences were found in root formation and shoot growth between the in vitro-derived shoots (the control) and cryo-derived ones when cultured in vitro. No significant differences were detected in survival and vegetative growth between the in vitro-derived plants (the control) and cryo-derived ones after re-establishment in greenhouse conditions. Inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) did not detect any polymorphic bands in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-establishment in greenhouse conditions. These data indicate rooting ability, vegetative growth and genetic stability are maintained in the cryo-derived kiwifruit plants recovered from the droplet-vitrification cryopreservation. Methylation sensitive amplification polymorphism (MSAP) detected 12.8% and 1.6% DNA methylation in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-established in greenhouse conditions, respectively. This droplet-vitrification was applied to five cultivars and three rootstocks belonging to A. chinensis var. deliciosa, A. chinensis var. chinensis, A. macrosperma, A. polygama and A. valvata. The highest (68.3%) and lowest (22.5%) shoot regrowth were obtained in A. macrosperma and A. chinensis var. chinensis 'Jinmi', respectively, with an average of 46.4% shoot regrowth obtained across the eight genotypes. The droplet-vitrification protocol described here can be considered the most applicable cryopreservation method so far reported for the genus Actinidia. Results reported here provide theoretical and technical supports for setting up cryo-banks of genetic resources of Actinidia spp.

Molecular cloning and functional characterization of AcGST1, an anthocyanin-related glutathione S-transferase gene in kiwifruit (Actinidia chinensis).

KEY MESSAGE: AcGST1, an anthocyanin-related GST, may functions as a carrier to transport anthocyanins from ER to tonoplast in kiwifruit. It was positively regulated by AcMYBF110 through directly binding to its promoter. Anthocyanins are synthesized in the cytoplasmic surface of the endoplasmic reticulum but accumulate predominantly in the vacuole. Previous studies in model and ornamental plants have suggested that a member of the glutathione S-transferase (GST) gene family is involved in sequestration of anthocyanins into the vacuole. However, little is known about anthocyanin-related GST protein in kiwifruit. Here, four putative AcGSTs were identified from the genome of the red-fleshed Actinidia chinensis cv 'Hongyang'. Expression analyses reveal only the expression of AcGST1 was highly consistent with anthocyanin accumulation. Molecular complementation of Arabidopsis tt19 demonstrates AcGST1 can complement the anthocyanin-less phenotype of tt19. Transient expression in Actinidia arguta fruits further confirms that AcGST1 is functional in anthocyanin accumulation in kiwifruit. In vitro assays show the recombinant AcGST1 increases the water solubility of cyanidin-3-O-galactoside (C3Gal) and cyanidin-3-O-xylo-galactoside (C3XG). We further show that AcGST1 protein is localized not only in the ER but also on the tonoplast, indicating AcGST1 (like AtTT19) may functions as a carrier protein to transport anthocyanins to the tonoplast in kiwifruit. Moreover, the promoter of AcGST1 can be activated by AcMYBF110, based on results from transient dual-luciferase assays and yeast one-hybrid assays. EMSAs show that AcMYBF110 binds directly to CAGTTG and CCGTTG motifs in the AcGST1 promoter. These results indicate that AcMYBF110 plays an important role in transcriptional regulation of AcGST1 and, therefore, in controlling accumulation of anthocyanins in kiwifruit.

Psychological intervention improves life quality of patients with laryngeal cancer.

BACKGROUND: The purpose of this study is to examine the effect of psychological intervention on the life quality of patients with laryngeal cancer. METHODS: Two hundred and ten patients with laryngeal cancer were randomly assigned to the study group receiving psychological intervention and control group receiving routine nursing care. The Hamilton Depression Scale (HAMD), Hamilton Anxiety Scale (HAMA), and the Europe Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) were used to evaluate life quality. RESULTS: There were no significant differences in the scores of HAMD, HAMA, and EORTC QLQ-C30 between the 2 groups before treatment (P>0.05). After treatment, the scores in the study group were significantly different to those in the control group (P<0.05). In addition, the satisfaction rate and compliance rate of patients in the study group were increased, compared to the control group (P<0.05). CONCLUSION: Psychological intervention is beneficial in improving life quality in patients with laryngeal cancer after surgery.

Characterization of Sox9-overexpressing human umbilical cord blood-derived mesenchymal stem cells-based engineered cartilage both in vitro and in vivo.

The source of seed cells is a critical factor for tissue engineering. The goal of this study was to evaluate the chondrogenesis of Sox9-overexpressing human umbilical cord mesenchymal stem cells (hUCMSCs) seeded onto bone matrix gelatin (BMG)/fibrin hybrid scaffolds both in vitro and in vivo. hUCMSCs were stably transfected with Sox9-expressing plasmid and grown on the three-dimensional BMG/fibrin hybrid scaffold for 8 weeks. Scanning electron microscopy and histochemistry were performed. The hUCMSC-loaded scaffolds were implanted into the subcutaneous layer of immunocompetent rats and chondrogenesis and host immune responses were monitored for 8 weeks. We found that hUCMSCs spread well and proliferated from 2 weeks after culturing. They produced abundant glycosaminoglycans and collagen II. At 8 weeks after implanting into rats, the hUCMSCs on the scaffolds formed cartilage-like tissue and displayed positive staining for toluidine blue, safranin O, Masson's trichrome, and collagen II. No significant changes in serum levels of lgG, lgA, lgM, C3, and C4 were observed after implantation of the hUCMSC-loaded scaffolds. Xenogeneic implantation of Sox9-overexpressing hUCMSCs embedded in the BMG/fibrin scaffolds promotes the formation of cartilage-like tissue without inducing evident host immune response. Therefore, Sox9-overexpressing hUCMSCs represent a promising cell candidate for cartilage tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1150-1155, 2017.

[The expression and clinical significance of pepsin and pepsinogen in patients with otitis media with effusion].

OBJECTIVE: To analyze the role and significance of pepsin and pepsinogen in the pathogenesis of OME in children. METHOD: Pediatric patients with otitis media aged 2-8 years who enrolled in our department of the hospital from May of 2012 to December of 2012 were set as experimental group (38 cases, 48 ears) which should be underwent tympanic membrane puncture/tube insertion. Meanwhile, pediatric patients waiting for cochlear implant without otitis media (10 ears), were set as control group. Middle ear lavage fluid and plasma samples from the two groups were collected and detected using enzyme-linked immune method for pepsin and pepsinogen. RESULT: The concentrations of pepsin and pepsinogen in the middle ear lavage fluid of OME group [(48.8 ± 415.99) ng/ml and 676.32 ± 336.71)ng/ml] were significantly higher than those in the control group [(8.20 ± 4.59)ng/ml and (77.27 ± 50.33) ng/ml] (P < 0.01). Meanwhile, the concentration of pepsinogen in the middle ear lavage of OME patients was significantly higher than that of plasma (P < 0.01). The concentration of pepsin in the middle ear lavage fluid from the dry ear subgroup was lower than those in the serum ear and mucous ear subgroups (P < 0.01), but there was no significant difference about concentrations of pepsinogen among the dry ear, serum ear and mucous ear subgroups (P > 0.05). CONCLUSION: Pepsin and pepsinogen in the middle ear cavity of OME patients maybe originated from laryngopharyngeal reflux (LPR), indicating that LPR is associated with the pathogenesis of OME in children.