Manufacturing, quality control, and GLP-grade preclinical study of nebulized allogenic adipose mesenchymal stromal cells-derived extracellular vesicles.

BACKGROUND: Human adipose stromal cells-derived extracellular vesicles (haMSC-EVs) have been shown to alleviate inflammation in acute lung injury (ALI) animal models. However, there are few systemic studies on clinical-grade haMSC-EVs. Our study aimed to investigate the manufacturing, quality control (QC) and preclinical safety of clinical-grade haMSC-EVs. METHODS: haMSC-EVs were isolated from the conditioned medium of human adipose MSCs incubated in 2D containers. Purification was performed by PEG precipitation and differential centrifugation. Characterizations were conducted by nanoparticle tracking analysis, transmission electron microscopy (TEM), Western blotting, nanoflow cytometry analysis, and the TNF-α inhibition ratio of macrophage [after stimulated by lipopolysaccharide (LPS)]. RNA-seq and proteomic analysis with liquid chromatography tandem mass spectrometry (LC-MS/MS) were used to inspect the lot-to-lot consistency of the EV products. Repeated toxicity was evaluated in rats after administration using trace liquid endotracheal nebulizers for 28 days, and respiratory toxicity was evaluated 24 h after the first administration. In vivo therapeutic effects were assessed in an LPS-induced ALI/ acute respiratory distress syndrome (ARDS) rat model. RESULTS: The quality criteria have been standardized. In a stability study, haMSC-EVs were found to remain stable after 6 months of storage at - 80°C, 3 months at - 20 °C, and 6 h at room temperature. The microRNA profile and proteome of haMSC-EVs demonstrated suitable lot-to-lot consistency, further suggesting the stability of the production processes. Intratracheally administered 1.5 × 108 particles/rat/day for four weeks elicited no significant toxicity in rats. In LPS-induced ALI/ARDS model rats, intratracheally administered haMSC-EVs alleviated lung injury, possibly by reducing the serum level of inflammatory factors. CONCLUSION: haMSC-EVs, as an off-shelf drug, have suitable stability and lot-to-lot consistency. Intratracheally administered haMSC-EVs demonstrated excellent safety at the tested dosages in systematic preclinical toxicity studies. Intratracheally administered haMSC-EVs improved the lung function and exerted anti-inflammatory effects on LPS-induced ALI/ARDS model rats.

The self-compatibility is acquired after polyploidization: a case study of Brassica napus self-incompatible trilinear hybrid breeding system.

Self-incompatibility plays a vital role in angiosperms, by preventing inbreeding depression and maintaining genetic diversity within populations. Following polyploidization, many angiosperm species transition from self-incompatibility to self-compatibility. Here, we investigated the S-locus in Brassicaceae and identified distinct origins for the sRNA loci, SMI and SMI2 (SCR Methylation Inducer 1 and 2), within the S-locus. The SMI loci were found to be widespread in Cruciferae, whereas the SMI2 loci were exclusive to Brassica species. Additionally, we discovered four major S-haplotypes (BnS-1, BnS-6, BnS-7, and BnS-1300) in rapeseed. Overexpression of BnSMI-1 in self-incompatible Brassica napus ('S-70S1300S6 ') resulted in a significant increase in DNA methylation in the promoter regions of BnSCR-6 and BnSCR-1300, leading to self-compatibility. Conversely, by overexpressing a point mutation of BnSmi-1 in the 'S-70S1300S6 ' line, we observed lower levels of DNA methylation in BnSCR-6 and BnSCR-1300 promoters. Furthermore, the overexpression of BnSMI2-1300 in the 'SI-326S7S6 ' line inhibited the expression of BnSCR-7 through transcriptional repression of the Smi2 sRNA from the BnS-1300 haplotype. Our study demonstrates that the self-compatibility of rapeseed is determined by S-locus sRNA-mediated silencing of SCR after polyploidization, which helps to further breed self-incompatible or self-compatible rapeseed lines, thereby facilitating the utilization of heterosis.

Interaction between phenylpropane metabolism and oil accumulation in the developing seed of Brassica napus revealed by high temporal-resolution transcriptomes.

