Mutation in BrFLS encoding flavonol synthase induced anthocyanin accumulation in Chinese cabbage.

KEY MESSAGE: BrFLS mutation promoted anthocyanin accumulation in Chinese cabbage, which was verified in four allelic mutants. Chinese cabbage is a major vegetable crop in Eastern Asia. Anthocyanin-rich vibrantly colored varieties are increasingly favored by consumers for their higher nutritional and aesthetic value compared to the typical green varieties of Chinese cabbage. Herein, we identified an anthocyanin accumulation mutant aam1 from a mutant library of EMS-mutagenized Chinese cabbage DH line 'FT', which appeared partial purple on leaves, bolting stems and floral buds. This anthocyanin accumulation trait was genetically controlled by a recessive nuclear gene, and through MutMap mapping and KASP genotyping, BraA10g030950.3C was identified as the candidate causal gene with a G202 to A202 non-synonymous SNP variation in exon 1. Three additional mutants allelic to aam1 were obtained via screening of similar-phenotype mutants from the mutant library, namely aam2/3/4, where the causal SNPs reside in the same gene as aam1, corroborating that the mutation of BraA10g030950.3C caused anthocyanin accumulation. BraA10g030950.3C encodes a flavonol synthase that catalyzes dihydroflavonols substrate into flavonols and is homologous to Arabidopsis FLS1 (AT5G08640), named BrFLS. Compared to wildtype, the expression level of BrFLS was significantly reduced in the mutants, while BrDFR, which is involved in the anthocyanin biosynthesis and competes with FLS for the common substrate dihydroflavonols, was increased. The flavonol synthase activity decreased, and dihydroflavonol 4-reductase activity was elevated. Differentially accumulated flavonoid metabolites were detected between wildtype and aam1, which were enriched primarily in flavonol and anthocyanin pathways. Our results revealed that mutations in the BrFLS gene could contribute to anthocyanin accumulation and provide a new target for Chinese cabbage color modification.

Involvement of CHRNA6 in the Immune Response in Lung Squamous Cell Carcinoma and its Potential as a Drug Target for the Disease.

BACKGROUND: Lung squamous cell carcinoma (LUSC) is a subtype of lung cancer with a poor prognosis and limited treatment options. Previous studies show that some components of the cholinergic pathway may play important roles in the tumorigenesis of lung cancer, including LUSC. OBJECTIVE: The purpose of this study is to investigate the involvement of cholinergic genes in immune infiltration in LUSC, and identify the key genes in the pathway and analyze their potential as targets for LUSC treatment and novel drugs. METHODS: We first screened the cholinergic genes associated with immune infiltration in LUSC based on transcriptomic samples and explored the correlation between the key genes and immune infiltrating cells and immune pathways. Then, we assessed the effect of immunotherapeutic response in the high and low-expression groups of key genes in vitro. And finally, we screened potential drugs for the treatment of LUSC. RESULTS: We found that the expression of CHRNA6, the gene encoding the α6 subunit of nicotinic acetylcholine receptors (nAChR), was significantly correlated with the proportion of immune infiltrating cells in LUSC, and the high expression level of the gene was associated with poor prognosis of the disease. Also, the proportion of Tregs, M1 macrophages, and resting mast cells was correlated with the expression of CHRNA6. In addition, LUSC patients with higher CHRNA6 expression levels had better immunotherapy responses. Furthermore, we found that the drugs, i.e., adavosertib, varbulin and pyrazoloacridine, had a strong affinity with CHRNA6, with adavosertib binding most stably with the protein. CONCLUSION: CHRNA6 may be associated with immune infiltration in LUSC and affects patient prognosis and immunotherapeutic response by regulating immune cells and immune pathways. In addition, adavosertib may be a potential drug for the treatment of LUSC.

The common genes involved in the pathogenesis of Alzheimer's disease and type 2 diabetes and their implication for drug repositioning.

