Characteristics of the Complete Genome of Ageratum Yellow Vein China Virus Infecting Sonchus oleraceus.

Sonchus (Sonchus oleraceus) originated from Europe and is now cultivated worldwide. The wild resources of sonchus are very abundant, and it has rich nutritional and medicinal value. In this study, 15 sonchus samples with typical symptoms showing leaf curling, vein thickening, and enations were collected from Guigang and Baise City of Guangxi, China. Diseased sonchus were identified by PCR detection, whole genome sequence amplification, and phylogenetic and recombination analysis. The results showed that all the samples were confirmed infected by begomoviruses, and three full-length viral genomes were obtained from 15 sonchus, named GG7-13, GG8-6, and BS63-5. The full genome lengths were 2,584, 2,735, and 2,746 nt, respectively. The nucleotide identities among the three isolates ranged from 92.67 to 99.93%. All of them shared the highest identities (greater than 91.69%) with other isolates of ageratum yellow vein China virus (AYVCNV) (available on GenBank). According to the guidelines of classification of begomoviruses, the virus isolates obtained in this study are different isolates of AYVCNV; a phylogenetic tree analysis showed that these isolates formed a large branch with three other Guangxi isolates of AYVCNV, indicating their close evolution. The genome structures of GG8-6 and BS63-5 are consistent with the monopartite genome virus of the begomoviruses, and both have six open reading frames (ORFs), while GG7-13 has a 151-nt deletion between C2 and C3, resulting in a mutant strain of only five ORFs. This study is the first report on S. oleraceus infected by ageratum yellow vein China virus.

First Report of Banana mild mosaic virus Infecting Banana in China.

Banana (Musa spp.) is an important fruit in tropical and subtropical regions and an essential food crop in some developing countries. China has a long history of banana cultivation and ranks second in global banana production, with a planting area exceeding 11 million hectares (FAOSTAT, 2023). Banana mild mosaic virus (BanMMV) is a flexuous filamentous virus infecting bananas and a banmivirus in the Betaflexiviridae family. Its infection often results in symptomless plants of Musa spp., and the virus has a worldwide distribution, which can explain its high prevalence (Kumar et al., 2015). BanMMV infection often causes transitory symptoms, such as mild chlorotic streaks and mosaics, on young leaves (Thomas, 2015). The mixed infection of BanMMV with other banana-infecting viruses such as banana streak viruses (BSV) and cucumber mosaic virus (CMV), can exacerbate the mosaic symptoms of BanMMV (Fidan et al., 2019). In October 2021, we collected twenty-six leaf samples of suspected viral disease of bananas from four cities (Huizhou, Qingyuan, Zhanjiang, and Yangjiang) in Guangdong province, two cities (Hekou and Jinghong) in Yunnan province, two cities (Yulin and Wuming) in Guangxi Zhuang autonomous region. After fully mixing these infected samples, we divided them into two pools and sent them to Shanghai Biotechnology Corporation (China) for metatranscriptome sequencing. Each sample contained about 5 g of leaves in total. Zymo-Seq RiboFree Total RNA Library Prep Kit (Zymo Research, USA) was used for ribosomal RNA depletion and library preparations. Illumina sequencing (Illumina NovaSeq 6000) was carried out by Shanghai Biotechnology Corporation (China). Paired-end (150 bp) sequencing of the RNA library was performed on an Illumina HiSeq 2000/2500 platform. Clean reads were assembled by a metagenomic de novo assembly using the CLC Genomics Workbench (version: 6.0.4). Then the non-redundant protein database in the National Center for Biotechnology Information (NCBI) was used for BLASTx annotation. A total of 79,528 contigs were generated from the clean reads (68,878,162) through de novo assembly. A contig of 7265 nucleotides (nts) showed the highest nucleotide sequence identity (90.08%) to the genome of BanMMV isolate EM4-2 (GenBank accession no. OL826745.1). We designed specific primers according to the BanMMV CP gene (Table S1), tested the twenty-six leaf samples collected from the above-mentioned eight cities, and found that only one sample of Fenjiao (Musa ABB Pisang Awak) in Guangzhou city was infected with this virus. The symptoms of banana leaves containing BanMMV were slight chlorosis and yellowing of leaf edges (Fig. S1). We failed to detect other banana viruses, such as BSV, CMV, and banana bunchy top virus (BBTV) in the BanMMV-infected banana leaves. RNA from the infected leaves was extracted, and the assembled contig was confirmed by overlapping PCR amplification across the whole sequence (Table S1). All ambiguous regions were amplified by PCR and RACE, and the products were subjected to Sanger sequencing. The complete genome of the virus candidate was 7310 nts in length, excluding the poly (A) tail. The sequence was deposited in GenBank under accession number ON227268 (isolate BanMMV-GZ from Guangzhou). A schematic representation of the genome organization of BanMMV-GZ is shown in Fig. S2. Its genome has five open reading frames (ORF) encoding RNA-dependent RNA polymerase (RdRp), three triple gene block proteins necessary for cell-to-cell movement (TGBp1 to TGBp3) and a coat protein (CP), similar to other BanMMV isolates (Kondo et al., 2021). Phylogenetic analyses of the complete nt sequence of the full genome and RdRp gene using the neighbor-joining (NJ) method also clearly placed the BanMMV-GZ firmly within all isolates of BanMMV (Fig. S3). To our knowledge, this is the first report of BanMMV infecting bananas in China, extending the geographical range of this viral disease around the world. Accordingly, larger-scale BanMMV investigations must be conducted to determine the distribution and prevalence of BanMMV in China.

