SR45a plays a key role in enhancing cotton resistance to Verticillium dahliae by alternative splicing of immunity genes.

Alternative splicing (AS) of pre-mRNAs increases the diversity of transcriptome and proteome and plays fundamental roles in plant development and stress responses. However, the prevalent changes in AS events and the regulating mechanisms of plants in response to pathogens remain largely unknown. Here, we show that AS changes are an important mechanism conferring cotton immunity to Verticillium dahliae (Vd). GauSR45a, encoding a serine/arginine-rich RNA binding protein, was upregulated expression and underwent AS in response to Vd infection in Gossypium australe, a wild diploid cotton species highly resistant to Vd. Silencing GauSR45a substantially reduced the splicing ratio of Vd-induced immune-associated genes, including GauBAK1 (BRI1-associated kinase 1) and GauCERK1 (chitin elicitor receptor kinase 1). GauSR45a binds to the GAAGA motif that is commonly found in the pre-mRNA of genes essential for PTI, ETI, and defense. The binding between GauSR45a and the GAAGA motif in the pre-mRNA of BAK1 was enhanced by two splicing factors of GauU2AF35B and GauU1-70 K, thereby facilitating exon splicing; silencing either AtU2AF35B or AtU1-70 K decreased the resistance to Vd in transgenic GauSR45a Arabidopsis. Overexpressing the short splicing variant of BAK1GauBAK1.1 resulted in enhanced Verticillium wilt resistance rather than the long one GauBAK1.2. Vd-induced far more AS events were in G. barbadense (resistant tetraploid cotton) than those in G. hirsutum (susceptible tetraploid cotton) during Vd infection, indicating resistance divergence in immune responses at a genome-wide scale. We provided evidence showing a fundamental mechanism by which GauSR45a enhances cotton resistance to Vd through global regulation of AS of immunity genes.

General Regulatory Factor7 regulates innate immune signalling to enhance Verticillium wilt resistance in cotton.

Sessile growing plants are always vulnerable to microbial pathogen attacks throughout their lives. To fend off pathogen invasion, plants have evolved a sophisticated innate immune system that consists of cell surface receptors and intracellular receptors. Somatic embryogenesis receptor kinases (SERKs) belong to a small group of leucine-rich repeat receptor-like kinases (LRR-RLKs) that function as co-receptors regulating diverse physiological processes. GENRAL REGULATORY FACTOR (GRF) proteins play an important role in physiological signalling transduction. However, the function of GRF proteins in plant innate immune signalling remains elusive. Here, we identified a GRF gene, GauGRF7, that is expressed both constitutively and in response to fungal pathogen infection. Intriguingly, silencing of GRF7 compromised plant innate immunity, resulting in susceptibility to Verticillium dahliae infection. Both transgenic GauGRF7 cotton and transgenic GauGRF7 Arabidopsis lines enhanced the innate immune response to V. dahliae infection, leading to high expression of two helper NLRs (hNLR) genes (ADR1 and NRG1) and pathogenesis-related genes, and increased ROS production and salicylic acid level. Moreover, GauGRF7 interacted with GhSERK1, which positively regulated GRF7-mediated innate immune response in cotton and Arabidopsis. Our findings revealed the molecular mechanism of the GRF protein in plant immune signaling and offer potential opportunities for improving plant resistance to V. dahliae infection.

PRMT1 accelerates cell proliferation, migration, and tumor growth by upregulating ZEB1/H4R3me2as in thyroid carcinoma.

