Microtubule-assisted mechanism for toxisome assembly in Fusarium graminearum

In Fusarium graminearum, a trichothecene biosynthetic complex known as the toxisome forms ovoid and spherical structures in the remodelled endoplasmic reticulum (ER) under mycotoxin-inducing conditions. Previous studies also demonstrated that disruption of actin and tubulin results in a significant decrease in deoxynivalenol (DON) biosynthesis in F. graminearum. However, the functional association between the toxisome and microtubule components has not been clearly defined. In this study we tested the hypothesis that the microtubule network provides key support for toxisome assembly and thus facilitates DON biosynthesis. Through fluorescent live cell imaging, knockout mutant generation, and protein–protein interaction assays, we determined that two of the four F. graminearum tubulins, α₁ and β₂ tubulins, are indispensable for DON production. We also showed that these two tubulins are directly associated. When the α₁–β₂ tubulin heterodimer is disrupted, the metabolic activity of the toxisome is significantly suppressed, which leads to significant DON biosynthesis impairment. Similar phenotypic outcomes were shown when F. graminearum wild type was treated with carbendazim, a fungicide that binds to microtubules and disrupts spindle formation. Based on our results, we propose a model where α₁–β₂ tubulin heterodimer serves as the scaffold for functional toxisome assembly in F. graminearum.     

干细胞改善糖尿病的分子机制及临床研究进展

糖尿病是各种因素导致的高血糖慢性代谢疾病,已发展成为流行疾病之一。化学抗糖药虽能控制血糖水平,延缓病程进展,但需长期服用;胰岛移植能从根本上治愈糖尿病,但胰岛来源不足,且需终生应用免疫抑制剂,故并没有得到广泛应用;干细胞是一类能够自我复制的细胞,具有多向分化潜能和旁分泌特性,近年来的研究证明,干细胞在糖尿病治疗方面有着积极的效果,被认为是有效治疗糖尿病的理想细胞类型。因此,就干细胞治疗糖尿病的分子机制和临床研究现状进行简要阐述。     

上海农作物种质资源数据库的设计和构建

随着农作物种质资源数量的不断增加,种质资源交流的日益频繁以及种质资源利用的逐步深入,农作物种质资源数据库的建设对农作物的应用研究和基础性研究至关重要。上海市农业生物基因中心借助于本中心保存的近21万份种质资源的信息,开发了一套上海农作物种质资源数据库,本文主要阐述了该数据库设计和构建的目的、原则和数据库的架构、运行环境以及主要功能模块。上海农作物种质资源数据库采用了B/S结构,平台系统利用多层逻辑架构实现了对农作物种质资源数据信息的收集、存储、查询、统计分析等功能。外部用户通过浏览器即可访问平台系统,浏览查询种质资源信息。内部管理员可使用平台的受限功能,需要认证成功后才可使用后台管理功能,实现对整个平台数据的管理。注册用户还可通过基因资源数据库提交引种申请,管理员结合管理信息系统实现对外供种全流程的网络化,借此可提高工作效率,增加工作流程的透明度。     

桔黄赛多孢菌胞外胰蛋白酶的酶学特性

桔黄赛多孢菌Scedosporium aurantiacum是慢性肺病患者的常见呼吸道定植菌,在免疫缺陷人群中可引起侵袭性感染,致死率高,但由于致病机理不明,目前仍然缺乏有效的防控手段。我们在前期研究中,通过差异蛋白组学及酶工程技术发现分泌胰蛋白酶是桔黄赛多孢菌的潜在毒力因子,目前对这种蛋白酶的遗传信息、结构及致病机制并不清楚。本研究用Superdex S-200分子筛和DEAE-Sepharose离子交换两种填料将这种蛋白酶分离纯化出来,通过酶谱验证了纯化效果。进一步研究发现,这种胰蛋白酶对bFSR、bLSTR和bEKK 3种底物的水解性能最佳,对zFR和bzLR的水解性能最差。酶解最快的反应所对应的K_m为6.09μmol/L,V_max为13.01μmol/L/s,K_cat为23.65/s;酶解最慢的反应所对应的K_m为29.94μmol/L,V_max为11.35μmol/L/s,K_cat为20.63/s。研究结果对于填补赛多孢菌毒力因子研究的空白、针对毒力因子开发新型的抗真菌药物和治疗方法都具有重要意义。     

Epidermal restriction confers robustness to organ shapes

The shape of comparable tissues and organs is consistent among individuals of a given species, but how this consistency or robustness is achieved remains an open question. The interaction between morphogenetic factors determines organ formation and subsequent shaping, which is ultimately a mechanical process. Using a computational approach, we show that the epidermal layer is essential for the robustness of organ geometry control. Specifically, proper epidermal restriction allows organ asymmetry maintenance, and the tensile epidermal layer is sufficient to suppress local variability in growth, leading to shape robustness. The model explains the enhanced organ shape variations in epidermal mutant plants. In addition, differences in the patterns of epidermal restriction may underlie the initial establishment of organ asymmetry. Our results show that epidermal restriction can answer the longstanding question of how cellular growth noise is averaged to produce precise organ shapes, and the findings also shed light on organ asymmetry establishment.     

