一次性竹筷浸出液对斑马鱼(Danio rerio) 胚胎发育的影响

目的观察市售某合格品牌一次性竹筷浸出液对斑马鱼胚胎发育的毒性效应。方法将一次性竹筷称重,用过滤自来水浸泡24h,配制5.0、7.5、11.2、16.8、25.0mg/mL5个不同浓度的一次性竹筷浸出液。选取成年健康斑马鱼将雌雄按1:2的比例进行交配产卵,分别用不同浓度的竹筷浸出液对受精卵(1枚受精卵/培养皿)进行暴露孵化实验,设空白对照。通过显微成像技术观察一次性竹筷浸出液暴露对斑马鱼胚胎发育形态、孵化率、仔鱼畸形率与仔鱼死亡率的影响。在总周期96 h内每隔12 h观察一次。结果对照组仔鱼以及浓度在11.2 mg/mL以下的各浸筷液组仔鱼无畸形,无死亡。高浓度浸出液(16.8mg/mL和25 mg/mL)中孵化的斑马鱼表现出胚胎发育明显延迟。虽然96 h总孵化率在不同组间无差异,但是高浓度竹筷浸出液中孵出的斑马鱼仔鱼出现高度畸形,96 h仔鱼畸形率分别为67.65%和61.29%;仔鱼死亡率的增加呈现剂量依赖性,分别为61.76%和100%。结论市售某合格品牌一次性竹筷浸出液可导致斑马鱼胚胎发育延迟、仔鱼畸形和早期死亡。     

Deletion of POMT2 in Zebrafish Causes Degeneration of Photoreceptors

Mutations in the extracellular matrix protein eyes shut homolog (EYS) are a common cause of retinitis pigmentosa, a blinding disease characterized by photoreceptor degeneration. EYS binds to matriglycan, a carbohydrate modification on O-mannosyl glycan substitutions of the cell-surface glycoprotein α-dystroglycan. Patients with mutations in enzymes required for the biosynthesis of matriglycan exhibit syndromic retinal atrophy, along with brain malformations and congenital muscular dystrophy. Protein O-mannosyltransferase 2 (POMT2) is an enzyme required for the synthesis of O-mannosyl glycans. To evaluate the roles of O-mannosyl glycans in photoreceptor health, we generated protein O-mannosyltransferase 2 (pomt2) mutant zebrafish by CRISPR. pomt2 mutation resulted in a loss of matriglycan and abolished binding of EYS protein to α-dystroglycan. Mutant zebrafish presented with hydrocephalus and hypoplasia of the cerebellum, as well as muscular dystrophy. EYS protein was enriched near photoreceptor connecting cilia in the wild-type, but its presence and proper localization was significantly reduced in mutant animals. The mutant retina exhibited mis-localization of opsins and increased apoptosis in both rod and cone photoreceptors. Immunofluorescence intensity of G protein subunit alpha transducin 2 (GNAT2) antibody (a general cone marker) and 1D4 antibody (a long double cone marker) in mutant retinas did not differ from wild-type retinas at 1-month post fertilization, but was reduced at 6 months post fertilization, indicating significant cone degeneration. These data suggest that POMT2-mediated O-mannosyl glycosylation is required for EYS protein localization to the connecting cilium region and photoreceptor survival.     

核桃粕源抗氧化活性肽的酶解制备及活性分析

目的：深度利用核桃粕蛋白。方法：采用分级分离法从核桃粕中提取蛋白质后,分别用胰蛋白酶、木瓜蛋白酶、中性蛋白酶、碱性蛋白酶和风味蛋白酶5种蛋白酶对其进行水解,并比较分析胰蛋白酶酶解物（trypsin hydrolysate,TH）、木瓜蛋白酶酶解物（papain hydrolysate,PH）、中性蛋白酶酶解物（dispase hydrolysate,DH）、碱性蛋白酶酶解物（alcalase hydrolysate,AH）和风味蛋白酶酶解物（flavourzyme hydrolysate,FPH）的抗氧化活性;以DPPH自由基清除率、ABTS自由基清除率和羟自由基清除率为检测指标分析不同浓度AH的体外抗氧化性;采用LC-MS/MS技术鉴定AH中所有肽的序列,并利用PeptideRanker、BIOPEP-UWM数据库和分子对接工具AutoDock1.5.6筛选潜在的核桃抗氧化活性肽;将上述两条肽分别与Keap1蛋白进行计算机模拟分子对接,并利用斑马鱼胚胎氧化损伤模型,对人工合成的两条抗氧化活性肽的抗氧化活性进行分析。结果：5种酶解物中抗氧化活性较好的AH对DPPH自由基、ABTS自由基和羟自由基的最佳清除率分别为（71.56±0.75）%,（60.63±0.45）%,（98.63±0.06）%。LC-MS/MS鉴定并通过计算机分析预测得到两条活性最优的肽,即：ALWPF和PLRWPF;两条抗氧化活性肽均能与Keap1蛋白的活性部位结合,其结合能分别为-9.2,-9.6 kJ/mol。ALWPF和PLRWPF肽处理斑马鱼胚胎的安全质量浓度范围分别为0～50,0～30 μg/mL。结论：从核桃粕中鉴定出两条抗氧化肽,其对斑马鱼胚胎具有保护作用。     