Thyroid carcinoma (TC) represents the most prevalent malignancy of the endocrine system. Protein arginine methyltransferase 1 (PRMT1) is a critical member of the protein arginine methyltransferase family in mammals and is involved in multiple biological processes. This study aimed to investigate the function of PRMT1 in TC. In the present study, human TC cell lines (8505C, CAL62, and BCPAP) and a normal human thyroid cell line Nthy­ori 3­1 were employed. Small interfering RNA targeting PRMT1 was used to knock down PRMT1 expression in 8505C cells, and PRMT1 overexpression plasmids were transfected into BCPAP cells. Cell viability was assessed using a CCK­8 and colony formation assays. Apoptosis was measured using flow cytometry and TUNEL assays. Cell migration was assessed using wound healing and Transwell assays. Reverse transcription­quantitative PCR was used to determine the mRNA expression levels of PRMT1. Western blotting was used to detect the protein expression levels of PRMT1, E­cadherin, vimentin, H4R3me2as, and zinc­finger E homeobox­binding 1 (ZEB1). Notably, PRMT1 expression was elevated in TC (P<0.01). PRMT1 knockdown inhibited TC cell proliferation and migration and concurrently enhanced migration. Furthermore, PRMT1 knockdown suppressed tumor growth and metastasis in a mouse model of TC. PRMT1 downregulation increased E­cadherin expression and decreased the expression of vimentin, H4R3me2as, and ZEB1 in the TC cells and the mouse model of TC. Conversely, PRMT1 overexpression had the opposite effect on TC malignant characteristics (P<0.05). These findings suggest that PRMT1 knockdown inhibited TC malignancy by downregulating H4R3me2as/ZEB1, thereby highlighting novel therapeutic targets and diagnostic markers for the management of TC.

Genetic dissection of lint percentage in short-season cotton using combined QTL mapping and RNA-seq.

KEY MESSAGE: In total, 17 QTLs for lint percentage in short-season cotton, including three stable QTLs, were detected. Twenty-eight differentially expressed genes located within the stable QTLs were identified, and two genes were validated by qRT-PCR. The breeding and use of short-season cotton have significant values in addressing the question of occupying farmlands with either cotton or cereals. However, the fiber yields of short-season cotton varieties are significantly lower than those of middle- and late-maturing varieties. How to effectively improve the fiber yield of short-season cotton has become a focus of cotton research. Here, a high-density genetic map was constructed using genome resequencing and an RIL population generated from the hybridization of two short-season cotton accessions, Dong3 and Dong4. The map contained 4960 bin markers across the 26 cotton chromosomes and spanned 3971.08 cM, with an average distance of 0.80 cM between adjacent markers. Based on the genetic map, quantitative trait locus (QTL) mapping for lint percentage (LP, %), an important yield component trait, was performed. In total, 17 QTLs for LP, including three stable QTLs, qLP-A02, qLP-D04, and qLP-D12, were detected. Three out of 11 non-redundant QTLs overlapped with previously reported QTLs, whereas the other eight were novel QTLs. A total of 28 differentially expressed genes associated with the three stable QTLs were identified using RNA-seq of ovules and fibers at different seed developmental stages from the parental materials. The two genes, Ghir_A02G017640 and Ghir_A02G018500, may be related to LP as determined by further qRT-PCR validation. This study provides useful information for the genetic dissection of LP and promotes the molecular breeding of short-season cotton.

GbCYP72A1 Improves Resistance to Verticillium Wilt via Multiple Signaling Pathways.

Verticillium dahliae is a fungal pathogen that causes Verticillium wilt (VW), which seriously reduces the yield of cotton owing to biological stress. The mechanism underlying the resistance of cotton to VW is highly complex, and the resistance breeding of cotton is consequently limited by the lack of in-depth research. Using quantitative trait loci (QTL) mapping, we previously identified a novel cytochrome P450 (CYP) gene on chromosome D4 of Gossypium barbadense that is associated with resistance to the nondefoliated strain of V. dahliae. In this study, the CYP gene on chromosome D4 was cloned together with its homologous gene on chromosome A4 and were denoted as GbCYP72A1d and GbCYP72A1a, respectively, according to their genomic location and protein subfamily classification. The two GbCYP72A1 genes were induced by V. dahliae and phytohormone treatment, and the findings revealed that the VW resistance of the lines with silenced GbCYP72A1 genes decreased significantly. Transcriptome sequencing and pathway enrichment analyses revealed that the GbCYP72A1 genes primarily affected disease resistance via the plant hormone signal transduction, plant-pathogen interaction, and mitogen-activated protein kinase (MAPK) signaling pathways. Interestingly, the findings revealed that although GbCYP72A1d and GbCYP72A1a had high sequence similarity and both genes enhanced the disease resistance of transgenic Arabidopsis, there was a difference between their disease resistance abilities. Protein structure analysis revealed that this difference was potentially attributed to the presence of a synaptic structure in the GbCYP72A1d protein. Altogether, the findings suggested that the GbCYP72A1 genes play an important role in plant response and resistance to VW.

