First Report of Calystegia hederacea as a new host of sweet potato leaf curl Hubei virus.

Calystegia hederacea (Convolvulaceae) is one of the most problematic perennial weeds widely distributed around or in crop fields. Our previous studies showed that C. hederacea is natural reservoir of sweet potato chlorotic stunt virus isolate CH (SPCSV-CH) and sweet potato latent virus (SPLV) (Liu et al. 2020; Zhao et al. 2022). To shed further light on the role of C. hederacea in the epidemiology of sweet potato viruses, in May 2021, a total of seven C. hederacea plants (five asymptomatic, one curling and one mild vein-clearing) were collected from two different sweet potato fields in Xinxiang city of Henan Province in China. Total RNA was prepared from a pool of the seven leaf samples using the EZNA Plant RNA Kit (Omega Bio-Tek, Norcross, GA). A library was constructed from the ribosomal-depleted RNA using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, MA, USA) and sequenced using the Illumina HiSeq platform (Novogene, Tianjin, China). A total of 139,057,020 paired-end clean reads of 150 bp were obtained after removing adaptor sequences and low-quality reads and used for de novo assembly using the Trinity (v2.2.0) software. Blast searches of the assembled contigs longer than 200 bp against NCBI nucleotide and protein sequence databases revealed the presence of 37 contigs (237 to 4885 bp) and 19 contigs (261 to 758 bp) with high nucleotide (nt) identity with SPLV and SPCSV-CH, respectively. The occurrence of SPLV and SPCSV-CH on C. hederacea was previously reported, and thus the contig sequences related to SPLV and SPCSV-CH were not subjected to further verification in this study. In addition, one contig (2,827 bp) with the highest nt sequence identity of 94.94% with sweet potato leaf curl Hubei virus (SPLCHbV, genus Begomovirus, family Geminiviridae, accession no. MK931304) was assembled from 16,592 reads, with average coverage depth of 740.5X. These results suggested the presence of SPLCHbV in C. hederacea. To further confirm the RNA sequencing result, each of the seven samples was tested by PCR using partially overlapping (italicized nucleotides) forward and reverse primers (SweeIn-F1, 5`-GGAGGAAGCTAAGTACGAGAATCAGTTAGAG-3`; SweeIn-R1, 5`-GCTTCCTCCTTGTGATTGTAAGTAACATGG-3`) that were designed based on the SPLCHbV-related contig for amplification of circular DNA viral genome (approximately 2.7 kb). Two symptomatic and three symptomless C. hederacea samples were SPLCHbV positive, indicating that virus-like symptoms of the two C. hederacea samples were probably not induced by SPLCHbV. Two of the five amplified products were completely sequenced and deposited to GenBank (accession nos. OQ551733 and OQ551734). Sequences analysis showed that the complete genome sequences of two SPLCHbV C. headrace isolates (2,763 nt and 2,761 nt) had 96.53% nt identity with each other and 95.92 to 97.70% nt identity with that of SPLCHbV isolate Shandong7-2017 (MK931304). In August 2021, fourteen C. hederacea plants (three symptomatic, 11 asymptomatic) collected from natural fields from Zhumadian and Pingdingshan cities in Henan Province, were tested by PCR using SweeIn-F1/R1 primers for SPLCHbV, showing that eight samples were SPLCHbV positive. SPLCHbV belongs to the sweepoviruses, a group of phylogenetically distinct begomoviruses infecting sweet potato, and was reported to infect sweet potato from many provinces of China (Wang et al., 2021). To the best of our knowledge, this is the first report of SPLCHbV infection in C. hederacea, which expands the natural host range of SPLCHbV.

Autophagy regulates X-ray radiation-induced premature senescence through STAT3-Beclin1-p62 pathway in lung adenocarcinoma cells.