First Report of Tobacco Mosaic Virus Infecting Papaya in China.

Papaya (Carica papaya. L) is widely cultivated in tropical and subtropical regions of China and has high nutritional and medicinal values. More than 11 species of papaya viruses have been recorded in the world, but the most destructive one for papaya production in China is papaya ringspot virus (PRSV) (Li, 2019). In order to control PRSV, a transgenic papaya cultivar, designated as 'Huanong No.1', carrying the nuclear inclusion b (Nib) cistron of PRSV Ys isolate, was successfully commercialized in 2006, and has shown a wide range of resistance to PRSV in China (Li et al. 2007). However, more than 10% of 'Huanong No.1' plants developed different virus-like symptoms on leaves, including mosaic, yellow mottle, and deformation in some plantations of Guangdong Province, China in 2020 (Suppl Figure 1a, b, and c). Based on observation of the symptomatic phenotypes, the field surveys indicated that the disease incidence ranged from 10% to 40%, resulting in significant loss of papaya fruit. The virus particles were purified from symptomatic papaya plants following Gooding and Hebert (1967) and rigid filamentous particles resembling tobacco mosaic virus (TMV) were observed by transmission electron microscopy. Purified virus samples were further utilized to mechanically inoculate healthy seedlings of papaya, Nicotiana glutinosa and N. tabacum K326. At 15 days after inoculation, the obvious symptoms of virus infection on different plants were observed. The diseased plants showed systemic mottling and mosaic in the papaya leaves (Suppl Figure 1d), necrotic spots on the leaves of N. glutinosa (Suppl Figure 1e), mosaic and mottling spots on leaves of N. tabacum K326 (Suppl Figure 1f). These symptoms produced on the hosts were exactly the same caused by TMV. In order to reconfirm the species of the infected virus, the total RNA was extracted from the single leaf of 30 diseased papaya plants using RNAiso Plus kit (Takara, Japan) and reverse transcription--polymerase chain reaction was performed using TMV coat protein cistron specific primers (TMV-CP-R: 5'-TCAAGTTGCAGGACCAGA-3' and TMV-CP-F 5'- ATGTCTTACAGTATCACTAC-3') as described previously (Srivastava et al. 2015). An expected 480-bp fragment was amplified from all of the samples. Sequence analysis revealed that the diseased papaya was infected with TMV, designated as Cpa-TMV. In order to understand the difference among TMV isolates on papaya and other host plants, the whole genomic sequence of TMV from papaya was obtained and analyzed. The total length of the genome of Cpa-TMV was 6395 bp, and the sequence was submitted to the NCBI database (GenBank no. OK149218). A neighbor-joining phylogenetic tree of 19 TMV isolates was constructed using MEGA X software. Phylogenetic analysis indicated that the 19 TMV isolates were divided into Clade I, II and III (Suppl Figure 2). Interestingly, Clade I was composed of 12 Chinese mainland isolates, which further was grouped into IA (Northern China) and IB (Southern China), while 6 isolates from other countries and 1 isolate (pet-TMV) from China Taiwan belong to Clade II and III. It is inferred that the TMV isolates from Chinese mainland are quite different from other countries and China Taiwan. This suggests that geographical differences between Northern and Southern China may lead to the gradual differentiation of TMV isolates and eventually induce those isolates to evolve into two subclades. To our knowledge, this is the first report of TMV infection on papaya under natural conditions. It is necessary to find effective methods to control TMV in transgenic papaya.