Membrane Localized GbTMEM214s Participate in Modulating Cotton Resistance to Verticillium Wilt.

Verticillium wilt (VW) is a soil-borne fungal disease caused by Verticillium dahliae Kleb, which leads to serious damage to cotton production annually in the world. In our previous study, a transmembrane protein 214 protein (TMEM214) gene associated with VW resistance was map-based cloned from Gossypium barbadense (G. barbadense). TMEM214 proteins are a kind of transmembrane protein, but their function in plants is rarely studied. To reveal the function of TMEM214s in VW resistance, all six TMEM214s were cloned from G. barbadense in this study. These genes were named as GbTMEM214-1_A/D, GbTMEM214-4_A/D and GbTMEM214-7_A/D, according to their location on the chromosomes. The encoded proteins are all located on the cell membrane. TMEM214 genes were all induced with Verticillium dahliae inoculation and showed significant differences between resistant and susceptible varieties, but the expression patterns of GbTMEM214s under different hormone treatments were significantly different. Virus-induced gene silencing analysis showed the resistance to VW of GbTMEM214s-silenced lines decreased significantly, which further proves the important role of GbTMEM214s in the resistance to Verticillium dahliae. Our study provides an insight into the involvement of GbTMEM214s in VW resistance, which was helpful to better understand the disease-resistance mechanism of plants.

Oncotype DX 21-gene test has a low recurrence score in both pure and mixed mucinous breast carcinoma.

The Oncotype DX 21-gene test can be used to predict chemotherapy efficacy in patients with estrogen receptor (ER)-positive and HER2-negative breast cancer; however, the data on the 21-gene recurrence score (RS) for mucinous breast carcinoma (MBC) are limited. The present study aimed to evaluate the distribution pattern and clinical value of the 21-gene RS in patients with MBC. A total of 38 pure MBC (PMBC) and 11 mixed MBC (MMBC) cases were retrospectively analyzed, and a total of 29 ER-positive and HER2-negative MBCs underwent the Oncotype DX 21-gene test. There were no statistically significant differences between the PMBCs and MMBCs in age, tumor size and molecular subtype; however, patients with MMBC showed a significantly higher incidence rate of nodal metastases compared with that in patients with PMBC (72.7 vs. 16.2%, respectively). Following surgery, 87.8 and 59.2% of the enrolled patients received endocrine therapy and chemotherapy, respectively. With a median follow-up of 65.6 months, the 5-year disease-free survival and overall survival rates were 97.0 and 100.0%, respectively. The 21-gene test revealed that the proportions of patients with MBC categorized into low (RS <18), intermediate (RS ≥18-30) and high (RS ≥30) risk groups were 51.7, 44.8 and 3.5%, respectively, and there was no statistically significant difference between the PMBC and MMBC cases. Notably, among the genes in the 21-gene RS testing, the expression levels of cathepsin V, progesterone receptor (PR) and CD68 were significantly higher in the PMBC group compared with that in the MMBC group. In conclusion, the current study demonstrated that patients with MBC had a favorable prognosis, and both PMBC and MMBC cases had a low- and intermediate-risk RS, which suggests that a considerable proportion of patients may be able to avoid chemotherapy. In addition, the high expression level of PR, based on the 21-gene test in PMBCs, indicated that they may have a more favorable response to endocrine therapy than MMBCs.

Effects of chronic exposure to nonylphenol at environmental concentration on thyroid function and thyroid hyperplasia disease in male rats.

