Identification of CaPCR1, an OFP gene likely involved in pointed versus concave fruit tip regulation in pepper (Capsicum annuum L.) using recombinant inbred lines.

KEY MESSAGE: CaPCR1 (Capana12g002165) was a candidate gene regulating fruit concave/pointed tip shape in pepper. The concave shape of the fruit tip in pepper plants is highly susceptible to drought and low temperature stresses, resulting in the appearance of a pointed tip fruit, which affects its commercial value. However, few studies on the process of fruit tip development and regulatory genes in pepper have been reported. Herein, the developmental process of the ovary before anthesis, especially changes in the shape of the ovary tip, was studied in detail. The results showed that the final fruit tip shape was consistent with the ovary tip shape before anthesis, and a concave tip shape gradually developed. F4 recombinant inbred lines (RILs) were constructed to map the genes regulating fruit tip shape through hybridization of the LRS and SBS pepper inbred lines. CaPCR1 (Capana12g002165), an OFP (OVATE Family Protein) family gene, was located in the candidate region on chr12. Three SNPs were found in the protein coding sequence of CaPCR1 between SBS and LRS, but only one SNP led to amino acid variation. Sequence variations, including base replacements, deletions and insertions, were also detected in the gene promoter region. The relative expression level of the CaPCR1 gene was significantly greater in the concave tip ovary than in the pointed tip ovary. qRT‒PCR analysis revealed that the CaPCR1 gene was expressed mainly in the gynoecium, placenta and green fruit pericarp, which was consistent with its function in ovary and fruit development. Taken together, these results suggested that CaPCR1 is a candidate gene involved in fruit tip shape determination in pepper.

Guide RNA scaffold variants enabled easy cloning of large gRNA cluster for multiplexed gene editing.

Cas9 protein-mediated gene editing has revolutionized genetic manipulation in most organisms. There are many cases where multiplexed gene editing is needed. Cas9 is capable of multiplex gene editing when expressed with multiple guide RNAs. Conventional cloning methods for multiplexed gene editing vector is not efficient due to repeated use of a single-guide RNA scaffold and inefficient ligation. In this study, we conducted structure-guided mutagenesis and random mutagenesis on the original sgRNA scaffold and identified a large number of functional sgRNA scaffold variants. With these scaffold variants and different tRNAs, fusion polymerase chain reaction protocol was developed to rapidly synthesize spacer-scaffold-tRNA-spacer units with up to 9 targets. In conjunction with golden gate cloning, gene editing vectors with up to 24 target sites were efficiently cloned in one-step cloning. One such gene editing vector targeting 12 genes in tomato were tested in stable transformation and 10 out of the 12 genes were found mutated in a single transgenic line. To facilitate the application of multiplexed gene editing using these scaffold variants and tRNAs from different species, a webserver was created to generate primer sets and provide template sequences for the synthesis of large sgRNA expression units based on the user-supplied target sequences and species.

[Significance of high expression of C5orf46 in gastric cancer and potential intervention of tarditional Chinese medicine based on bioinformatics, molecular docking, and cell experiments].

This study aims to investigate the expression, prognosis, and clinical significance of C5orf46 in gastric cancer and to study the interaction between the active components of C5orf46 and tarditional Chinese medicine. The ggplot2 package was utilized for differential expression analysis of C5orf46 in gastric cancer tissues and normal tissues. The survival package was used for survival analysis, univariate regression analysis, and multivariate regression analysis. Nomogram analysis was used to assess the connection between C5orf46 expression in gastric cancer and overall survival. The abundance of tumor-infiltrating lymphocytes was calculated by GSVA package. Coremine database, Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) database, and PubChem database were used to search the potential components corresponding to C5orf46 gene and tarditional Chinese medicine. Molecular docking was performed to explore the binding affinity of potential components to C5orf46. Cell experiments were performed to explore the expression of C5orf46 gene in cells of the blank group, model group, and drug administration groups. As compared with normal tissues, C5orf46 expression was higher in gastric cancer tissues, which had more significant predictive effects in the early stages(T2, N0, and M0). The more advanced the tumor node metastasis(TNM) stage, the higher the C5orf46 expression and the lower the probability of survival of patients with gastric cancer. The expression of C5orf46 positively correlated with the helper T cells1 in gastric cancer and the macrophage infiltration level in gastric cancer, and negatively correlated with B cells, central memory T cells, helper T cells 17, and follicular helper T cells. Seven potential components of C5orf46 were obtained, and three active components were obtained after the screening, which matched five tarditional Chinese medicines, namely, Sojae Semen Nigrum, Jujubae Fructus, Trichosanthis Fructus, Silybi Fructus, and Bambusae Concretio Silicea. Molecular docking revealed that sialic acid and adeno-sine monophosphate(AMP) had a good binding ability to C5orf46. The results of real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot showed that, as compared with the model group, the mRNA and protein expression levels of C5orf46 were significantly lower in the drug administration groups. The lowest expression level was found at the concentration of 40 µmol·L~(-1). The results of this study provide ideas for the clinical development of traditional Chinese medicine compounds for the treatment of gastric cancer as well as other cancers.