Genetic diversity analysis of papaya leaf distortion mosaic virus isolates infecting transgenic papaya "Huanong No. 1" in South China.

The commercialized genetically modified papaya "Huanong No. 1" has been utilized to successfully control the destructive virus-papaya ringspot virus (PRSV) in South China since 2006. However, another new emerging virus, papaya leaf distortion mosaic virus (PLDMV), was found in some PRSV-resistant transgenic plants in Guangdong and Hainan provinces of South China through a field investigation from 2012 to 2019. The survey results showed that "Huanong No. 1" papaya plants are susceptible to PLDMV, and the disease prevalence in Hainan Province is generally higher than that in Guangdong Province. Twenty representative isolates were selected to inoculate "Huanong No. 1," and all of the inoculated plants showed obvious disease symptoms similar to those in the field, indicating that PLDMV is a new threat to widely cultivated transgenic papaya in South China. Phylogenetic analysis of 111 PLDMV isolates in Guangdong and Hainan based on the coat protein nucleotide sequences showed that PLDMV isolates can be divided into two groups. The Japan and Taiwan China isolates belong to group I, whereas the Guangdong and Hainan isolates belong to group II and can be further divided into two subgroups. The Guangdong and Hainan isolates are far different from the Japan and Taiwan China isolates and belong to a new lineage. Further analysis showed that the Guangdong and Hainan isolates had a high degree of genetic differentiation, and no recombination was found. These isolates deviated from neutral evolution and experienced population expansion events in the past, which might still be unstable. The results of this study provide a theoretical basis for clarifying the evolutionary mechanism and population genetics of the virus and for preventing and controlling the viral disease.

Kartogenin hydrolysis product 4-aminobiphenyl distributes to cartilage and mediates cartilage regeneration.

Rationale The small molecule Kartogenin (KGN) promotes cartilage regeneration in osteoarthritis (OA) by activating stem cells differentiation, but its pharmacological mode-of-action remains unclear. KGN can be cleaved into 4-aminobiphenyl (4-ABP) and phthalic acid (PA) following enzymolysis of an amide bond. Therefore, this study investigated whether 4-ABP or PA exerted the same action as KGN. Methods KGN, 4-ABP and PA were analyzed in cartilage of mice after oral, intravenous or intra-articular administration of KGN by liquid chromatography-mass spectrometry method. Their effect on proliferation and chondrogenic differentiation of mesenchymal stem cells (MSC) was evaluated in vitro. Furthermore, their effect on cartilage preservation was tested in mice OA model induced by destabilization of medial meniscus. OA severity was quantified using OARSI histological scoring. Transcriptional analysis was used to find the possible targets of the chemicals, which were further validated. Results We demonstrated that while oral or intra-articular KGN delivery effectively ameliorated OA phenotypes in mice, only 4-ABP was detectable in cartilage. 4-ABP could induce chondrogenic differentiation and proliferation of MSC in vitro and promote cartilage repair in OA mouse models mainly by increasing the number of CD44+/CD105+ stem-cell and prevention of matrix loss. These effect of 4-ABP was stronger than that of KGN. Transcriptional profiling of 4-ABP-stimulated MSC suggested that RPS6KA2 and the PI3K-Akt pathway were 4-ABP targets; 4-ABP could activate the PI3K-Akt pathway to promote MSC proliferation and repair OA injury, which was blocked in RPS6KA2-knockdown MSC or RPS6KA2-deficient mice.Conclusion 4-ABP bio-distribution in cartilage promotes proliferation and chondrogenic differentiation of MSC, and repairs osteoarthritic lesions via PI3K-Akt pathway activation.

Characterization of Papaya ringspot virus isolates infecting transgenic papaya 'Huanong No.1' in South China.

In 2006, the release and cultivation of the genetically modified papaya cultivar 'Huanong No.1' successfully controlled the destructive papaya ringspot disease caused by Papaya ringspot virus (PRSV) in South China. However, some transgenic papaya plants from Guangdong and Hainan are found infected by PRSV. In this study, Field investigation was carried out and susceptible transgenic papaya samples were collected during 2012-2016. Twenty representative isolates were artificially inoculated into Cucurbita pepo and commercialised 'Huanong No.1' papaya, and results indicated that the plants showed obvious disease symptoms. Phylogenetic analysis of CP genes of 120 PRSV-infected isolates showed that PRSV can be divided into three groups. Isolates from Guangdong and Hainan belong to Group III, which is further divided into two subgroups. The isolates collected in this study have greatly diverged from the previously reported dominant strains Ys, Vb and Sm in South China, indicating that they belong to a new lineage. Further analysis showed a highly genetic differentiation between isolates, and 27.1% of the isolates were identified as recombinants on the basis of CP nucleotide sequences. These results indicate that the genetic variation of PRSV and the formation of the new virus lineage may explain the loss of transgenic papaya resistance in South China.