BACKGROUND: Brassica napus is an important oilseed crop providing high-quality vegetable oils for human consumption and non-food applications. However, the regulation between embryo and seed coat for the synthesis of oil and phenylpropanoid compounds remains largely unclear. RESULTS: Here, we analyzed the transcriptomes in developing seeds at 2-day intervals from 14 days after flowering (DAF) to 64 DAF. The 26 high-resolution time-course transcriptomes are clearly clustered into five distinct groups from stage I to stage V. A total of 2217 genes including 136 transcription factors, are specifically expressed in the seed and show high temporal specificity by being expressed only at certain stages of seed development. Furthermore, we analyzed the co-expression networks during seed development, which mainly included master regulatory transcription factors, lipid, and phenylpropane metabolism genes. The results show that the phenylpropane pathway is prominent during seed development, and the key enzymes in the phenylpropane metabolic pathway, including TT5, BAN, and the transporter TT19, were directly or indirectly related to many key enzymes and transcription factors involved in oil accumulation. We identified candidate genes that may regulate seed oil content based on the co-expression network analysis combined with correlation analysis of the gene expression with seed oil content and seed coat content. CONCLUSIONS: Overall, these results reveal the transcriptional regulation between lipid and phenylpropane accumulation during B. napus seed development. The established co-expression networks and predicted key factors provide important resources for future studies to reveal the genetic control of oil accumulation in B. napus seeds.

Circ_0078607 increases platinum drug sensitivity via miR-196b-5p/GAS7 axis in ovarian cancer.

Platinum-based chemotherapy is one of the predominant strategies for treating ovarian cancer (OC), however, platinum resistance greatly influences the therapeutic effect. Circular RNAs (circRNAs) have been found to participate in the pathogenesis of platinum resistance. Our aim was to explore the involvement of circ_0078607 in OC cell cisplatin (DDP) resistance and its potential mechanisms. Circ_0078607, miR-196b-5p, and growth arrest-specific 7 (GAS7) levels were assessed by qPCR. Circ_0078607 stability was assessed by ribonuclease R digestion and actinomycin D treatment. Cell viability of various conic of DDP treatment was measured by CCK-8. The cell proliferation was determined by CCK-8 and colony formation assay. Western blotting was performed for determining GAS7, ABCB1, CyclinD1 and Bcl-2 protein levels. The direct binding between miR-196b-5p and circ_0078607 or GAS7 was validated by dual-luciferase reporter and RIP assay. DDP resistance in vivo was evaluated in nude mice. Immunohistochemistry staining for detecting Ki67 expression in xenograft tumours. Circ_0078607 and GAS7 was down-regulated, but miR-196b-5p was up-regulated in OC samples and DDP-resistant cells. Overexpression of circ_0078607 inhibited DDP resistance, cell growth and induced apoptosis in DDP-resistant OC cells. Mechanistically, circ_0078607 sequestered miR-196b-5p to up-regulate GAS7. MiR-196b-5p mimics reversed circ_0078607 or GAS7 overexpression-mediated enhanced sensitivity. Finally, circ_0078607 improved the sensitivity of DDP in vivo. Circ_0078607 attenuates DDP resistance via miR-196b-5p/GAS7 axis, which highlights the therapeutic potential of circ_0078607 to counter DDP resistance in OC.

Stigma receptors control intraspecies and interspecies barriers in Brassicaceae.

Flowering plants have evolved numerous intraspecific and interspecific prezygotic reproductive barriers to prevent production of unfavourable offspring1. Within a species, self-incompatibility (SI) is a widely utilized mechanism that rejects self-pollen2,3 to avoid inbreeding depression. Interspecific barriers restrain breeding between species and often follow the SI × self-compatible (SC) rule, that is, interspecific pollen is unilaterally incompatible (UI) on SI pistils but unilaterally compatible (UC) on SC pistils1,4-6. The molecular mechanisms underlying SI, UI, SC and UC and their interconnections in the Brassicaceae remain unclear. Here we demonstrate that the SI pollen determinant S-locus cysteine-rich protein/S-locus protein 11 (SCR/SP11)2,3 or a signal from UI pollen binds to the SI female determinant S-locus receptor kinase (SRK)2,3, recruits FERONIA (FER)7-9 and activates FER-mediated reactive oxygen species production in SI stigmas10,11 to reject incompatible pollen. For compatible responses, diverged pollen coat protein B-class12-14 from SC and UC pollen differentially trigger nitric oxide, nitrosate FER to suppress reactive oxygen species in SC stigmas to facilitate pollen growth in an intraspecies-preferential manner, maintaining species integrity. Our results show that SRK and FER integrate mechanisms underlying intraspecific and interspecific barriers and offer paths to achieve distant breeding in Brassicaceae crops.