Propionate-producing Veillonella parvula regulates the malignant properties of tumor cells of OSCC.

Oral squamous cell carcinoma (OSCC), main head and neck squamous cell carcinomas (HNSCCs), remains a global health concern with unknown pathogenesis. Veillonella parvula NCTC11810 was observed to decrease in saliva microbiome of OSCC patients in this study and the aim was to detect the novel role of Veillonella parvula NCTC11810 in regulating the biological characteristics of OSCC through TROP2/PI3K/Akt pathway. Oral microbial community changes of OSCC patients were detected by 16S rDNA gene sequencing technology. CCK8 assay, Transwell assay, and Annexin V-FITC/PI staining were used for proliferation, invasion, and apoptosis analysis of OSCC cell lines. Expression of proteins were determined by Western blotting analysis. Veillonella parvula NCTC11810 showed decreased in saliva microbiome of TROP2 high-expressed OSCC patients. Culture supernatant of Veillonella parvula NCTC11810 promoted the apoptosis and inhibited the proliferation and invasion ability of HN6 cells, while sodium propionate (SP), the main metabolite of Veillonella parvula NCTC11810, played a similar role through the inhibition of TROP2/PI3K/Akt pathway. Studies above supported the proliferation-inhibiting, invasion-inhibiting, and apoptosis-promoting function of Veillonella parvula NCTC11810 in OSCC cells which provided new insights into oral microbiota and their metabolite as a therapeutic method for OSCC patients with TROP2 high expressing.

A New Biotin Labeling and High-Molecular-Weight RNA Northern Method and Its Application in Viral RNA Detection.

Viruses cause severe crop losses. Studying the interaction between viruses and plants is very important for development of control measures. Northern blot is a well-accepted but very challenging technique to monitor the infection of viruses. Here, we modified the high-molecular-weight (hmw)RNA Northern blot experiment process, utilizing vertical electrophoresis to separate the RNA with denatured agarose gel. This protocol is compatible with regular equipment for Western blots and small RNA Northern blots and requires less input of total RNA. A new method to label the probe with biotin was also developed, which requires commonly used T4 DNA polymerase and detects viral RNA with high sensitivity. These new protocols made hmwRNA Northern blot cost-effective and easy-to-operate, very suitable for studying virus-host interactions.

Global trends of researches on radioactive enteritis: A bibliometric and visualization study.

BACKGROUND: Radiation enteritis (RE) caused by radiation therapy, can seriously affect human health. Recently, studies on RE have been growing rapidly, but there are no bibliometric studies on RE. This study aims to explore the development trends and research hotspots of RE. METHODS: Academic papers on the Web of Science were retrieved on the topic of "radioactive enteritis" from the establishment of the database to December 2020. Countries, institutions, and subjects selected in this field were visualized using Citespace, HistCite, and Vosviewer. The annual trends in publications, distribution, co-authorship status, and research hotspots were analyzed. RESULTS: The authors ranked first in terms of publication amount were Delaney, Francois, Milliat, and Vozenin-Brotons. The United States had the highest number of posts, followed by China, France, the United Kingdom, and Spain. CONCLUSION: Future research in the field of RE will focus on double-blind clinical trials of RE, and the related mechanisms, such as oxidative stress and apoptosis.

Mechanism of Xiaoying Daotan decoction in treating Hashimoto's thyroiditis based on the Notch/Treg/Th17 pathway.