One-step Derivation of Functional Mesenchymal Stem Cells from Human Pluripotent Stem Cells.

Mesenchymal stem cells (MSCs) are invaluable cell sources for understanding stem cell biology and potential application in tissue engineering and regenerative medicine. The current issues of MSCs that demand to be further addressed are limited donors, tissue sources and limited capacity of ex vivo expansion. Here, we describe a simple and easy protocol for generating functional mesenchymal stem cells from human pluripotent stem cells (hPSCs) via one-step low glucose medium switch strategy in feeder-free culture system. In this protocol, human induced pluripotent stem cells (hiPSCs) and H9 human embryonic stem cells (hESCs) were successfully differentiated into MSCs, named hiPSC-MSCs and hESC-MSCs, respectively. The derived hiPSC-MSCs and hESC-MSCs exhibited common MSC characteristics as MSCs derived from human bone marrow (hBM-MSCs), including expressing MSC surface markers and possessing capability of tri-lineage differentiation in vitro (adipogenesis, osteogenesis and chondrogenesis). As compared with other available protocols, our protocol can be applied to generate a large number of MSCs from hPSCs with high efficiency, low-cost manner, moreover, not involving embryoid body, mouse feeder-cell, flow sorting, and pathway inhibitors (such as SB203580 and SB431542). We believe that this protocol could provide a robust platform to reach the future demand for producing the industrial scale of MSC from hPSCs for autologous cell-based therapy.

[EFFECTS OF BONE MARROW MESENCHYMAL STEM CELLS TRANSPLANTATION FOR TREATING RAT SPINAL CORD INJURY AND CYTOKINE EXPRESSION AT INJURY SITES].

OBJECTIVE: To investigate the effects of bone marrow mesenchymal stem cells (BMSCs) transplantation for treating spinal cord injury (SCI) in rat and the cytokine expression changes in the local injury tissues. METHODS: BMSCs were separated from Sprague Dawley (SD) rat and cultured with the whole bone marrow culture method. rAd-EGFP was used to transfect the 5th generation BMSCs for green fluorescent protein (GFP) label. Twelve SD rats were randomly divided into experimental group (n = 6) and control group (n = 6). After the T10 SCI model was established with Allen's impact device in 2 groups, 1 x 1096) GFP-labeled BMSCs and PBS were administered by subarachnoid injection in situ in experimental group and control group, respectively. Basso-Beattie-Bresnahan (BBB) score was used to detect the motor function at immediat, 1, 2, 3, 4, and 5 weeks after SCI. At 5 weeks, the spinal cord tissues were harvested for the histological and immunofluorescent staining examinations to measure the expressions of neural marker molecules, including Nestin, glial fibrillary acidic protein (GFAP), and neuron-specific nuclear protein (NeuN). Cytokine was analyzed with antibody array. RESULTS: At 5 weeks, 2 rats died of urinary tract infection in 2 groups respectively, the other rats survived to the end of experiment. BBB score of experimental group was significantly higher than that of control group at 1, 2, 3, 4, and 5 weeks (P < 0.05). At 5 weeks, histological results showed that there were many cells with regular arrangement in the experimental group; there were less cells with irregular arrangement in the control group. Compared with the control group, Nestin and NeuN expressions significantly increased (P < 0.05), and GFAP expression significantly decreased (P < 0.05) in the experimental group. Leptin and ciliary neurotrophic factor levels were higher in the experimental group than the control group, but granulocyte-macrophage colony-stimulating factor, tumor necrosis factor ß, interleukin 1 ß, and tissue inhibitor of metalloproteinases 1 levels were lower in the experimental group than the control group. CONCLUSION: BMSCs transplantation can improve survival and regeneration of nerve cells and enhances the recovery of nerve function by regulating secretion of cytokines from grafted BMSCs.

Use of Ferritin Expression, Regulated by Neural Cell-Specific Promoters in Human Adipose Tissue-Derived Mesenchymal Stem Cells, to Monitor Differentiation with Magnetic Resonance Imaging In Vitro.