Induction of Male Sterility by Targeted Mutation of a Restorer-of-Fertility Gene with CRISPR/Cas9-Mediated Genome Editing in Brassica napus L.

Brassica napus L. (canola, oil seed rape) is one of the world's most important oil seed crops. In the last four decades, the discovery of cytoplasmic male-sterility (CMS) systems and the restoration of fertility (Rf) genes in B. napus has improved the crop traits by heterosis. The homologs of Rf genes, known as the restoration of fertility-like (RFL) genes, have also gained importance because of their similarities with Rf genes. Such as a high non-synonymous/synonymous codon replacement ratio (dN/dS), autonomous gene duplications, and a possible engrossment in fertility restoration. B. napus contains 53 RFL genes on chromosomes A9 and C8. Our research aims to study the function of BnaRFL11 in fertility restoration using the CRISPR/Cas9 genome editing technique. A total of 88/108 (81.48%) T0 lines, and for T1, 110/145 (75%) lines carried T-DNA insertions. Stable mutations were detected in the T0 and T1 generations, with an average allelic mutation transmission rate of 81%. We used CRISPR-P software to detect off-target 50 plants sequenced from the T0 generation that showed no off-target mutation, signifying that if the designed sgRNA is specific for the target, the off-target effects are negligible. We also concluded that the mutagenic competence of the designed sgRNAs mediated by U6-26 and U6-29 ranged widely from 31% to 96%. The phenotypic analysis of bnarfl11 revealed defects in the floral structure, leaf size, branch number, and seed production. We discovered a significant difference between the sterile line and fertile line flower development after using a stereomicroscope and scanning electron microscope. The pollen visibility test showed that the pollen grain had utterly degenerated. The cytological observations of homozygous mutant plants showed an anther abortion stage similar to nap-CMS, with a Orf222, Orf139, Ap3, and nad5c gene upregulation. The bnarfl11 shows vegetative defects, including fewer branches and a reduced leaf size, suggesting that PPR-encoding genes are essential for the plants' vegetative and reproductive growth. Our results demonstrated that BnaRFL11 has a possible role in fertility restoration. The current study's findings suggest that CRISPR/Cas9 mutations may divulge the functions of genes in polyploid species and provide agronomically desirable traits through a targeted mutation.

The dynamics of lncRNAs transcription in interspecific F1 allotriploid hybrids between Brassica species.

Interspecific hybridization is the intrinsic forces behind genome evolution. Long non-coding RNAs (lncRNAs) are important for plant biological processes regulation. However, it is unclear that these non-coding fractions are impacted by interspecific hybridization. Here we examined the profiles of lncRNAs by comparing them with coding genes in Brassica napus, three accessions of Brassica rapa, and their F1 hybrids. 6206 high-confidential lncRNAs were identified in F 1 hybrids and their parentals, and the lncRNAs transcriptome in the F1 hybrids was reprogrammed by the genome shock. Notably, genome-wide unbalanced of lncRNAs were observed between An and Ar subgenomes, ELD (Expression Level Dominance) was biased toward the An -genome in F1 hybrids, and ELD of non-conserved lncRNAs was more than conserved lncRNAs. Our findings demonstrate that the reprogramed lncRNAs acts as important role in enhancing plant plasticity, leading to the acquisition of desirable traits in polyploid Brassica species.

Gentianella acuta improves TAC-induced cardiac remodelling by regulating the Notch and PI3K/Akt/FOXO1/3 pathways.

