Nanocomposite scaffold seeded with mesenchymal stem cells for bone repair

The mechanical property of bone tissue scaffolds is one of the most important aspects in bone tissue engineering that has remained problematic. In our previous study, we fabricated a three‐dimensional scaffold from nano‐hydroxyapatite/gelatin (nHA/Gel) and investigated its efficiency in promoting bone regeneration both in vitro and in vivo. In the present study, the effect of adding silicon carbide (SiC) on the mechanical and biological behaviors of the nHA/Gel/SiC and bone regeneration in vivo were determined. nHA and SiC were synthesized and characterized by the X‐ray diffraction pattern and transmission electron microscope image. Layer solvent casting, freeze drying, and lamination techniques were applied to prepare these scaffolds. Then, the biocompatibility and cell adhesion behavior of the synthesized nHA/Gel/SiC scaffolds were investigated. For in vivo studies, rats were categorized into three groups: blank defect, blank scaffold, and rat bone marrow mesenchymal stem cells (rBM‐MSCs)/scaffold. After 1, 4, and 12 weeks post‐injury, the rats were sacrificed and the calvaria were harvested. Sections with a thickness of 5 µm thickness were prepared and stained with hematoxylin–eosin and Masson's Trichrome, and immunohistochemistry was performed. Our results showed that SiC effectively increased the mechanical properties of the nHA/Gel/SiC scaffold. No significant differences were observed in biocompatibility, cell adhesion, and cytotoxicity of the nHA/Gel/SiC in comparison with the nHA/Gel nanocomposite. Based on histological and immunohistochemical studies, both osteogenesis and collagenization were significantly higher in the rBM‐MSCs/scaffold group, quantitatively and qualitatively. The present study strongly suggests the potential of SiC as an alternative strategy to improve the mechanical and biological properties of bone tissue engineering scaffolds, and shows that the pre‐seeded nHA/Gel/SiC scaffold with rBM‐MSCs improves osteogenesis in the engineered bone implant.     

Salicylic acid influences the protease activity and posttranslation modifications of the secreted peptides in the moss Physcomitrella patens

Plant secretome comprises dozens of secreted proteins. However, little is known about the composition of the whole secreted peptide pools and the proteases responsible for the generation of the peptide pools. The majority of studies focus on target detection and characterization of specific plant peptide hormones. In this study, we performed a comprehensive analysis of the whole extracellular peptidome, using moss Physcomitrella patens as a model. Hundreds of modified and unmodified endogenous peptides that originated from functional and nonfunctional protein precursors were identified. The plant proteases responsible for shaping the pool of endogenous peptides were predicted. Salicylic acid (SA) influenced peptide production in the secretome. The proteasome activity was altered upon SA treatment, thereby influencing the composition of the peptide pools. These results shed more light on the role of proteases and posttranslational modification in the “active management” of the extracellular peptide pool in response to stress conditions. It also identifies a list of potential peptide hormones in the moss secretome for further analysis.     

Haemophilus parasuis infection in 3D4/21 cells induces autophagy through the AMPK pathway

Haemophilus parasuis (H. parasuis) is a common commensal in the upper respiratory tract of pigs, but causes Glässer's disease in stress conditions. To date, many studies focused on the immune evasion and virulence of H. parasuis; very few have focused on the role autophagy played in H. parasuis infection, particularly in porcine alveolar macrophages (PAMs). In this study, a PAM cell line, 3D4/21 cells were used to study the role of autophagy in H. parasuis infection. 3D4/21 cells tandemly expressing GFP, mCherry, and LC3 were infected with H. parasuis serovar 5 (Hps5). Western blot analysis and confocal and transmission electron microscopy showed that H. parasuis infection effectively induces autophagy. Using Hps strains of varying virulence (Hps4, Hps5, and Hps7) and UV‐inactivated Hps5, we demonstrated that autophagy is associated with the internalisation of living virulent strains into cells. In 3D4/21 cells pretreated with rapamycin and 3‐MA then infected by Hps4, Hps5, and Hps7, we demonstrated that autophagy affects invasion of H. parasuis in cells. AMPK signal results showed that Hps5 infection can upregulate the phosphorylation level of AMPK, which is consistent with the autophagy development. 3D4/21 cells pretreated with AICAR or Compound C then infected by Hps5 revealed that the autophagy induced by Hps5 infection is associated with the AMPK pathway. Our study contributes to the theoretical basis for the study of H. parasuis pathogenesis and development of novel drugs target for prevention Glässer's disease.     