PURPOSE: Ionizing radiation (IR) can induce autophagy and premature senescence; however, the link between them has not been clarified. Our research has shown that X-ray irradiation induces premature senescence in lung adenocarcinoma cells, and its occurrence partially depends on the signal transducer and activator of transcription 3 (STAT3). STAT3 can bind to the promoter region of Beclin1 and regulate its expression. Therefore, it is speculated that there may be a close link between premature senescence and autophagy induced by ionizing radiation in lung adenocarcinoma cells. p62 plays a regulatory role in both autophagy and premature senescence, and it is also an irreplaceable molecule that causes the senescence -associated secretory phenotype (SASP) and a substrate for selective autophagy. This study focused on STAT3, Beclin1 and p62 to clarify the regulatory relationship between IR-induced autophagy and premature senescence. MATERIALS AND METHODS: After exposure to 4 Gy X-rays, a ß-galactosidase staining kit was used to detect the positive rate of premature senescence. STAT3 was overexpressed by pcDNA3.0-STAT3 transfection, and was inhibited by AG490 and rapamycin. Lung adenocarcinoma cells were transduced with the adenovirus vector GV119-Beclin1 to knockdown the expression of Beclin1, or treated with ATM and ATR inhibitors to inhibit premature senescence. Western blotting was used to examine alterations in the radiation response proteins STAT3 and p-STAT3, senescence-related proteins p62 and GATA4, autophagy-related proteins Beclin1, and LC3-II/LC3-I. The mRNA expression levels of SASP factors, including IL-6 and IL-8, were examined by real-time polymerase chain reaction. RESULTS: The activity of SA-ß-gal increased significantly (p < .05), and the expression of p62 decreased significantly at 72 h after 4 Gy X-ray irradiation, accompanied by the increased expression of STAT3, p-STAT3, Beclin1, and the LC3-II/LC3-I ratio. Up- or down-regulation of STAT3 expression was followed by an increase or decrease in Beclin1 expression. After treatment with ATM and ATR inhibitors, there were no significant changes in Beclin1 expression or LC3-II/LC3-I ratio in A549 cells after 4 Gy X-ray irradiation. The p62 expression, the percentage of the SA-ß-gal-positive staining cells, and the expression of IL-6 and IL-8 mRNA in cells transduced with GV119-Beclin1 were also decreased significantly after 4 Gy X-ray irradiation compared with that of the 0 Gy group. CONCLUSION: Radiation induces premature senescence and autophagy in lung adenocarcinoma cells. Autophagy regulates X-ray radiation-induced premature senescence through the STAT3-Beclin1-p62 pathway in lung adenocarcinoma cells.

Calystegia hederacea: a new natural host of Sweet potato latent virus in China.

Sweet potato is a global root crop, with a worldwide production of 91.5 million tons in 2019 (FAOSTAT, 2019). However, virus diseases cause significant yield losses and quality decline in sweet potato. Up to now, over 30 different viruses have been identified in sweet potato (Clark et al. 2012). Expanding knowledge of the host range of sweet potato viruses will provide a benefit for the understanding of virus occurrence and designing appropriate virus control measures. In August 2019, ten Calystegia hederacea and two Convolvulus arvensis (Convolvulaceae) weed plants with or without symptoms of leaf yellowing symptoms were collected from various virus disease-affected sweet potato fields in four cities (Jiaozuo, Xinxiang, Zhengzhou and Kaifeng) of Henan Province for virus detection. The leaves of these plants were harvested and pooled for total RNA extraction using a Plant Total RNA Purification Kit (GMbiolab, Taichung, Taiwan). A library for high-throughput sequencing (HTS) was constructed and sequenced using the Illumina HiSeq 2000 platform by BGI Tech (Shenzhen, China). Clean reads (n = 100,570,346), each 150 bp in length, were de novo assembled using CLC Genomics Workbench 9.5 (Qiagen, USA). The assembled contigs were analyzed against the viral reference genome database in GenBank using the BLASTN and BLASTX searches. Three contigs related to sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus, family Closteroviridae) were identified (Liu et al. 2021). In addition, a total of 20 contigs, ranging from 1,019 to 9,859 bp in length with an average depth of coverage of 1439.26, showed 74.80-87.59% nucleotide (nt) sequence identities with corresponding sequences of sweet potato latent virus (SPLV, genus Potyvirus, family Potyviridae). The sequence of the 9,859-bp contig covering nearly complete genome sequence for SPLV, was deposited in GenBank (accession no.OL625609). These results demonstrated the presence of genetically diverse isolates of SPLV in the pooled samples. To further confirm the HTS result, each of the 12 samples were tested by RT-PCR using SPLV primers (SPLV-F1: 5'-AATGCCAAGGCTACAAGGAGT-3' and SPLV-R1: 5'-CAAGTAGTGTGTGTATGTTCC-3') that targets a partial conserved region of the coat protein gene in SPLV and SPCSV primers designed based on three contigs (ctg1-F1/R1, ctg2-F1/R1, and ctg3-F1/R1) (Liu et al. 2021), respectively. As a result, four symptomless C. hederacea samples tested positive for SPLV, yielding the expected approximately 500 bp PCR fragment, and one leaf yellowing C. hederacea sample tested positive for SPCSV (Liu et al. 2021). The sequences obtained from two of the four amplicons of SPLV (MZ089700 and OM056706) showed 90.2 and 89.8% nt (100 and 99.4% amino acid) identities with the corresponding sequences of the SPLV isolate Shaanxi1 from sweet potato (HQ844148). In 2021, a further 45 C. hederacea plants collected from Shangqiu (n = 6), Xinxiang (n =30) and Pingdingshan (n = 9) cities in Henan Province, were screened by RT-PCR with SPLV-F1/R1 primers, giving an incidence of 33.33%. SPLV is an important potyvirus infecting sweet potato. SPLV is asymptomatic in most sweet potato cultivars in single infection but is able to mediate synergistic viral disease in co-infection with SPCSV (Untiveros et al. 2007). To the best of our knowledge, this is the first report of SPLV in C. hederacea. The finding reported here indicated that C. hederacea may act as a reservoir of SPLV and possible infection source for the sweet potato crop.