BACKGROUND: The prevalences of Alzheimer's disease (AD) and type 2 diabetes (T2D) continuously increase with the aging of world population. Clinical and epidemiological studies indicate that T2D is an important risk factor for AD. However, the mechanisms underlying the linkage of the two disorders are still not fully elucidated. The aim of this study is to explore the molecular mechanisms of their comorbidity and potential drug targets for AD treatment. METHODS: We first compiled comprehensive lists of genes associated with AD and T2D, respectively. Then, we investigated the signatures of the shared genes and screened for interactions between the hub genes. Subsequently, we used Autodock Vina to perform molecular docking to predict new drug candidates. Lastly, structure and dynamics of docking results were examined by molecular dynamics simulation to verify drug reliability. RESULTS: We obtained 917 AD-associated genes, 631 T2D-associated genes and 175 shared genes between the two disorders for subsequent analyses. Functional analysis revealed that metabolic process, lipid and atherosclerosis, AMPK signaling pathway, insulin resistance, chemokines and cytokines were enriched in the shared genes. In addition, 50 central hub genes were identified, including IL6, TNF, INS, IL1B, AKT1, VEGFA, IL10, TP53, PTGS2, TLR4, and others. Finally, we predicted new drug candidates (verdoheme and stannsoporfin) that could be potentially used for AD treatment. CONCLUSIONS: Our study confirmed that there are important shared genes and pathways between AD and T2D, which may provide clues to reveal the molecular mechanism underlying the pathophysiology of the two diseases and help us to discover novel drug candidates for the treatment of AD. The results may also provide clues into identification of new targets and strategies for prevention and therapy of T2D that predisposes to AD.

BrCPS1 Function in Leafy Head Formation Was Verified by Two Allelic Mutations in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

The formation of the leafy heads of Chinese cabbage is an important agricultural factor because it directly affects yield. In this study, we identified two allelic non-heading mutants, nhm4-1 and nhm4-2, from an ethyl methanesulfonate mutagenic population of a heading Chinese cabbage double haploid line "FT." Using MutMap, Kompetitive Allele-Specific PCR genotyping, and map-based cloning, we found that BraA09g001440.3C was the causal gene for the mutants. BraA09g001440.3C encodes an ent-copalyl diphosphate synthase 1 involved in gibberellin biosynthesis. A single non-synonymous SNP in the seventh and fourth exons of BraA09g001440.3C was responsible for the nhm4-1 and nhm4-2 mutant phenotypes, respectively. Compared with the wild-type "FT," the gibberellin content in the mutant leaves was significantly reduced. Both mutants showed a tendency to form leafy heads after exogenous GA3 treatment. The two non-heading mutants and the work presented herein demonstrate that gibberellin is related to leafy head formation in Chinese cabbage.

Single-Cell Transcriptomics Unveils the Dedifferentiation Mechanism of Lung Adenocarcinoma Stem Cells.

Lung adenocarcinoma (LUAD) is a major subtype of lung cancer, and its prognosis is still poor due to therapy resistance, metastasis, and recurrence. In recent years, increasing evidence has shown that the existence of lung cancer stem cells is responsible for the propagation, metastasis, therapy resistance, and recurrence of the tumor. During their transition to cancer stem cells, tumor cells need to inhibit cell differentiation and acquire invasive characteristics. However, our understanding of the property and role of such lung cancer stem cells is still limited. In this study, lung adenocarcinoma cancer stem cells (LCSCs) were enriched from the PC-9 cell line in a serum-free condition. PC-9 cells grew into spheres and showed higher survival rates when exposed to gefitinib: the drug used for the treatment of LUAD. Additionally, we found that the canonical stemness marker protein CD44 was significantly increased in the enriched LCSCs. Then, LCSCs were inoculated into the groin of nude mice for 1.5 months, and tumors were detected in the animals, indicating the strong stemness of the cells. After that, we performed single-cell RNA sequencing (scRNA-seq) on 7320 LCSCs and explored the changes in their transcriptomic signatures. We identified cell populations with a heterogeneous expression of cancer stem marker genes in LCSCs and subsets with different degrees of differentiation. Further analyses revealed that the activation of the FOXM1 (oncoprotein) transcription factor is a key factor in cell dedifferentiation, which enables tumor cells to acquire an epithelial-mesenchymal transition phenotype and increases the LCSC surface marker CD44. Moreover, we found that the combination of CD44, ABCG2, and ALCAM was a specific marker for LCSCs. In summary, this study identified the potential factors and molecular mechanisms underlying the stemness properties of LUAD cancer cells; it could also provide insight into developing novel and effective therapeutic approaches.

RTN3 - ASC interaction: The potential mechanism behind diabetes-induced cortical neuritic dystrophy.

Recent studies have found that people with diabetes are more vulnerable to cognitive dysfunction, particularly Alzheimer's disease (AD). Previous studies revealed that Reticulon 3 (RTN3) oligomers could induce cortical neuritic dystrophy (CND) in the brains of diabetic rats. However, it is not clear how diabetes induces RTN3 aggregation. In this study, we examined in vivo and in vitro diabetes models to explore the underlying effects of RTN3-mediated neurite dystrophy. The results showed that the binding ability of ASC and RTN3 was significantly increased during diabetes- or high glucose-induced neuritic dystrophy, and ASC siRNA or an anti-inflammatory drug (CP 424174) could inhibit neuritic dystrophy in vitro. These results suggest that the ASC and RTN3 interaction is involved in diabetes-induced CND, and anti-inflammatory therapy might be an effective way to prevent and inhibit diabetes-induced CND.

