IL-33-Dependent Endothelial Activation Contributes to Apoptosis and Renal Injury in Orientia tsutsugamushi-Infected Mice

Endothelial cells (EC) are the main target for Orientia tsutsugamushi infection and EC dysfunction is a hallmark of severe scrub typhus in patients. However, the molecular basis of EC dysfunction and its impact on infection outcome are poorly understood. We found that C57BL/6 mice that received a lethal dose of O. tsutsugamushi Karp strain had a significant increase in the expression of IL-33 and its receptor ST2L in the kidneys and liver, but a rapid reduction of IL-33 in the lungs. We also found exacerbated EC stress and activation in the kidneys of infected mice, as evidenced by elevated angiopoietin (Ang) 2/Ang1 ratio, increased endothelin 1 (ET-1) and endothelial nitric oxide synthase (eNOS) expression. Such responses were significantly attenuated in the IL-33^-/- mice. Importantly, IL-33^-/- mice also had markedly attenuated disease due to reduced EC stress and cellular apoptosis. To confirm the biological role of IL-33, we challenged wild-type (WT) mice with a sub-lethal dose of O. tsutsugamushi and gave mice recombinant IL-33 (rIL-33) every 2 days for 10 days. Exogenous IL-33 significantly increased disease severity and lethality, which correlated with increased EC stress and activation, increased CXCL1 and CXCL2 chemokines, but decreased anti-apoptotic gene BCL-2 in the kidneys. To further examine the role of EC stress, we infected human umbilical vein endothelial cells (HUVEC) in vitro. We found an infection dose-dependent increase in the expression of IL-33, ST2L soluble ST2 (sST2), and the Ang2/Ang1 ratio at 24 and 48 hours post-infection. This study indicates a pathogenic role of alarmin IL-33 in a murine model of scrub typhus and highlights infection-triggered EC damage and IL-33-mediated pathological changes during the course of Orientia infection.     

Two flavonoid glucosyltransferases from Petunia hybrida: molecular cloning,biochemical properties and developmentally regulated expression

Two flavonoid glucosyltransferases, UDP-glucose:flavonoid 3-O-glucosyltransferase (3-GT) and UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT), are responsible for the glucosylation of anthocyani(di)ns to produce stable molecules in the anthocyanin biosynthetic pathway. The cDNAs encoding 3-GT and 5-GT were isolated from Petunia hybrida by hybridization screening with heterologous probes. The cDNA clones of 3-GT, PGT8, and 5-GT, PH1, encode putative polypeptides of 448 and 468 amino acids, respectively. A phylogenetic tree based on amino acid sequences of the family of glycosyltransferases from various plants shows that PGT8 belongs to the 3-GT subfamily and PH1 belongs to the 5-GT subfamily. The function of isolated cDNAs was identified by the catalytic activities for 3-GT and 5-GT exhibited by the recombinant proteins produced in yeast. The recombinant PGT8 protein could convert not only anthocyanidins but also flavonols into the corresponding 3-O-glucosides. In contrast, the recombinant PH1 protein exhibited a strict substrate specificity towards anthocyanidin 3-acylrutinoside, comparing with other 5-GTs from Perilla frutescens and Verbena hybrida, which showed broad substrate specificities towards several anthocyanidin 3-glucosides. The mRNA expression of both 3-GT and 5-GT increased in the early developmental stages of P. hybrida flower, reaching the maximum at the stage before flower opening. Southern blotting analysis of genomic DNA indicates that both 3-GT and 5-GT genes exist in two copies in P. hybrida, respectively. The results are discussed in relation to the molecular evolution of flavonoid glycosyltransferases.     

Improvement of Cephalosporin C Production by Recombinant DNA Integration in <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis>

Cephalosporins are widely used as anti-infectious β-lactam antibiotics in clinic. For the purpose of increasing the yield of cephalosporin C (CPC) fermentation, especially in an industrial strain, A. chrysogenum genes cefEF and cefG, which encode the ultimate and penultimate steps in CPC biosynthesis, cefT, which encodes a CPC efflux pump, and vgb, which encodes a bacterial hemoglobin gene were transformed in various combinations into an industrial strain of A. chrysogenum. Both PCR and Southern blotting indicated that the introduced genes were integrated into the chromosome of A. chrysogenum. Carbon monoxide difference spectrum absorbance assay was performed and the result showed that Vitreoscilla hemoglobin was successfully expressed in A. chrysogenum and had biological activity. HPLC analysis of fermentation broth of recombinant A. chrysogenum showed that most transformants had a higher CPC production level than the parental strain. Multiple transformants containing an additional copy of cefG showed a significant increase in CPC production. However, cefT showed little effect on CPC production in this high producer. The highest improvement of CPC titer was observed in the mutant with an extra copy of cefG + cefEF + vgb whose CPC production was increased by 116.3%. This was the first report that three or more genes were introduced simultaneously into A. chrysogenum. Our results also demonstrated that the combination of these genes had a synergy effect in a CPC high producer.     