Sulfite as the substrate of C-sulfonate metabolism of α, ß-unsaturated carbonyl containing andrographolide: analysis of sulfite in rats' intestinal tract and the reaction kinetics of andrographolide with sulfite.

One-sixth of the currently known natural products contain α, ß-unsaturated carbonyl groups. Our previous studies reported a rare C-sulfonate metabolic pathway. Sulfonate groups were linked to the ß-carbon of α, ß-unsaturated carbonyl-based natural compounds through this pathway. However, the mechanism of this type of metabolism is still not fully understood, especially whether it is formed through enzyme-mediated biotransformation or direct sulfite addition. In this work, the enzyme-mediated and non-enzymatic pathways were studied. First, the sulfite content in rat intestine was determined by LC-MS/MS. The results showed that the amount of sulfite in rat intestinal contents was from 41.5 to 383 µg·g-1, whereas the amount of sulfite in rat feed was lower than the lower limit of quantitation (20 µg·g-1). Second, the reaction kinetics of sulfite-andrographolide reactions in phosphate buffer solutions (pH 6-8) was studied. The half-lives of andrographolide ranged from minutes to hours. This was suggested that the C-sulfonate reaction of andrographolide was very fast. Third, the C-sulfonate metabolites of andrographolide were both detected when andrographolide and L-cysteine-S-conjugate andrographolide were incubated with the rat small intestine contents or sulfite, indicating that the sulfite amount in rat intestine contents was high enough to react with andrographolide, which assisted a significant portion of andrographolide metabolism. Finally, the comparison of andrographolide metabolite profiles among liver homogenate (with NADPH), liver S9 (with NADPH), small intestine contents homogenate (with no NADPH), and sulfite solution incubations showed that the C-sulfonate metabolites were predominantly generated in the intestinal tract by non-enzymatic pathway. In summary, sulfite can serve as a substrate for C-sulfonate metabolism, and these results identified non-enzymatically nucleophilic addition as the potential mechanism for C-sulfonate metabolism of compounds containing α, ß-unsaturated carbonyl moiety.

Regioselective [3 + 2] Cycloaddition Reaction of 3-Alkynoates with Seyferth-Gilbert Reagent.

A Et3N-triggered regioselective [3 + 2] cycloaddition reaction of 3-alkynoates with Seyferth-Gilbert reagent has been developed to furnish a series of trisubstituted pyrazole-3-phosphonates. A one-pot cycloaddition/alkylation sequence further offered access to the corresponding fully substituted pyrazoles.