RAB31 marks and controls an ESCRT-independent exosome pathway

Exosomes are generated within the multivesicular endosomes (MVEs) as intraluminal vesicles (ILVs) and secreted during the fusion of MVEs with the cell membrane. The mechanisms of exosome biogenesis remain poorly explored. Here we identify that RAB31 marks and controls an ESCRT-independent exosome pathway. Active RAB31, phosphorylated by epidermal growth factor receptor (EGFR), engages flotillin proteins in lipid raft microdomains to drive EGFR entry into MVEs to form ILVs, which is independent of the ESCRT (endosomal sorting complex required for transport) machinery. Active RAB31 interacts with the SPFH domain and drives ILV formation via the Flotillin domain of flotillin proteins. Meanwhile, RAB31 recruits GTPase-activating protein TBC1D2B to inactivate RAB7, thereby preventing the fusion of MVEs with lysosomes and enabling the secretion of ILVs as exosomes. These findings establish that RAB31 has dual functions in the biogenesis of exosomes: driving ILVs formation and suppressing MVEs degradation, providing an exquisite framework to better understand exosome biogenesis.Subject terms: Small GTPases, Endosomes, Multivesicular bodies, Lysosomes, ESCRT     

中国传统大菊叶片形态的数量化定义与分类

中国传统大菊品种叶片形态变异丰富,然而至今仍未对其进行科学的定义和分类,无法有效利用这些形态性状进行品种鉴定和叶形遗传解析。利用数量化分析方法对植物形态进行定义和分类,是植物性状遗传解析的前提。对436个中国传统大菊品种的24个叶形性状进行重新定义及观测,通过相关性分析确定了8个相对独立的性状,用变异系数及主成分分析等...     

Seasonal expressions of GPR41 and GPR43 in the colon of the wild ground squirrels (Spermophilus dauricus)

G-protein-coupled receptor 41 (GPR41) and G-protein-coupled receptor 43 (GPR43) are important short-chain fatty acids (SCFAs) receptors. Previous studies indicated that GPR41 and GPR43 are involved in the secretion of gastrointestinal peptides, and glucose and lipid metabolism, and are closely related to obesity and type II diabetes, and other diseases. The purpose of the study was to explore the relationship of the GPR41 and GPR43 with seasonal breeding, and provide new prospects for further exploring the nutritional needs of breeding. We identified the localization and expression levels of GPR41 and GPR43 in the colon of the wild ground squirrels (Spermophilus dauricus) both in the breeding season and non-breeding season. The histological results revealed that the lumen diameter of the colon had obvious seasonal changes, and the diameter of the colonic lumen in the non-breeding season was larger than that in the breeding season. Immunohistochemical staining suggested that GPR41 and GPR43 are expressed in the simple layer columnar epithelium. In addition, compared with the breeding season, the mRNA and protein expression levels of GPR41 and GPR43 in the colon were higher during the non-breeding season. In general, these results indicated that GPR41 and GPR43 might play a certain role in regulating seasonal breeding.Key words: GPR41, GPR43, colon, wild ground squirrel, seasonal breeding     

TRIM21 suppresses CHK1 activation by preferentially targeting CLASPIN for K63-linked ubiquitination

Expression of the E3 ligase TRIM21 is increased in a broad spectrum of cancers; however, the functionally relevant molecular pathway targeted by TRIM21 overexpression remains largely unknown. Here, we show that TRIM21 directly interacts with and ubiquitinates CLASPIN, a mediator for ATR-dependent CHK1 activation. TRIM21-mediated K63-linked ubiquitination of CLASPIN counteracts the K6-linked ubiquitination of CLASPIN which is essential for its interaction with TIPIN and subsequent chromatin loading. We further show that overexpression of TRIM21, but not a TRIM21 catalytically inactive mutant, compromises CHK1 activation, leading to replication fork instability and tumorigenesis. Our findings demonstrate that TRIM21 suppresses CHK1 activation by preferentially targeting CLASPIN for K63-linked ubiquitination, providing a potential target for cancer therapy.     

Lipid nanoparticle-encapsulated mRNA antibody provides long-term protection against SARS-CoV-2 in mice and hamsters

Monoclonal antibodies represent important weapons in our arsenal to against the COVID-19 pandemic. However, this potential is severely limited by the time-consuming process of developing effective antibodies and the relative high cost of manufacturing. Herein, we present a rapid and cost-effective lipid nanoparticle (LNP) encapsulated-mRNA platform for in vivo delivery of SARS-CoV-2 neutralization antibodies. Two mRNAs encoding the light and heavy chains of a potent SARS-CoV-2 neutralizing antibody HB27, which is currently being evaluated in clinical trials, were encapsulated into clinical grade LNP formulations (named as mRNA-HB27-LNP). In vivo characterization demonstrated that intravenous administration of mRNA-HB27-LNP in mice resulted in a longer circulating half-life compared with the original HB27 antibody in protein format. More importantly, a single prophylactic administration of mRNA-HB27-LNP provided protection against SARS-CoV-2 challenge in mice at 1, 7 and even 63 days post administration. In a close contact transmission model, prophylactic administration of mRNA-HB27-LNP prevented SARS-CoV-2 infection between hamsters in a dose-dependent manner. Overall, our results demonstrate a superior long-term protection against SARS-CoV-2 conferred by a single administration of this unique mRNA antibody, highlighting the potential of this universal platform for antibody-based disease prevention and therapy against COVID-19 as well as a variety of other infectious diseases.Subject terms: Biological techniques, Immunology     