The aim of this work was to determine whether chronic exposure to nonylphenol (NP), a representative substance of environmental endocrine disruptors (EEDs), at environmental concentration would have toxic effects on thyroid function and thyroid hyperplasia disease. Two hundred SPF Sprague-Dawley rats were divided into five groups (n = 40 per group): blank control group (corn oil), low-dose NP exposure group (0.4 mg/kg/d), medium-dose NP exposure group (4 mg/kg/d), high-dose NP exposure group (40 mg/kg/d), and estradiol control group (E2: 30 µg/kg/d). The rats were treated by gavage for 34 weeks, which were sampled twice (17 weeks and 34 weeks respectively). NP accumulation in the thyroid tissue (F = 52.93, P < 0.001) and serum (F = 5.54, P = 0.00) continuously increased in a significant dose-effect relationship. After NP exposure, the serum FT3 levels exhibited a dose-dependent increasing trend (F = 4.68, P = 0.01), while the serum FT4 level showed an opposite trend (F = 3.93, P= 0.01). Compared with the control group, hyperechoic areas (i.e., calcification points) were observed in the high-dose group. Follicular epithelial stratification was extremely severe, the monolayer cubic epithelial cells became flat, and the area of single follicles was even smaller in the high-dose group. In the high-dose NP group, there were numerous mitochondria that were severely swollen. The rough endoplasmic reticulum was abundant, with obvious expansion and vesiculation. The relative expression of ERα (F = 5.29, P = 0.00), ERß (F = 10.17, P = 0.00), TRα (F = 7.71, P = 0.00), TRß (F = 3.52.17, P = 0.02) and HMGB1 (F = 10.16, P = 0.01) proteins in the thyroid tissue in each NP exposure group was increased compared with the control group, and the relative expression of proteins increased if the exposure time was prolonged under the same exposure dose. Chronic exposure to NP at environmental concentration could have toxic effects on thyroid function, and induce thyroid hyperplasia disease in male rats.

Genome-Wide Introgression and Quantitative Trait Locus Mapping Reveals the Potential of Asian Cotton (Gossypium arboreum) in Improving Upland Cotton (Gossypium hirsutum).

Gossypium arboreum (2n=2x=26, A2), the putative progenitor of the At-subgenome of Gossypium hirsutum (2n=4x=52, AD), is a repository of genes of interesting that have been eliminated during evolution/domestication of G. hirsutum. However, its valuable genes remain untapped so far due to species isolation. Here, using a synthetic amphiploid (AADDA2A2) previously reported, we developed a set of 289 G. arboreum chromosome segment introgression lines (ILs) in G. hirsutum by expanding the backcrossing population and through precise marker-assisted selection (MAS) although complex chromosomal structural variations existed between parents which severely hindered introgression. Our results showed the total coverage length of introgressed segments was 1,116.29 Mb, representing 78.48% of the At-subgenome in the G. hirsutum background, with an average segment-length of 8.69 Mb. A total of 81 co- quantitative trait loci (QTLs) for yield and fiber quality were identified by both the RSTEP-ADD-based QTL mapping and the genome-wide association study (GWAS) analysis, with 1.01-24.78% of the phenotypic variance explained. Most QTLs for boll traits showed negative additive effects, but G. arboreum still has the potential to improve boll-number traits in G. hirsutum. Most QTLs for fiber quality showed negative additive effects, implying these QTLs were domesticated in G. hirsutum compared with G. arboreum and, a small quantity of fiber quality QTLs showing positive additive effects, conversely; however, indicates that G. arboreum has the underlying genes of enhancing fiber quality of G. hirsutum. This study provides new insights into the breeding genetic potential of G. arboreum, lays the foundation for further mining favorable genes of interest, and provides guidance for inter-ploidy gene transference from relatives into cultivated crops.

Relationship Orientation, Justice Perception, and Opportunistic Behavior in PPP Projects: An Empirical Study From China.

An equal and high-quality partnership between public and private sectors is essential to the sustainable development of public-private partnership (PPP) projects. However, in the special social circumstance in China, the public sector has a strong voice in PPP projects. According to the existing research on PPP project failure, the government's dishonest performance and negative cooperative attitude and the private sector's speculative behavior of concealing information will lead to termination or even failure of project. The attitude and behavior that reflect the relationship orientation of public sector may determine whether the private sector adopts an opportunistic behavior. However, few studies have revealed the mechanism of relationship orientation on opportunism in PPP projects. This paper proposes the connotation of the public sector's relationship orientation and designs a measurement scale from three aspects: emotional relationship orientation, instrumental relationship orientation, and rent-seeking relationship orientation. Based on the data from large construction enterprises, financial institutions and investors, and scholars with practical experience in PPP projects, this paper explores the mechanism of the public sector's relationship orientation on the private sector's justice perception and opportunistic behavior by using the structural equation model (SEM). The results show that the public sector's relationship orientation significantly affects the formation and development of the private sector's justice perception and opportunistic behavior, justice perception plays a mediating role in the process of relationship orientation acting on opportunistic behavior, and the instrumental relationship orientation is more conducive to reducing the opportunistic behavior. The results provide new ideas for changing the public sector's concept and attitude and regulating behavior in PPP projects.