Tasselseed5 encodes a cytochrome C oxidase that functions in sex determination by affecting jasmonate catabolism in maize

Maize (Zea mays L.) is a monoecious grass plant in which mature male and female florets form the tassel and ear, respectively. Maize is often used as a model plant to study flower development. Several maize tassel seed mutants, such as the recessive mutants tasselseed1 (ts1) and tasselseed2 (ts2), exhibit a reversal in sex determination, which leads to the generation of seeds in tassels. The phenotype of the dominant mutant, Tasselseed5 (Ts5), is similar to that of ts2. Here, we positionally cloned the underlying gene of Ts5 and characterized its function. We show that the GRMZM2G177668 gene is overexpressed in Ts5. This gene encodes a cytochrome C oxidase, which catalyzes the transformation of jasmonoyl‐L‐isoleucine (JA‐Ile) to 12OH‐JA‐Ile during jasmonic acid catabolism. Consistent with this finding, no JA‐Ile peak was detected in Ts5 tassels during the sex determination period, unlike in the wild type. Transgenic maize plants overexpressing GRMZM2G177668 exhibited a tassel‐seed phenotype similar to that of Ts5. These results indicate that the JA‐Ile peak in tassels is critical for sex determination and that the Ts5 mutant phenotype results from the disruption of this peak in tassels during sex determination.     

条形柄锈菌吸器特异效应蛋白Hasp68抑制寄主免疫并与小麦组织蛋白酶B互作

作为活体营养专性寄生真菌,条形柄锈菌（小麦条锈病）在侵染过程中通过形成吸器向寄主细胞释放效应蛋白,干扰寄主的防卫反应,促进其侵染与致病。因此,条形柄锈菌效应蛋白的鉴定与功能研究对揭示其毒性机理具有重要意义。本实验室前期完成了条形柄锈菌CYR31生理小种吸器转录组分析,从中鉴定得到一个吸器特异诱导表达分泌蛋白Hasp68,利用农杆菌侵染在烟草细胞中瞬时表达该基因,能够抑制小鼠促细胞凋亡蛋白Bax诱导的细胞程序性死亡,鉴定为条形柄锈菌候选效应蛋白。Hasp68基因全长318bp,编码105_aa,N-端包含20_aa的信号肽,无保守结构域。BlastX分析表明Hasp68为条形柄锈菌特有效应蛋白,在其他真菌中无同源蛋白,且在条形柄锈菌16个菌系中呈较低的序列多态性,表明其在条形柄锈菌的进化过程中相对保守。借助荧光假单胞菌EtHAn的三型分泌系统,在小麦细胞中过表达Hasp68能够抑制由非致病细菌引起的PTI（PAMP-triggered immunity）相关胼胝质的积累;同时,也能抑制小麦与无毒条形柄锈菌互作中ETI（effector-triggered immunity）相关的活性氧爆发和过敏性坏死反应,表明效应蛋白Hasp68具有抑制寄主免疫反应的功能。利用酵母双杂交系统筛选Hasp68在小麦中的互作蛋白,发现其与组织蛋白酶B（cathepsin B）TaCTSB互作,双分子荧光技术进一步验证二者在烟草细胞中共表达存在互作,初步揭示了效应蛋白Hasp68的互作靶标。     

Dynamics of NK,CD8 and Tfh cell mediated the production of cytokines and antiviral antibodies in Chinese patients with moderate COVID‐19

Recent studies have demonstrated a marked decrease in peripheral lymphocyte levels in patients with coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Few studies have focused on the changes of NK, T‐ and B‐cell subsets, inflammatory cytokines and virus‐specific antibodies in patients with moderate COVID‐19. A total of 11 RT‐PCR‐confirmed convalescent patients with COVID‐19 and 11 patients with non‐SARS‐CoV‐2 pneumonia (control patients) were enrolled in this study. NK, CD8⁺ T, CD4⁺ T, Tfh‐like and B‐cell subsets were analysed using flow cytometry. Cytokines and SARS‐CoV‐2‐specific antibodies were analysed using an electrochemiluminescence immunoassay. NK cell counts were significantly higher in patients with COVID‐19 than in control patients (P = 0.017). Effector memory CD8⁺ T‐cell counts significantly increased in patients with COVID‐19 during a convalescent period of 1 week (P = 0.041). TIM‐3⁺ Tfh‐like cell and CD226⁺ Tfh‐like cell counts significantly increased (P = 0.027) and decreased (P = 0.022), respectively, during the same period. Moreover, ICOS⁺ Tfh‐like cell counts tended to decrease (P = 0.074). No abnormal increase in cytokine levels was observed. The high expression of NK cells is important in innate immune response against SARS‐CoV‐2. The increase in effector memory CD8⁺ T‐cell counts, the up‐regulation of inhibitory molecules and the down‐regulation of active molecules on CD4⁺ T cells and Tfh‐like cells in patients with COVID‐19 would benefit the maintenance of balanced cellular and humoural immune responses, may prevent the development of severe cases and contribute to the recovery of patients with COVID‐19.     