A risk‐based inventory of invasive plant species of Queensland,Australia: Regional,ecological and floristic insights

Invasive alien plant species threaten agriculture and biodiversity globally and require ongoing management to minimise impacts. However, the large number of invasive species means that a risk‐based approach to prioritisation is needed, taking into account the spatial scale of management decisions and myriad of available information. Here, we developed a risk‐based inventory of invasive plants in Queensland, Australia, using both current species distribution/abundance and the severity of their impacts. Our assessment followed a comprehensive data collection process including a scoping of local government pest management plans, herbarium records, the published literature and structured elicitation of expert knowledge during a series of regional stakeholder workshops. From ~300 plant species that were identified as established and/or emerging invaders in the State, only one‐third were considered by practitioners to pose significant risks across regions to be considered management priorities. We aggregated regional species lists into a statewide priority list and analysed the data set (107 species) for historical, geographical, floristic and ecological patterns. Regions on the mainland eastern seaboard of the State share similar invasive plant communities, suggesting that these regions may form a single management unit, unlike the western/inland and the extreme far north (Torres Strait Islands) regions, which share fewer invasive plant species. Positive correlations were detected between invasiveness and time since introduction for some but not all plant life forms. Stakeholders identified research and management priorities for the invasive plant list, including biological control options, public awareness/education, effective herbicide use, ecology/taxonomy and risk analysis. In the course of the exercise, a statewide invasive plant priority list of high‐, medium‐ and low‐impact scores for policy, research and management was compiled. Finally, our approach to invasive plant species prioritisation highlighted that planning and policy documents are not necessarily reflected at the grass‐root level in terms of species identity and management priorities.     

CRISPR/Cas9介导的LATS1/2敲除抑制卵巢癌细胞ES-2增殖

Hippo信号通路在哺乳动物肝脏发育、动态平衡、再生和疾病中发挥非常重要的作用。大肿瘤抑制基因1/2（large tumor suppressor 1/2, LATS1/2）激酶是Hippo信号通路的关键激酶,可以磷酸化YES相关蛋白（yes-associated protein,YAP）,从而调节YAP的核质定位和降解。本文采用CRISPR/Cas9方法构建慢病毒介导的Last1/2基因敲除的载体,通过包装、感染和嘌呤霉素筛选,获得LATS1/2部分敲除的人卵巢癌ES-2和H08910细胞,免疫印迹方法检测LATS1/2表达明显减少。细胞增殖实验检测LATS1/2缺失明显抑制ES-2和HO8910细胞增殖。软琼脂克隆形成实验表明,LATS1/2缺失抑制卵巢癌ES-2细胞的克隆形成能力。细胞划痕和Transwell实验证明,LATS1/2缺失明显抑制卵巢癌ES-2细胞迁移。流式细胞检测发现,LATS1/2敲除促进卵巢癌ES-2细胞凋亡并影响细胞周期。裸鼠成瘤实验表明,LATS1/2缺失明显抑制体内肿瘤组织增殖。分子机制研究表明, LATS1/2敲除促进卵巢癌ES-2细胞中胶原I型α1（collagen type I α1,ColIα1）基因表达量增加,在卵巢癌ES-2细胞中同时敲除LATS1/2和COL1A1,可以促进细胞克隆形成。综上结果,人卵巢癌ES-2细胞中LATS1/2缺失能促进COL1A1表达增加, 从而抑制细胞增殖、转移和克隆形成,并影响细胞周期和促进细胞凋亡。     

基因前定点敲入FLAG标签表达的FLAG-SND1蛋白参与胞内应激颗粒的形成

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-Cas9 nuclease) 基因编辑技术是近年来新兴的一种可以实现基因特异性敲除和敲入的技术。本文利用CRISPR/Cas9基因编辑系统,将3×FLAG标签定点敲入HeLa细胞SND1基因前方,使细胞内源性表达的SND1蛋白带有3×FLAG标签,并观察SND1与应激颗粒及加工体的定位情况。设计针对SND1基因起始密码子ATG附近的sgRNA,以px459为表达载体,构建出重组真核表达质粒。设计含有3×FLAG及待插入位置上下游150 bp同源臂的序列,经公司合成获得重组质粒。将2个质粒共同转染HeLa细胞,使用嘌呤霉素筛选阳性细胞,挑取单克隆后培养。Western 印迹表明,细胞表达3×FLAG-SND1融合蛋白质。提取细胞基因组DNA进行测序。测序无误获得稳定株后,用流式细胞术检测细胞周期和凋亡,发现与WT细胞相比无显著性差异。同时,使用0.5 mmol/L亚砷酸钠处理,细胞发生氧化应激,eIF2α蛋白磷酸化增加,胞浆中出现应激颗粒,SND1与应激颗粒标志蛋白TIAR存在共定位现象,但不存在与加工体蛋白DCP1α的共定位。     

Resistance outside the substrate envelope: hepatitis C NS3/4A protease inhibitors

Direct acting antivirals have dramatically increased the efficacy and tolerability of hepatitis C treatment, but drug resistance has emerged with some of these inhibitors, including nonstructural protein 3/4?A protease inhibitors (PIs). Although many co-crystal structures of PIs with the NS3/4A protease have been reported, a systematic review of these crystal structures in the context of the rapidly emerging drug resistance especially for early PIs has not been performed. To provide a framework for designing better inhibitors with higher barriers to resistance, we performed a quantitative structural analysis using co-crystal structures and models of HCV NS3/4A protease in complex with natural substrates and inhibitors. By comparing substrate structural motifs and active site interactions with inhibitor recognition, we observed that the selection of drug resistance mutations correlates with how inhibitors deviate from viral substrates in molecular recognition. Based on this observation, we conclude that guiding the design process with native substrate recognition features is likely to lead to more robust small molecule inhibitors with decreased susceptibility to resistance.