The purpose of this study was to establish a method for monitoring the neural differentiation of stem cells using ferritin transgene expression, under the control of a neural-differentiation-inducible promoter, and magnetic resonance imaging (MRI). Human adipose tissue-derived mesenchymal stem cells (hADMSCs) were transduced with a lentivirus containing the human ferritin heavy chain 1 (FTH1) gene coupled to one of three neural cell-specific promoters: human synapsin 1 promoter (SYN1p, for neurons), human glial fibrillary acidic protein promoter (GFAPp, for astrocytes), and human myelin basic protein promoter (MBPp, for oligodendrocytes). Three groups of neural-differentiation-inducible ferritin-expressing (NDIFE) hADMSCs were established: SYN1p-FTH1, GFAPp-FTH1, and MBPp-FTH1. The proliferation rate of the NDIFE hADMSCs was evaluated using a Cell Counting Kit-8 assay. Ferritin expression was assessed with western blotting and immunofluorescent staining before and after the induction of differentiation in NDIFE hADMSCs. The intracellular iron content was measured with Prussian blue iron staining and inductively coupled plasma mass spectrometry. R2 relaxation rates were measured with MRI in vitro. The proliferation rates of control and NDIFE hADMSCs did not differ significantly (P > 0.05). SYN1p-FTH1, GFAPp-FTH1, and MBPp-FTH1 hADMSCs expressed specific markers of neurons, astrocytes, and oligodendrocytes, respectively, after neural differentiation. Neural differentiation increased ferritin expression twofold, the intracellular iron content threefold, and the R2 relaxation rate two- to threefold in NDIFE hADMSCs, resulting in notable hypointensity in T2-weighted images (P < 0.05). These results were cross-validated. Thus, a link between neural differentiation and MRI signals (R2 relaxation rate) was established in hADMSCs. The use of MRI and neural-differentiation-inducible ferritin expression is a viable method for monitoring the neural differentiation of hADMSCs.

Transplantation of induced pluripotent stem cells improves functional recovery in Huntington's disease rat model.

UNLABELLED: The purpose of this study was to determine the functional recovery of the transplanted induced pluripotent stem cells in a rat model of Huntington's disease with use of 18F-FDG microPET/CT imaging. METHODS: In a quinolinic acid-induced rat model of striatal degeneration, induced pluripotent stem cells were transplanted into the ipsilateral lateral ventricle ten days after the quinolinic acid injection. The response to the treatment was evaluated by serial 18F-FDG PET/CT scans and Morris water maze test. Histological analyses and Western blotting were performed six weeks after stem cell transplantation. RESULTS: After induced pluripotent stem cells transplantation, higher 18F-FDG accumulation in the injured striatum was observed during the 4 to 6-weeks period compared with the quinolinic acid-injected group, suggesting the metabolic recovery of injured striatum. The induced pluripotent stem cells transplantation improved learning and memory function (and striatal atrophy) of the rat in six week in the comparison with the quinolinic acid-treated controls. In addition, immunohistochemical analysis demonstrated that transplanted stem cells survived and migrated into the lesioned area in striatum, and most of the stem cells expressed protein markers of neurons and glial cells. CONCLUSION: Our findings show that induced pluripotent stem cells can survive, differentiate to functional neurons and improve partial striatal function and metabolism after implantation in a rat Huntington's disease model.

Gadolinium promoted proliferation in mouse embryo fibroblast NIH3T3 cells through Rac and PI3K/Akt signaling pathways.

Nephrogenic systemic fibrosis (NSF) is a fibrosing disorder disease developed in patients with underlying renal insufficiency following exposure to gadolinium-based contrast agents (GBCAs). Previous studies have demonstrated that GdCl3 can promote NIH3T3 fibroblast cell proliferation, which provide a new clue to the role of GBCAs in the development of NSF. In the present study, we further clarify the molecular mechanism of Gd-promoted proliferation. The results showed that intervention with the Rac inhibitor NSC23766 abrogated Gd-promoted proliferation. The levels of active Rac1 significantly increased in Gd-treated cells detected by pull-down assays. In addition, the phosphorylation of Akt was significantly elevated in the treatment group, which was blocked by NSC23766. NSC23766 also reduced the migration of NIH3T3 cells enhanced by Gd. Moreover, the F-actin cytoskeleton was strengthened and the mitotic cell numbers was significantly increased after exposure to Gd. These results suggest that Rac and PI3K/Akt signaling pathways, as well as integrin-mediated signal pathway may play important roles in Gd-induced cell proliferation. In addition, under serum-free condition, Gd could decrease ROS accumulation and increase NIH3T3 cell survival.