Repellent,Insecticidal and Antimicrobial Activities of Leaf Essential Oils from Three Eucalyptus Species

Developing effective and eco‐friendly antimicrobials and pesticides has become a highly important issue. The repellent, insecticidal and antimicrobial activity of essential oils (EOs) isolated by hydrodistillation from dried leaves of the three Eucalyptus species (E. cloeziana, E. umbellata and E. benthamii) were investigated. During GC/MS analysis, α‐pinene (47.36 %), 1,8‐cineol (38.53 %) and α‐pinene (35.31 %) were identified as major components of E. cloeziana, E. umbellata and E. benthamii, respectively. The EOs from E. cloeziana exhibited the longest effective protection time (465 min, at 50.0 % w/w) for humans among the EOs studied. The effective protection time was 30 min and 300 min at concentrations of 12.5 % (w/w) and 25.0 % (w/w), respectively. Fumigating insecticidal activity of EOs from three Eucalyptus species was tested by airtight fumigation in conical flask, which indicated that essential oils had a highly and rapidly insecticidal activity on Culex pipiens quinquefasciatus. The antimicrobial activity of EOs was evaluated by using disc diffusion and agar dilution methods. There was no significant difference in the antibacterial activity of EOs from E. cloeziana and E. umbellate and they had the same MICs (20 mL/L) on Staphylococcus aureus, Salmonella typhi, Bacillus subtilis and Escherichia coli. E. benthamii had the worst microbial inhibitory effect among the three Eucalyptus essential oils and the MIC value for the test species is 40 mL/L except for Rhodotorula Harrison (10 mL/L).     

Allopurinol reduces oxidative stress and activates Nrf2/p62 to attenuate diabetic cardiomyopathy in rats

Allopurinol (ALP) attenuates oxidative stress and diabetic cardiomyopathy (DCM), but the mechanism is unclear. Activation of nuclear factor erythroid 2‐related factor 2 (Nrf2) following the disassociation with its repressor Keap1 under oxidative stress can maintain inner redox homeostasis and attenuate DCM with concomitant attenuation of autophagy. We postulated that ALP treatment may activate Nrf2 to mitigate autophagy over‐activation and consequently attenuate DCM. Streptozotocin‐induced type 1 diabetic rats were untreated or treated with ALP (100 mg/kg/d) for 4 weeks and terminated after heart function measurements by echocardiography and pressure‐volume conductance system. Cardiomyocyte H9C2 cells infected with Nrf2 siRNA or not were incubated with high glucose (HG, 25 mmol/L) concomitantly with ALP treatment. Cell viability, lactate dehydrogenase, 15‐F2t‐Isoprostane and superoxide dismutase (SOD) were measured with colorimetric enzyme‐linked immunosorbent assays. ROS, apoptosis, was assessed by dihydroethidium staining and TUNEL, respectively. The Western blot and qRT‐PCR were used to assess protein and mRNA variations. Diabetic rats showed significant reductions in heart rate (HR), left ventricular eject fraction (LVEF), stroke work (SW) and cardiac output (CO), left ventricular end‐systolic volume (LVVs) as compared to non‐diabetic control and ALP improved or normalized HR, LVEF, SW, CO and LVVs in diabetic rats (all P < .05). Hearts of diabetic rats displayed excessive oxidative stress manifested as increased levels of 15‐F2t‐Isoprostane and superoxide anion production, increased apoptotic cell death and cardiomyocytes autophagy that were concomitant with reduced expressions of Nrf2, heme oxygenase‐1 (HO‐1) and Keap1. ALP reverted all the above‐mentioned diabetes‐induced biochemical changes except that it did not affect the levels of Keap1. In vitro, ALP increased Nrf2 and reduced the hyperglycaemia‐induced increases of H9C2 cardiomyocyte hypertrophy, oxidative stress, apoptosis and autophagy, and enhanced cellular viability. Nrf2 gene silence cancelled these protective effects of ALP in H9C2 cells. Activation of Nrf2 subsequent to the suppression of Keap1 and the mitigation of autophagy over‐activation may represent major mechanisms whereby ALP attenuates DCM.