Cardiac remodelling mainly manifests as excessive myocardial hypertrophy and fibrosis, which are associated with heart failure. Gentianella acuta (G. acuta) is reportedly effective in cardiac protection; however, the mechanism by which it protects against cardiac remodelling is not fully understood. Here, we discuss the effects and mechanisms of G. acuta in transverse aortic constriction (TAC)-induced cardiac remodelling in rats. Cardiac function was analysed using echocardiography and electrocardiography. Haematoxylin and eosin, Masson's trichrome, and wheat germ agglutinin staining were used to observe pathophysiological changes. Additionally, real-time quantitative reverse transcription polymerase chain reaction and western blotting were used to measure protein levels and mRNA levels of genes related to myocardial hypertrophy and fibrosis. Immunofluorescence double staining was used to investigate the co-expression of endothelial and interstitial markers. Western blotting was used to estimate the expression and phosphorylation levels of the regulatory proteins involved in autophagy and endothelial-mesenchymal transition (EndMT). The results showed that G. acuta alleviated cardiac dysfunction and remodelling. The elevated levels of myocardial hypertrophy and fibrosis markers, induced by TAC, decreased significantly after G. acuta intervention. G. acuta decreased the expression of LC3 II and Beclin1, and increased p62 expression. G. acuta upregulated the expression of CD31 and vascular endothelial-cadherin, and prevented the expression of α-smooth muscle actin and vimentin. Furthermore, G. acuta inhibited the PI3K/Akt/FOXO1/3a pathway and activated the Notch signalling. These findings demonstrated that G. acuta has cardioprotective effects, such as alleviating myocardial fibrosis, inhibiting hypertrophy, reducing autophagy, and blocking EndMT by regulating the PI3K/Akt/FOXO1/3a and Notch signalling pathways.

Nebulized exosomes derived from allogenic adipose tissue mesenchymal stromal cells in patients with severe COVID-19: a pilot study.

BACKGROUND: Existing clinical studies supported the potential efficacy of mesenchymal stromal cells as well as derived exosomes in the treatment of COVID-19. We aimed to explore the safety and efficiency of aerosol inhalation of the exosomes derived from human adipose-derived MSCs (haMSC-Exos) in patients with COVID-19. METHODS: The MEXCOVID trial is a phase 2a single-arm, open-labelled, interventional trial and patients were enrolled in Jinyintan Hospital, Wuhan, China. Eligible 7 patients were assigned to receive the daily dose of haMSCs-Exos (2.0 × 108 nano vesicles) for consecutively 5 days. The primary outcomes included the incidence of prespecified inhalation-associated events and serious adverse events. We also observed the demographic data, clinical characteristics, laboratory results including lymphocyte count, levels of D-dimer and IL-6 as well as chest imaging. RESULTS: Seven severe COVID-19 related pneumonia patients (4 males and 3 females) were enrolled and received nebulized haMSC-Exos. The median age was 57 year (interquartile range (IQR), 43 year to 70 year). The median time from onset of symptoms to hospital admission and administration of nebulized haMSC-Exos was 30 days (IQR, 15 days to 40 days) and 54 d (IQR, 34 d to 69 d), respectively. All COVID-19 patients tolerated the haMSC-Exos nebulization well, with no evidence of prespecified adverse events or clinical instability during the nebulization or during the immediate post-nebulization period. All patients presented a slight increase of serum lymphocyte counts (median as 1.61 × 109/L vs. 1.78 × 109/L). Different degrees of resolution of pulmonary lesions after aerosol inhalation of haMSC-Exos were observed among all patients, more obviously in 4 of 7 patients. CONCLUSIONS: Our trial shows that a consecutive 5 days inhalation dose of clinical grade haMSC-Exos up to a total amount of 2.0 × 109 nano vesicles was feasible and well tolerated in seven COVID-19 patients, with no evidence of prespecified adverse events, immediate clinical instability, or dose-relevant toxicity at any of the doses tested. This safety profile is seemingly followed by CT imaging improvement within 7 days. Further trials will have to confirm the long-term safety or efficacy in larger population. TRIAL REGISTRATION: MEXCOVID, NCT04276987.

Lin28B-high breast cancer cells promote immune suppression in the lung pre-metastatic niche via exosomes and support cancer progression.

The formation of pre-metastatic niche is a key step in the metastatic burden. The pluripotent factor Lin28B is frequently expressed in breast tumors and is particularly upregulated in the triple negative breast cancer subtype. Here, we demonstrate that Lin28B promotes lung metastasis of breast cancer by building an immune-suppressive pre-metastatic niche. Lin28B enables neutrophil recruitment and N2 conversion. The N2 neutrophils are then essential for immune suppression in pre-metastatic lung by PD-L2 up-regulation and a dysregulated cytokine milieu. We also identify that breast cancer-released exosomes with low let-7s are a prerequisite for Lin28B-induced immune suppression. Moreover, Lin28B-induced breast cancer stem cells are the main sources of low-let-7s exosomes. Clinical data further verify that high Lin28B and low let-7s in tumors are both indicators for poor prognosis and lung metastasis in breast cancer patients. Together, these data reveal a mechanism by which Lin28B directs the formation of an immune-suppressive pre-metastatic niche.