Sorafenib and radioiodine-refractory differentiated thyroid cancer (RR-DTC): a systematic review and meta-analysis.

BACKGROUND: Except conventional treatments, research on medical approach for radioiodine-refractory differentiated thyroid cancer (RR-DTC) was considered particularly challenging. Sorafenib, a novel biological agent, has been widely studied in the treatment of RR-DTC for years. We performed a systematic review and meta-analysis to explore the efficiency and safety of treating RR-DTC patients with sorafenib. METHODS: An electronic search on PubMed/Medline and Embase was carried out to search associated articles. Fixed-effects or random-effects models were chose according to the heterogeneity. RESULTS: A total of 15 eligible studies (636 patients) were included. As shown by the only randomised clinical trial-DECISION, sorafenib significantly improved progression-free survival (PFS) compared with placebo in patients with progressive RR-DTC. The pooled analysis indicated that there were 26% patients (95% CI: 0.19-0.34) achieved partial response (PR), and 44% patients (98% CI: 0.39-0.48) achieved stable disease (SD). The most frequent adverse effects (AEs) observed included hand-foot syndrome (HFS), diarrhoea, fatigue, alopecia, weight loss (WS) and rash, the incidence of all grades AEs for which were 71%, 60%, 59%, 55%, 51% and 50%, respectively. There were 68% patients (252/368), who had a dose reduction because of the drug toxicities and AEs. CONCLUSIONS: Sorafenib could improve PFS in patients with progressive RR-DTC, comparing with placebo. Due to the resistance to conventional treatments, sorafenib is considered as a promising treatment for RR-DTC by most physicians specialised in this field. However, the use of sorafenib should be cautious due to a high incidence of AEs caused by the agent. More effective agents with less toxicities are warranted.

Genome-wide quantitative trait loci reveal the genetic basis of cotton fibre quality and yield-related traits in a Gossypium hirsutum recombinant inbred line population.

Cotton is widely cultivated globally because it provides natural fibre for the textile industry and human use. To identify quantitative trait loci (QTLs)/genes associated with fibre quality and yield, a recombinant inbred line (RIL) population was developed in upland cotton. A consensus map covering the whole genome was constructed with three types of markers (8295 markers, 5197.17 centimorgans (cM)). Six fibre yield and quality traits were evaluated in 17 environments, and 983 QTLs were identified, 198 of which were stable and mainly distributed on chromosomes 4, 6, 7, 13, 21 and 25. Thirty-seven QTL clusters were identified, in which 92.8% of paired traits with significant medium or high positive correlations had the same QTL additive effect directions, and all of the paired traits with significant medium or high negative correlations had opposite additive effect directions. In total, 1297 genes were discovered in the QTL clusters, 414 of which were expressed in two RNA-Seq data sets. Many genes were discovered, 23 of which were promising candidates. Six important QTL clusters that included both fibre quality and yield traits were identified with opposite additive effect directions, and those on chromosome 13 (qClu-chr13-2) could increase fibre quality but reduce yield; this result was validated in a natural population using three markers. These data could provide information about the genetic basis of cotton fibre quality and yield and help cotton breeders to improve fibre quality and yield simultaneously.

Identification of genomewide single-nucleotide polymorphisms associated with presummer, summer and autumn bolls in upland cotton.