OsSPO11-1 is essential for both homologous chromosome pairing and crossover formation in rice

Spo11 is a homolog of a subunit of archaebacterial topoisomerase, which catalyzes DNA double-strand breaks and initiates homologous chromosome recombination. In the present study, we silenced the SPO11-1 gene in rice (Oryza sativa) using RNAi. Rice plants with loss-of-function of OsSPO11-1 have no apparent growth defects during vegetative development, but homologous chromosome pairing and recombination are significantly obstructed. Telomeres can be assembled as bouquet during the zygotene stage of the OsSPO11-1-deficient plants, just as that in wild type. Although the two axial-associated proteins, REC8 and PAIR2, are loaded onto the chromosomes, the depletion of PAIR2 from the chromosomes is much later than in wild type. The central element of the synaptonemal complex (SC), ZEP1, does not load onto the chromosomes normally, implying that SC formation is disturbed severely. The crossover protein, MER3, isn't efficiently assembled onto chromosomes and the lack of bivalent suggests that crossovers are also affected in the absence of OsSPO11-1. Thus, OsSPO11-1 is essential for both homologous chromosomes pairing and crossover formation during meiosis in rice.     

Identification of zRAP55,a gene preponderantly expressed in Stages I and II oocytes of zebrafish

In an in silico search for gonand specific expressed genes, we have identified zRAP55 which is enriched in the ovary of zebrafish . zRAP55 encodes a protein of 382 amino acids with a highly conserved Lsm domain. zRAP55 protein shares more than 56% identities with that of other vertebrate species. RT-PCR results show that it is predominantly expressed in the ovary. In situ hybridization and immunohistochemistry studies reveal that zRAP55 is ubiquitously dispersed throughout the cytoplasm of stages I and II oocytes, whereas no expression is observed in stages III and IV oocytes. As an RNA associated protein, zRAP55 might function in the control of protein translation at the early stages of oogenesis in zebrafish.     

R-藻红蛋白的结构、功能及其应用

R-藻红蛋白是最重要类型的藻红蛋白,为许多藻类的前级捕光色素蛋白,在光的激发下,能发出桔红色荧光。现对R-藻红蛋白的三维结构与功能的关系、R-藻红蛋白离体的光学活性在肿瘤光动力学治疗(PDT)中作为光敏剂和荧光免疫检测等领域作为荧光探针分子的应用进行综述。     

An Integrated Approach Combining Chemical Analysis and an In Vivo Bioassay to Assess the Estrogenic Potency of a Municipal Solid Waste Landfill Leachate in Qingdao

Various adverse effects related to landfill leachate have made leachates an important issue in past decades, and it has been demonstrated that landfill leachate is an important source of environmental estrogens. In this study, we employed chemical analysis of some already evaluated estrogenic substances, in combination with a bioassay using several specific biomarkers (e.g., plasma vitellogenin and sex steroids, enzyme activity of gonad gamma-glutamyl transpeptidase, and gonadosomatic index) to evaluate the estrogenic activities in outlets from different stages of the leachate treatment process. The results indicated that 5 environmental estrogens (4-t-octylphenol, bisphenol A, di-ethyl phthalate, di-n-butyl phthalate, and diethylhexyl phthalate) were detected by a gas chromatography-mass spectrometry, and the concentrations in leachate samples were 6153 ng/L, 3642 ng/L, 2139 ng/L, 5900 ng/L, and 9422 ng/L, respectively. Leachate (1∶200 diluted) induced the synthesis of plasma vitellogenin and led to decreased enzyme activity of gonad gamma-glutamyl transpeptidase and gonadosomatic index in male goldfish (Carassius auratus) after a 28-day exposure, while increased circulating 17β-estradiol level was also observed in males exposed to treated effluent. Although the target EEs were partially removed with removal rates varying from 87.2% to 99.77% by the “membrane bioreactor+reverse osmosis+aeration zeolite biofilter” treatment process, the treated effluent is still estrogenic to fish. The method combined chemical techniques with the responses of test organisms allowing us to identify the group of estrogen-like chemicals so that we were able to evaluate the overall estrogenic effects of a complex mixture, avoiding false negative assessments.     

Circ_0075829 facilitates the progression of pancreatic carcinoma by sponging miR‐1287‐5p and activating LAMTOR3 signalling

Pancreatic cancer (PC) is a leading cause of cancer‐related mortality globally. Though increasing evidence has demonstrated that circular RNAs (circRNAs) are linked to the development and progression of cancers, the biological functions of circRNAs in PC remain largely unexplored so far. Based on previous studies, Hsc_circ_0075829 (circ_0075829) was screened out and then further identified in PC clinical specimens and cell lines by real‐time PCR. After the stability tests, a series of in vitro and in vivo functional experiments were performed to investigate the role of circ_0075829 in PC development. Furthermore, fluorescent in situ hybridization (FISH), bioinformatics tools, dual‐luciferase assays and rescue experiments were conducted to clarify the regulatory mechanisms of circ_0075829 in SW1990 and BxPC‐3 cells. Compared with paracancerous tissues, the expression of circ_0075829 was increased in PC tissues, which was positively correlated with the clinical features of PC. Knockdown of circ_0075829 significantly suppressed the proliferative, migratory and invasive rates of SW1990 and BxPC‐3 cells both in vitro and in vivo. Bioinformatics analysis and dual‐luciferase reporter gene assay indicated that circ_0075829 could bind to miR‐1287‐5p. Mechanism research and rescue experiments demonstrated that circ_0075829 could regulate the LAMTOR3/p‐ERK signalling pathway via sponging miR‐1287‐5p in PC cell lines. Our data reveal that the circ_0075829 could facilitate the proliferation and metastasis of PC through circ_0075829/miR‐1287‐5p/LAMTOR3 axis.