Transcriptome shock in interspecific F1 allotriploid hybrids between Brassica species.

Interspecific hybridization drives the evolution of angiosperms and can be used to introduce novel alleles for important traits or to activate heterosis in crop breeding. Hybridization brings together gene expression networks from two different species, potentially causing global alterations of gene expression in the F1 plants which is called 'transcriptome shock'. Here, we explored such a transcriptome shock in allotriploid Brassica hybrids. We generated interspecific F1 allotriploid hybrids between the allotetraploid species Brassica napus and three accessions of the diploid species Brassica rapa. RNA-seq of the F1 hybrids and the parental plants revealed that 26.34-30.89% of genes were differentially expressed between the parents. We also analyzed expression level dominance and homoeolog expression bias between the parents and the F1 hybrids. The expression-level dominance biases of the Ar, An, and Cn subgenomes was genotype and stage dependent, whereas significant homoeolog expression bias was observed among three subgenomes from different parents. Furthermore, more genes were involved in trans regulation than in cis regulation in allotriploid F1 hybrids. Our findings provide new insights into the transcriptomic responses of cross-species hybrids and hybrids showing heterosis, as well as a new method for promoting the breeding of desirable traits in polyploid Brassica species.

Genetic and Molecular Characterization of a Self-Compatible Brassica rapa Line Possessing a New Class II S Haplotype.

Most flowering plants have evolved a self-incompatibility (SI) system to maintain genetic diversity by preventing self-pollination. The Brassica species possesses sporophytic self-incompatibility (SSI), which is controlled by the pollen- and stigma-determinant factors SP11/SCR and SRK. However, the mysterious molecular mechanism of SI remains largely unknown. Here, a new class II S haplotype, named BrS-325, was identified in a pak choi line '325', which was responsible for the completely self-compatible phenotype. To obtain the entire S locus sequences, a complete pak choi genome was gained through Nanopore sequencing and de novo assembly, which provided a good reference genome for breeding and molecular research in B. rapa. S locus comparative analysis showed that the closest relatives to BrS-325 was BrS-60, and high sequence polymorphism existed in the S locus. Meanwhile, two duplicated SRKs (BrSRK-325a and BrSRK-325b) were distributed in the BrS-325 locus with opposite transcription directions. BrSRK-325b and BrSCR-325 were expressed normally at the transcriptional level. The multiple sequence alignment of SCRs and SRKs in class II S haplotypes showed that a number of amino acid variations were present in the contact regions (CR II and CR III) of BrSCR-325 and the hypervariable regions (HV I and HV II) of BrSRK-325s, which may influence the binding and interaction between the ligand and the receptor. Thus, these results suggested that amino acid variations in contact sites may lead to the SI destruction of a new class II S haplotype BrS-325 in B. rapa. The complete SC phenotype of '325' showed the potential for practical breeding application value in B. rapa.

Characterization of a Common S Haplotype BnS-6 in the Self-Incompatibility of Brassica napus.

Self-incompatibility (SI) is a pollen-stigma recognition system controlled by a single and highly polymorphic genetic locus known as the S-locus. The S-locus exists in all Brassica napus (B. napus, AACC), but natural B. napus accessions are self-compatible. About 100 and 50 S haplotypes exist in Brassica rapa (AA) and Brassica oleracea (CC), respectively. However, S haplotypes have not been detected in B. napus populations. In this study, we detected the S haplotype distribution in B. napus and ascertained the function of a common S haplotype BnS-6 through genetic transformation. BnS-1/BnS-6 and BnS-7/BnS-6 were the main S haplotypes in 523 B. napus cultivars and inbred lines. The expression of SRK in different S haplotypes was normal (the expression of SCR in the A subgenome affected the SI phenotype) while the expression of BnSCR-6 in the C subgenome had no correlation with the SI phenotype in B. napus. The BnSCR-6 protein in BnSCR-6 overexpressed lines was functional, but the self-compatibility of overexpressed lines did not change. The low expression of BnSCR-6 could be a reason for the inactivation of BnS-6 in the SI response of B. napus. This study lays a foundation for research on the self-compatibility mechanism and the SI-related breeding in B. napus.