BACKGROUND: The study created mice model of Hashimoto's thyroiditis (HT) and induced thyroid inflammatory cell lines, exploring the mechanism of Xiaoying Daotan decoction on HT. METHODS: Divided HT mice models into model group (0.2 mL saline), Western medicine group (0.2 mL levothyroxine sodium tablets), traditional Chinese medicine group (0.2 mL Xiaoyin Daotan prescription), and Notch protein inhibition group (0.2 mL Xiaoyin Daotan prescription). After treatment, serum Notch protein expression and T cell (Treg)/T helper cell 17 (Th17) cytokines levels were detected through Enzyme linked immunosorbent assay (ELISA). Use real-time qualitative polymerase chain reaction detected Notch protein expression. Thyroid inflammatory cell lines were induced and divided into 5 groups: blank group, iNotch group (knocking down the Notch protein gene of thyroid inflammatory cells), NC group (Notch protein carrier negative control group), iNotch + DS group and DS group (knocking down the Notch protein gene of thyroid inflammatory cells). The cells were treated with serum containing Xiaoying Daotan decoction. After culture, detected Notch protein expression level and Treg/Th17 cytokine level in each group. RESULTS: For the animal experiment, the serum Notch protein expression, the serum levels of key activating proteins Signal Transducer and Activator of Transcription 3 (STAT3), RAR-related orphan receptor gamma T (RORγt), and interleukin (IL)-22 of Th17 cells of mice in the model group was significantly higher than that of the other groups. Compared with the model group and Western medicine group, the serum transforming growth factor-ß (TGF-ß) level of the mice in the traditional Chinese medicine group and the Notch protein inhibition group was significantly higher. All the differences were statistically significant (P<0.05). For the cell experiment, the ß-actin value of Notch protein in thyroid inflammatory cell genes was significantly downregulated and the key activation protein of Treg was significantly upregulated in iNotch + DS group and DS group compared with the other 3 groups. Levels of Th17 key activating proteins STAT3, IL-17, and IL-22 in the iNotch group, iNotch + DS group, and DS group were lower than those of the blank group and NC group, both with statistically significant difference (P<0.001). CONCLUSIONS: The mechanism of Xiaoying Daotan decoction on HT could be related to the immune inflammatory response of the Treg/Th17 cell axis mediated by the Notch protein pathway.

Full-length mRNA sequencing and gene expression profiling reveal broad involvement of natural antisense transcript gene pairs in pepper development and response to stresses.

Pepper is an important vegetable with great economic value and unique biological features. In the past few years, significant development has been made toward understanding the huge complex pepper genome; however, pepper functional genomics has not been well studied. To better understand the pepper gene structure and pepper gene regulation, we conducted full-length mRNA sequencing by PacBio sequencing and obtained 57 862 high-quality full-length mRNA sequences derived from 18 362 previously annotated and 5769 newly detected genes. New gene models were built that combined the full-length mRNA sequences and corrected approximately 500 fragmented gene models from previous annotations. Based on the full-length mRNA, we identified 4114 and 5880 pepper genes forming natural antisense transcript (NAT) genes in-cis and in-trans, respectively. Most of these genes accumulate small RNAs in their overlapping regions. By analyzing these NAT gene expression patterns in our transcriptome data, we identified many NAT pairs responsive to a variety of biological processes in pepper. Pepper formate dehydrogenase 1 (FDH1), which is required for R-gene-mediated disease resistance, may be regulated by nat-siRNAs and participate in a positive feedback loop in salicylic acid biosynthesis during resistance responses. Several cis-NAT pairs and subgroups of trans-NAT genes were responsive to pepper pericarp and placenta development, which may play roles in capsanthin and capsaicin biosynthesis. Using a comparative genomics approach, the evolutionary mechanisms of cis-NATs were investigated, and we found that an increase in intergenic sequences accounted for the loss of most cis-NATs, while transposon insertion contributed to the formation of most new cis-NATs. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at http://bigd.big.ac.cn/gsa Accession number, CRA001412.

A role for small RNA in regulating innate immunity during plant growth.

Plant genomes encode large numbers of nucleotide-binding (NB) leucine-rich repeat (LRR) immune receptors (NLR) that mediate effector triggered immunity (ETI) and play key roles in protecting crops from diseases caused by devastating pathogens. Fitness costs are associated with plant NLR genes and regulation of NLR genes by micro(mi)RNAs and phased small interfering RNAs (phasiRNA) is proposed as a mechanism for reducing these fitness costs. However, whether NLR expression and NLR-mediated immunity are regulated during plant growth is unclear. We conducted genome-wide transcriptome analysis and showed that NLR expression gradually increased while expression of their regulatory small RNAs (sRNA) gradually decreased as plants matured, indicating that sRNAs could play a role in regulating NLR expression during plant growth. We further tested the role of miRNA in the growth regulation of NLRs using the tobacco mosaic virus (TMV) resistance gene N, which was targeted by miR6019 and miR6020. We showed that N-mediated resistance to TMV effectively restricted this virus to the infected leaves of 6-week old plants, whereas TMV infection was lethal in 1- and 3-week old seedlings due to virus-induced systemic necrosis. We further found that N transcript levels gradually increased while miR6019 levels gradually decreased during seedling maturation that occurs in the weeks after germination. Analyses of reporter genes in transgenic plants showed that growth regulation of N expression was post-transcriptionally mediated by MIR6019/6020 whereas MIR6019/6020 was regulated at the transcriptional level during plant growth. TMV infection of MIR6019/6020 transgenic plants indicated a key role for miR6019-triggered phasiRNA production for regulation of N-mediated immunity. Together our results demonstrate a mechanistic role for miRNAs in regulating innate immunity during plant growth.