Presummer, summer, and autumn bolls (PSB, SB and AB, respectively) in cotton are related to both maturity and yield. Therefore, studying their genetic basis is important for breeding purposes. In this study, we developed an association analysis panel consisting of 169 upland cotton accessions. The panel was phenotyped for PSB, SB and AB across four environments and genotyped using a Cotton SNP80K array. Single-nucleotide polymorphisms (SNPs) associated with these three traits were identified by a genomewide association study. A total of 53,848 high-quality SNPs were screened, and 91 significant trait-associated SNPs were detected. Of the 91 SNPs 33 were associated with PSB, 21 with SB and 37 with AB. Three SNPs for PSB (TM10410, TM13158 and TM21762) and five for AB (TM13730, TM13733, TM13834, TM29666 and TM43214) were repeatedly detected in two environments or by two methods. These eight SNPs exhibited high phenotypic variation of more than 10%, thus allowing their use formarker-assisted selection. The candidate genes for target traits were also identified. These findings provide a theoretical basis for the improvement of early maturity and yield in cotton breeding programmes.

[Isolation of homogeneous polysaccharide from Poria cocos and effect of its sulfated derivatives on migration of human breast cancer MDA-MB-231 cells].

SATB1 plays a crucial role in the invasion and metastasis of breast cancer,and inhibition of SATB1 expression can effectively control breast cancer metastasis. In this study,homogeneous polysaccharides were isolated from Poria cocos and their sulfated derivatives were prepared to screen out the polysaccharide compositions with inhibitory effects on SATB1 expression. Smal-molecule components were removed from P. cocos by ethanol extraction,and P. cocos crude polysaccharide PPS was obtained by water extraction and ethanol precipitation. Then PPS was successively separated by DEAE Sepharose fast flow anion-exchange and Superdex-75 gel permeation chromatographic steps to give PPSW-1. The structure of PPSW-1 was identified and its sulfated derivatives were prepared. Then their inhibitory effects on human breast cancer MDA-MB-231 cells were investigated. A kind of polysaccharide,PPSW-1 with inhibitory effect on human breast cancer MDA-MB-231 cells,was obtained from P. cocos,with a relative molecular weight of 3. 06×104,and structure of 1,6-branched 1,3-α-D-galactan. PPSW-1 and its sulfated derivative Sul-W-1 showed good inhibitory effect on cells migration,and the water solubility of Sul-W-1 was better than that of PPSW-1. In addition,it was found that polysaccharide of P. cocos and its sulfated derivative can inhibit expression of SATB1. In this study,a kind of homogeneous polysaccharide with inhibitory effect on human breast cancer MDA-MB-231 cells was isolated from P. cocos,and its sulfated derivative with similar efficacy but better solubility was prepared,laying the foundation for the substance basis study of P. cocos.

Overexpression of LASS2 inhibits proliferation and causes G0/G1 cell cycle arrest in papillary thyroid cancer.

BACKGROUND: The aim of this study was to investigate the role of LAG1 longevity-assurance homologue 2 (LASS2) in papillary thyroid cancer (PTC). METHODS: Immunohistochemistry staining was conducted to explore the expression levels of LASS2 in PTC tissues and adjacent normal thyroid tissues and nodular goiter tissues. Western blotting and RT-qPCR were performed to explore the expression levels of LASS2 in three PTC cell lines (TPC-1, K1, BCPAP). An Adv-LASS2-GFP recombinant adenovirus vector was constructed and transduced into BCPAP cells. Then CCK-8 assay, colony formation assay, cell cycle distribution, and apoptosis were performed. Western blotting was used to examine the expression of p21, cyclin D1, cyclin-dependent kinase 4, p53 and p-p53. RESULTS: LASS2 was downregulated in PTC tissues compared with adjacent thyroid tissues or nodular goiter tissues. In addition, the expression of LASS2 was found to be associated with TNM stage and lymph node metastasis. BCPAP cells expressed the lowest LASS2 compared to TPC-1 cells or K1 cells. Overexpression of LASS2 significantly inhibited proliferation, promoted apoptosis and caused G0/G1 cell cycle arrest in BCPAP cells. Furthermore, overexpression of LASS2 significantly increased the expression of p21, inhibited the expression of cyclin D1 and cyclin-dependent kinase 4, and increased the expression of p-p53, but did not effect the expression of p53 in BCPAP cells. CONCLUSION: Our findings indicate that overexpression of LASS2 inhibits PTC cell proliferation, promotes apoptosis and causes G0/G1 cell cycle arrest via a p53-dependent pathway. Thus, LASS2 may serve as a novel biomarker in PTC.