Preclinical efficacy and clinical safety of clinical-grade nebulized allogenic adipose mesenchymal stromal cells-derived extracellular vesicles.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) turn out to be a promising source of cell-free therapy. Here, we investigated the biodistribution and effect of nebulized human adipose-derived MSC-EVs (haMSC-EVs) in the preclinical lung injury model and explored the safety of nebulized haMSC-EVs in healthy volunteers. DiR-labelled haMSC-EVs were used to explore the distribution of nebulized haMSC-EVs in the murine model. Pseudomonas aeruginosa-induced murine lung injury model was established, and survival rate, as well as WBC counts, histology, IL-6, TNF-α and IL-10 levels in bronchoalveolar lavage fluid (BALF) were measured to explore the optimal therapeutic dose of haMSC-EVs through the nebulized route. Twenty-four healthy volunteers were involved and received the haMSC-EVs once, ranging from 2 × 108 particles to 16 × 108 particles (MEXVT study, NCT04313647). Nebulizing haMSC-EVs improved survival rate to 80% at 96 h in P. aeruginosa-induced murine lung injury model by decreasing lung inflammation and histological severity. All volunteers tolerated the haMSC-EVs nebulization well, and no serious adverse events were observed from starting nebulization to the 7th day after nebulization. These findings suggest that nebulized haMSC-EVs could be a promising therapeutic strategy, offering preliminary evidence to promote the future clinical applications of nebulized haMSC-EVs in lung injury diseases.

Risk Factors and Outcomes for Preoperative Asymptomatic Pulmonary Embolism in Patients Aged 60 Years and Over with Hip Fracture.

OBJECTIVE: To investigate the risk factors for, and outcomes of, preoperative asymptomatic pulmonary embolism (PE) in patients ≥60 years old following delayed operation for hip fracture. METHODS: From March 2017 to December 2018, 90 patients aged ≥60 years with hip fracture who suffered a delay in surgery were recruited to this prospective study following admission to our hospital. Computed tomography pulmonary angiography (CTPA) was used to detect preoperative asymptomatic PE and calculated its incidence. Time from injury to admission, baseline characteristics, medical comorbidities, and blood biomarker levels were evaluated as potential risk factors. Logistic regression analysis was used to identify risk factors. Mortality and major bleeding events were recorded and compared between individuals with PE and without. Data were analyzed by t-test, Mann-Whitney U test, χ2 test, Fisher's exact test, and logistic regression analysis. RESULTS: The incidence of preoperative asymptomatic PE was 18.9% (17/90 patients). In the univariate analysis, the risk factors for preoperative asymptomatic PE were male sex, hypertension, cerebrovascular accident, smoking, plasma D-dimer level, potassium level, urea level, creatinine level, and cysteine level. Multivariate logistic regression analysis showed that the risk of preoperative asymptomatic PE was higher in patients with hypertension (odds ratio [OR] = 10.048; 95% confidence interval [CI], 1.118-90.333), cerebrovascular accident (OR = 20.135; 95% CI, 1.875-216.164), smoking (OR = 48.741; 95% CI, 4.155-571.788), high plasma D-dimer levels (OR = 1.200; 95% CI, 1.062-157.300), and high plasma potassium levels (OR = 12.928; 95% CI, 1.062-157.300). All patients were followed up for 21.0 months (range, 2 to 36 months). Mortality within the first year postoperatively was higher in patients with PE (29.41% vs 9.59%, P = 0.046). CONCLUSIONS: In view of the high incidence of preoperative asymptomatic PE and the inferior prognosis in individuals with PE, routine CTPA examination for preoperative asymptomatic PE could be useful for patients aged ≥60 years with hip fracture for whom surgery is delayed.

The Brassica napus GATA transcription factor BnA5.ZML1 is a stigma compatibility factor.