Mapping of a Pale Green Mutant Gene and Its Functional Verification by Allelic Mutations in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Leaves are the main organ for photosynthesis, and variations in leaf color affect photosynthesis and plant biomass formation. We created two similar whole-plant pale green mutants (pem1 and pem2) from the double haploid (DH) Chinese cabbage line "FT" through ethyl methanesulfonate (EMS) mutagenesis of seeds. Photosynthetic pigment contents and net photosynthetic rates were significantly lower in the mutants than in the wild-type "FT," and the chloroplast thylakoid endomembrane system was poor. Genetic analysis showed that the mutated phenotypes of pem1 and pem2 were caused by a single nuclear gene. Allelism tests showed that pem1 and pem2 were alleles. We mapped Brpem1 to a 64.25 kb region on chromosome A10, using BSR-Seq and map-based cloning of 979 F2 recessive individuals. Whole-genome re-sequencing revealed a single nucleotide polymorphism (SNP) transition from guanine to adenosine on BraA10g021490.3C in pem1, causing an amino acid shift from glycine to glutamic acid (G to E); in addition, BraA10g021490.3C in pem2 was found to have a single nucleotide substitution from guanine to adenosine, causing an amino acid change from E to lysine (K). BraA10g021490.3C is a homolog of the Arabidopsisdivinyl chlorophyllide a 8-vinyl-reductase (DVR) gene that encodes 3,8-divinyl protochlorophyllide a 8-vinyl reductase, which is a key enzyme in chlorophyll biosynthesis. Enzyme activity assay and chlorophyll composition analysis demonstrated that impaired DVR had partial loss of function. These results provide a basis to understand chlorophyll metabolism and explore the mechanism of a pale green phenotype in Chinese cabbage.

The mutation of ent-kaurene synthase, a key enzyme involved in gibberellin biosynthesis, confers a non-heading phenotype to Chinese cabbage (Brassica rapa L. ssp. pekinensis).

The presence of a leafy head is a vital agronomic trait that facilitates the evaluation of the yield and quality of Chinese cabbage. A non-heading mutant (nhm1) was identified in an ethyl methanesulfonate mutagenesis population of the heading Chinese cabbage double haploid line FT. Segregation analysis revealed that a single recessive gene, Brnhm1, controlled the mutant phenotype. Using MutMap, Kompetitive allele-specific PCR, and cloning analyses, we demonstrated that BraA07g042410.3C, which encodes an ent-kaurene synthase involved in the gibberellin biosynthesis pathway, is the nhm1 mutant candidate gene. A single-nucleotide mutation (C to T) in the fourth exon of BraA07g042410.3C caused an amino acid substitution from histidine to tyrosine. Compared to that of the wild-type FT, BraA07g042410.3C in the leaves of the nhm1 mutant had lower levels of expression. In addition, gibberellin contents were lower in the mutant than in the wild type, and the mutant plant phenotype could be restored to that of the wild type after exogenous GA3 treatment. These results indicate that BraA07g042410.3C caused the non-heading mutation. This is the first study to demonstrate a relationship between gibberellin content in the leaves and leafy head formation in Chinese cabbage. These findings facilitate the understanding of the mechanisms underlying leafy head development in Chinese cabbage.

Fine mapping of a leaf flattening gene Bralcm through BSR-Seq in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

Leaf flattening influences plant photosynthesis, thereby affecting product yield and quality. Here, we obtained a stably inherited leaf crinkled mutant (lcm), derived from the Chinese cabbage doubled haploid (DH) 'FT' line using EMS mutagenesis combined with isolated microspore culture. The crinkled phenotype was controlled by a single recessive nuclear gene, namely Bralcm, which was preliminarily mapped to chromosome A01 by bulked segregant analysis RNA-seq, and further between markers SSRS-1 and IndelD-20 using 1,575 recessive homozygous individuals in F2 population by a map-based cloning method. The target region physical distance was 126.69 kb, containing 23 genes; the marker SSRMG-4 co-segregated with the crinkled trait. Further, we found SSRMG-4 to be located on BraA01g007510.3C, a homolog of AHA2, which encodes H+-ATPase2, an essential enzyme in plant growth and development. Sequence analysis indicated a C to T transition in exon 7 of BraA01g007510.3C, resulting in a Thr (ACT) to Ile (ATT) amino acid change. Genotyping revealed that the leaf crinkled phenotype fully co-segregated with this SNP within the recombinants. qRT-PCR demonstrated that BraA01g007510.3C expression in lcm mutant leaves was dramatically higher than that in wild-type 'FT'. Thus, BraA01g007510.3C is a strong candidate gene for Bralcm, and AHA2 is possibly associated with leaf flattening in Chinese cabbage.