乙型脑炎病毒DNA初免-蛋白加强策略在小鼠模型上的免疫效应评价

为了评价基因Ⅰ型乙型脑炎病毒prM-E DNA疫苗与prM和EⅢ融合抗原亚单位疫苗采用DNA初免-蛋白加强免疫策略对小鼠的免疫效果,本研究将prM-E融合基因插入到pVAX1真核表达载体中,构建重组表达载体prM-E-pVAX1作为DNA疫苗进行初免,利用原核表达系统获得的prM和EⅢ融合抗原作为亚单位疫苗进行加强免疫。将32只4−6周龄雌性BALB/c小鼠随机分成4组,设置prM-E-pVAX1 DNA疫苗组、DNA初免-蛋白加强免疫组、prM和EⅢ融合抗原亚单位疫苗组及pVAX1载体对照组,通过ELISA检测血清中特异性抗体水平;通过噬斑减少中和试验滴定中和抗体滴度;通过细胞因子表达丰度和淋巴细胞增殖试验分析不同疫苗免疫组诱导产生的细胞免疫反应。结果表明,用DNA初免-蛋白加强策略免疫的小鼠诱导产生的中和抗体滴度略高于prM和EⅢ融合抗原亚单位疫苗免疫组,显著高于prM-E-pVAX1 DNA疫苗免疫组。DNA初免-蛋白加强策略在小鼠模型中诱导产生了有效的Th1/Th2型免疫反应,特别是显著诱导了Th1型细胞免疫反应。本研究为预防流行性乙型脑炎提供了新的免疫策略和理论参考依据。     

The Role of DNA Methylation Reprogramming During Sex Determination and Transition in Zebrafish

DNA methylation is a prevalent epigenetic modification in vertebrates, and it has been shown to be involved the regulation of gene expression and embryo development. However, it remains unclear how DNA methylation regulates sexual development, especially in species without sex chromosomes. To determine this, we utilized zebrafish to investigate DNA methylation reprogramming during juvenile germ cell development and adult female-to-male sex transition.We reveal that primordial germ cells(PGCs) undergo significant DNA methylation reprogramming during germ cell development, and the methylome of PGCs is reset to an oocyte/ovary-like pattern at 9 days post fertilization(9 dpf). When DNA methyltransferase(DNMT) activity in juveniles was blocked after 9 dpf, the zebrafish developed into females. We also show that Tet3 is involved in PGC development. Notably, we find that DNA methylome reprogramming during adult zebrafish sex transition is similar to the reprogramming during the sex differentiation from 9 dpf PGCs to sperm. Furthermore, inhibiting DNMT activity can prevent the female-to-male sex transition, suggesting that methylation reprogramming is required for zebrafish sex transition. In summary, DNA methylation plays important roles in zebrafish germ cell development and sexual plasticity.     

The specific binding activities of human urinary radioiodinated colony-stimulating factor-1 to various human tissue cells

1. Colony-stimulating factor (CSF-1) was isolated from a large volume of fresh normal human urine by 5 steps of purification and enrichment. 2. The purification factor is 100,000 fold and the purified compound exhibits a 2.16 x 10(7) U/mg of protein sp. act. 3. The isolated CSF-1 is a sialoglycoprotein with 41.5% of carbohydrate. The almost complete removal of this carbohydrate moiety (up to 91%) was achieved by incubation with trifluoromethane sulfonic acid. 4. The deglycosylated CSF-1 (DG-CSF-1) possesses an apparent Mr 38,000 compared to native CSF-1 with an initial Mr 57,000 (Goa et al., 1988). 5. The features of the interaction of radio-iodinated [125I]CSF-1 with single cell suspensions from various human tissues (bone marrow, spleen, blood, peritoneal cavity, alveolar lavage, lymph node and thymus), were studied. 6. The binding activity of peritoneal macrophages was the highest among the cells examined and erythrocytes, thymus and blood granulocytes showed no CSF-1 binding. 7. On incubation with [125I]CSF-1 at 0 degrees C, cellular binding of [125I]CSF-1 reached a stable maximum within 16 hr. This is in contrast to the association behaviour at higher temperature. 8. At 37 degrees C, cellular associated [125I]CSF-1 levels reached, within 90 min, an unstable maximum which was up to 10 times less than that occurring under the same conditions at 0 degree C. From the Scatchard plot analysis, we obtained the affinity constant and the number of receptor(s). 9. The binding site is sensitive to trypsin. 10. The receptor alone, (labelled by cross-linking to [125I]CSF-1 with di-succinylimidyl-suberate), is a polypeptide with an approx. Mr 110,000. 11. Our results showed that the receptor of CSF-1 is a tyrosin-kinase.