Self-incompatibility (SI) is a genetic mechanism that rejects self-pollen and thus prevents inbreeding in some hermaphroditic angiosperms. In the Brassicaceae, SI involves a pollen-stigma recognition system controlled by a single locus known as the S locus, which consists of two highly polymorphic genes that encode S-locus cysteine-rich protein (SCR) and S-receptor kinase (SRK). When self-pollen lands on the stigma, the S-haplotype-specific interaction between SCR and SRK triggers SI. Here, we show that the GATA transcription factor BnA5.ZML1 suppresses SI responses in Brassica napus and is induced after compatible pollination. The loss-of-function mutant bna5.zml1 displays reduced self-compatibility. In contrast, overexpression of BnA5.ZML1 in self-incompatible stigmas leads to a partial breakdown of SI responses, suggesting that BnA5.ZML1 is a stigmatic compatibility factor. Furthermore, the expression levels of SRK and ARC1 are up-regulated in bna5.zml1 mutants, and they are down-regulated in BnA5.ZML1 overexpressing lines. SRK affects the cellular localization of BnA5.ZML1 through direct protein-protein interaction. Overall, our findings highlight the fundamental role of BnA5.ZML1 in SI responses in B. napus, establishing a direct interaction between BnA5.ZML1 and SRK in this process.

Generation of Transgenic Self-Incompatible Arabidopsis thaliana Shows a Genus-Specific Preference for Self-Incompatibility Genes.

Brassicaceae species employ both self-compatibility and self-incompatibility systems to regulate post-pollination events. Arabidopsis halleri is strictly self-incompatible, while the closely related Arabidopsis thaliana has transitioned to self-compatibility with the loss of functional S-locus genes during evolution. The downstream signaling protein, ARC1, is also required for the self-incompatibility response in some Arabidopsis and Brassica species, and its gene is deleted in the A. thaliana genome. In this study, we attempted to reconstitute the SCR-SRK-ARC1 signaling pathway to restore self-incompatibility in A. thaliana using genes from A. halleri and B. napus, respectively. Several of the transgenic A. thaliana lines expressing the A. halleri SCR13-SRK13-ARC1 transgenes displayed self-incompatibility, while all the transgenic A. thaliana lines expressing the B. napus SCR1-SRK1-ARC1 transgenes failed to show any self-pollen rejection. Furthermore, our results showed that the intensity of the self-incompatibility response in transgenic A. thaliana plants was not associated with the expression levels of the transgenes. Thus, this suggests that there are differences between the Arabidopsis and Brassica self-incompatibility signaling pathways, which perhaps points to the existence of other factors downstream of B. napus SRK that are absent in Arabidopsis species.

Polarity Protein AF6 Controls Hepatic Glucose Homeostasis and Insulin Sensitivity by Modulating IRS1/AKT Insulin Pathway in an SHP2-Dependent Manner.

Insulin resistance is a major contributing factor in the development of metabolic disease. Although numerous functions of the polarity protein AF6 (afadin and MLLT4) have been identified, a direct effect on insulin sensitivity has not been previously described. We show that AF6 is elevated in the liver tissues of dietary and genetic mouse models of diabetes. We generated liver-specific AF6 knockout mice and show that these animals exhibit enhanced insulin sensitivity and liver glycogen storage, whereas overexpression of AF6 in wild-type mice by adenovirus-expressing AF6 led to the opposite phenotype. Similar observations were obtained from in vitro studies. In addition, we discovered that AF6 directly regulates IRS1/AKT kinase-mediated insulin signaling through its interaction with Src homology 2 domain-containing phosphatase 2 (SHP2) and its regulation of SHP2's tyrosine phosphatase activity. Finally, we show that knockdown of hepatic AF6 ameliorates hyperglycemia and insulin resistance in high-fat diet-fed or db/db diabetic mice. These results demonstrate a novel function for hepatic AF6 in the regulation of insulin sensitivity, providing important insights about the metabolic role of AF6.

Loss of Wwox drives metastasis in triple-negative breast cancer by JAK2/STAT3 axis.

Loss of WW domain-containing oxidoreductase (Wwox) expression has been observed in breast cancer (BC). However, its regulatory effects are largely unknown, especially in triple-negative breast cancer (TNBC). Herein, gene expression profiling revealed that JAK/STAT3 pathway was one of the most differentially modulated pathways in basal-like BC cells. The lower expression of Wwox was significantly correlated with high activation of STAT3 in basal-like cells and TNBC tissues. Overexpression of Wwox markedly inhibited proliferation and metastasis of BC cells by suppressing STAT3 activation, which is to interact with JAK2 to inhibit JAK2 and STAT3 phosphorylation. Furthermore, Wwox limited STAT3 binding to the interleukin-6 promoter, repressing expression of the IL-6 cytokine. Altogether, our data established that Wwox suppresses BC cell metastasis and proliferation by JAK2/STAT3 pathway. Targeting of Wwox with STAT3 could offer a promising therapeutic strategy for TNBC.