Role of BrSDG8 on bolting in Chinese cabbage (Brassica rapa).

KEY MESSAGE: Mapping and resequencing of two allelic early bolting mutants ebm5-1 and ebm5-2 revealed that the BrSDG8 gene is related to bolting in Chinese cabbage (Brassica rapa ssp. pekinensis). Bolting influences the leafy head formation and seed yield of Chinese cabbage therefore being an important agronomic trait. Herein, two allelic early bolting mutants, ebm5-1 and ebm5-2, stably inherited in Chinese cabbage were obtained from wild-type 'FT' seeds by ethyl methane sulfonate mutagenesis. Both mutants flowered significantly earlier than 'FT,' and genetic analysis revealed that the early bolting of the two mutants was controlled by one recessive nuclear gene. With BSR-seq, the mutations originating lines ebm5-1 and ebm5-2 were located to the same region in chromosome A07. Using the 1741 F2 individuals with the ebm5-1 phenotype as the mapping population, this region was narrowed to 56.24 kb between markers InDel18 and InDel45. A single-nucleotide polymorphism (SNP) was aligned to the BraA07g040740.3C (BrSDG8) region by whole-genome resequencing of ebm5-1 mutant and 'FT.' BrSDG8 is a homolog of Arabidopsis thaliana SDG8 encoding a histone methyltransferase affecting H3K4 trimethylation in FLOWERING LOCUS C chromatin. Comparative sequencing established that the SNP occurred on BrSDG8 17th exon in ebm5-1. Genotype analysis showed full co-segregation of the early bolting phenotype with this SNP. Cloning of allelic mutant ebm5-2 indicated that it harbors a deletion mutation on the 12th exon of BrSDG8. Quantitative real-time PCR analysis indicated that BrSDG8 expression level was observably lower in mutant ebm5-1 than in 'FT.' Overall, the present results provide strong evidence that BrSDG8 mutation leads to early bolting in Chinese cabbage, thereby providing a basis to understand the molecular mechanisms underlying this phenotype.

Integrated metabolomic and transcriptomic analysis of the anthocyanin regulatory networks in Salvia miltiorrhiza Bge. flowers.

BACKGROUND: The objectives of this study were to reveal the anthocyanin biosynthesis metabolic pathway in white and purple flowers of Salvia miltiorrhiza using metabolomics and transcriptomics, to identify different anthocyanin metabolites, and to analyze the differentially expressed genes involved in anthocyanin biosynthesis. RESULTS: We analyzed the metabolomics and transcriptomics data of S. miltiorrhiza flowers. A total of 1994 differentially expressed genes and 84 flavonoid metabolites were identified between the white and purple flowers of S. miltiorrhiza. Integrated analysis of transcriptomics and metabolomics showed that cyanidin 3,5-O-diglucoside, malvidin 3,5-diglucoside, and cyanidin 3-O-galactoside were mainly responsible for the purple flower color of S. miltiorrhiza. A total of 100 unigenes encoding 10 enzymes were identified as candidate genes involved in anthocyanin biosynthesis in S. miltiorrhiza flowers. Low expression of the ANS gene decreased the anthocyanin content but enhanced the accumulation of flavonoids in S. miltiorrhiza flowers. CONCLUSIONS: Our results provide valuable information on the anthocyanin metabolites and the candidate genes involved in the anthocyanin biosynthesis pathways in S. miltiorrhiza.

Wear Evolution of the Glass Fiber-Reinforced PTFE under Dry Sliding and Elevated Temperature.

The wear evolution of the glass fiber reinforced Polytetrafluoroethylene (PTFE) sliding against duplex steel at elevated temperature was investigated using the interrupted wear tests coupling with the worn surface observations. The morphological changes of the PTFE composite during the sliding were related to the variation of the tribological properties to analyze the underlying wear mechanisms. Results show that the coefficient of friction and wear rate change with the increase of temperature. During the sliding, three regions can be identified regardless of the temperature. The high temperature is beneficial to the formation of tribo-film. The sequence of wear evolution is PTFE removal, load transfer to glass fiber, and minor formation of tribo-film for the low temperature condition. For high temperatures, the wear behaviors are more complicated. The different phenomena include the third body abrasion, flake delamination of PTFE matrix, scratching and reformation of transfer film on the counterface, and the filling of the large scale PTFE groove. These behaviors may dominate the different stages in the stable region, but occur simultaneously and cause the dynamic steady wear. As a result, the wear rate at 200 °C is slightly fluctuant.