Skeletal Class III Malocclusion Is Associated with ADAMTS2 Variants and Reduced Expression in a Familial Case

Skeletal Class III malocclusion with maxillary deficiency is a severe maxillofacial disease with unclear pathogenic mechanisms. We recruited a Han Chinese family who was clinically diagnosed with skeletal Class III malocclusion and maxillary deficiency. Using whole exome sequencing, a missense variant in ADAMTS2 ({"type":"entrez-nucleotide","attrs":{"text":"NM_014244","term_id":"1519241845","term_text":"NM_014244"}}NM_014244: c.3506G>T: p.G1169V) was identified and predicted as deleterious by in silico tools. We also found ADAMTS2 variants associated with deficient maxillary development in a cohort. ADAMTS2 expression in HEK293 cells showed significant decrease due to the variant, which was also consistent in dental pulp stem cells from the proband and a healthy control. In the adamts2-knockdown zebrafish model, the length and width of the ethmoid plate, as well as the length of the palatoquadrate became significantly shorter than the control group (p < 0.001), while there was no significant difference in the length and width of the mandible. The expression of Sox3, which was required in early embryonic craniofacial development, was significantly downregulated in the adamts2-knockdown zebrafish embryos. Bioinformatic and cellular studies showed that the decreased expression of ADAMTS2 may inhibit downstream ErbB signaling pathway transduction and restrain subsequent osteogenesis in human adult mesenchymal stromal cells. Collectively, these data showed that ADAMTS2 (c.3506G>T: p.G1169V) may confer susceptibility to risk of skeletal Class III malocclusion with maxillary deficiency.     

Phylogenetic and Expression Analysis of Fos Transcription Factors in Zebrafish

Members of the FOS protein family regulate gene expression responses to a multitude of extracellular signals and are dysregulated in several pathological states. Whilst mouse genetic models have provided key insights into the tissue-specific functions of these proteins in vivo, little is known about their roles during early vertebrate embryonic development. This study examined the potential of using zebrafish as a model for such studies and, more broadly, for investigating the mechanisms regulating the functions of Fos proteins in vivo. Through phylogenetic and sequence analysis, we identified six zebrafish FOS orthologues, fosaa, fosab, fosb, fosl1a, fosl1b, and fosl2, which show high conservation in key regulatory domains and post-translational modification sites compared to their equivalent human proteins. During embryogenesis, zebrafish fos genes exhibit both overlapping and distinct spatiotemporal patterns of expression in specific cell types and tissues. Most fos genes are also expressed in a variety of adult zebrafish tissues. As in humans, we also found that expression of zebrafish FOS orthologs is induced by oncogenic BRAF-ERK signalling in zebrafish melanomas. These findings suggest that zebrafish represent an alternate model to mice for investigating the regulation and functions of Fos proteins in vertebrate embryonic and adult tissues, and cancer.     

eif4ebp3l—A New Affector of Zebrafish Angiogenesis and Heart Regeneration?

The eukaryotic initiation factor 4E binding protein (4E-BP) family is involved in translational control of cell proliferation and pro-angiogenic factors. The zebrafish eukaryotic initiation factor 4E binding protein 3 like (eif4ebp3l) is a member of the 4E-BPs and responsible for activity-dependent myofibrillogenesis, but whether it affects cardiomyocyte (CM) proliferation or heart regeneration is unclear. We examined eif4ebp3l during zebrafish vascular development and heart regeneration post cryoinjury in adult zebrafish. Using morpholino injections we induced silencing of eif4ebp3l in zebrafish embryos, which led to increased angiogenesis at 94 h post fertilization (hpf). For investigation of eif4ebp3l in cardiac regeneration, zebrafish hearts were subjected to cryoinjury. Regenerating hearts were analyzed at different time points post-cryoinjury for expression of eif4ebp3l by in situ hybridization and showed strongly decreased eif4ebp3l expression in the injured area. We established a transgenic zebrafish strain, which overexpressed eif4ebp3l under the control of a heat-shock dependent promotor. Overexpression of eif4ebp3l during zebrafish heart regeneration caused only macroscopically a reduced amount of fibrin at the site of injury. Overall, these findings demonstrate that silencing of eif4ebp3l has pro-angiogenic properties in zebrafish vascular development and when eif4ebp3l is overexpressed, fibrin deposition tends to be altered in zebrafish cardiac regeneration after cryoinjury.