Tissue Culture of Red Sandalwood (Pterocarpus santalinus)

[Objectives] This study was conducted to develop a rapid propagation method for red sandalwood ( Pterocarpus santalinus ) by tissue culture.[Methods] The well-grown red sandalwood seed embryos were inoculated into three kinds of culture media after aseptic treatment,and the aseptic explants were obtained and inoculated into six kinds of media for light culture.[Results] In the best disinfection schemes of red sandalwood,disinfecting with HgCl 2 for 8 min achieved the highest germination and survival rates;when the medium for inducing red sandalwood explants was MS+0.2 mg/L IBA,the induction rate reached a maximum value;and when the culture medium for inducing stem segments of aseptic red sandalwood plantlets was MS+3.0 mg/L 6-BA+0.3 mg/L IBA,the growth of the stem segments achieved the best effect.The optimal medium for inducing red sandalwood explants was MS+IBA 0.2 mg/L,and the optimal medium for inducing stem segments of red sandalwood was MS+3.0 mg/L 6-BA+0.3 mg/L IBA.[Conclusions] The results of this study have a large reproductive coefficient,simple process and low cost,which have outstanding value for promoting the breeding and promotion of red sandalwood seedlings.

利用CRISPR/Cas9 AAV系统构建纹状体Slc20a2基因敲除小鼠模型

原发性家族性脑钙化症(primary familial brain calcification,PFBC)是慢性进展性的神经系统遗传病,临床症状主要包括运动障碍、认知障碍及精神障碍等,其致病机制尚未完全明确。研究表明SLC20A2是该病最主要的致病基因。由于Slc20a2基因全身性敲除小鼠模型会导致胎儿生长受限,为更好地研究PFBC发病机制,本研究应用CRISPR/Cas9技术构建了纹状体Slc20a2基因条件性敲除小鼠模型。首先,针对Slc20a2基因编码区,设计3条靶向exon3的sgRNA(single guide RNA),通过构建质粒、转染细胞、Surveyor assay等实验验证sgRNA的活性。其次,选取活性较高的sgRNA重组包装AAV-Cre病毒,应用立体定位将AAV病毒定点注射于小鼠纹状体。体外实验结果表明设计的3条sgRNA均能够有效地介导Cas9切割靶DNA。细胞免疫荧光实验结果证实AAV-Cre病毒具有Cre重组酶活性。最后,通过小鼠脑部组织免疫组化、TA-克隆、高通量测序及Western blot方法检测Slc20a2基因敲除效率,发现实验组小鼠纹状体组织Slc20a2表达明显降低。本研究成功设计了3条能够敲除Slc20a2的功能sgRNA,并应用CRISPR/Cas9技术成功构建了纹状体Slc20a2基因条件性敲除小鼠,为研究PFBC的发病机制提供了有效的动物模型。

CGG repeat expansion in LOC642361/NUTM2B-AS1 typically presents as oculopharyngodistal myopathy

CGG repeat expansions in LOC642361/NUTM2B-AS1 have recently been identified as a cause of oculopharyngeal myopathy with leukoencephalopathy.However,since only three patients from a single family were reported,it remains unknown whether their clinicopathological features are typical for CGG repeat expansions in LOC642361/NUTM2B-AS1.Here,using repeat-primed-polymerase chain reaction and long-read sequencing,we identify 12 individuals from 3 unrelated families with CGG repeat expansions in LOC642361/NUTM2B-AS1,typically presenting with oculopharyngodistal myopathy.The CGG repeat expansions range from 161 to 669 repeat units.Most of the patients present with ptosis,restricted eye movements,dysphagia,dysarthria,and diffuse limb muscle weakness.Only one patient shows T2-weighted hyperintensity in the cerebellar white matter surrounding the deep cerebellar nuclei on brain magnetic resonance imaging.Muscle biopsies from three patients show a myopathic pattern and rimmed vacuoles.Analyses of muscle biopsies suggest that CGG repeat expansions in LOC642361/NUTM2B-AS1 may deleteriously affect aggrephagic capacity,suggesting that RNA toxicity and mitochondrial dysfunction may contribute to pathogenesis.Our study thus expands the phenotypic spectrum for the CGG repeat expansion of LOC642361/NUTM2B-AS1 and indicates that this genetic variant typically manifests as oculopharyngodistal myopathy with chronic myopathic changes with rimmed vacuoles and filamentous intranuclear inclusions in muscle fibers.

Euphorbia factor L2 induces apoptosis in A549 cells through the mitochondrial pathway

Euphorbia factor L2, a lathyrane diterpenoid isolated from caper euphorbia seed(the seeds of Euphorbia lathyris L.), has been traditionally applied to treat cancer. This article focuses on the cytotoxic activity of Euphorbia factor L2 against lung carcinoma A549 cells and the mechanism by which apoptosis is induced. We analyzed the cytotoxicity and related mechanism of Euphorbia factor L2 with an MTT assay, an annexin V-FITC/PI test, a colorimetric assay, and immunoblotting. Euphorbia factor L2 showed potent cytotoxicity to A549 cells. Euphorbia factor L2 led to an increase in reactive oxygen species(ROS) generation,a loss of mitochondrial electrochemical potential, release of cytochrome c, activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase, suggesting that Euphorbia factor L2 induced apoptosis through a mitochondrial pathway. The cytotoxic activity of Euphorbia factor L2 in A549 cells and the related mechanisms of apoptotic induction provide support for the further investigation of caper euphorbia seeds.

Exosomes with low miR-34c-3p expression promote invasion and migration of non-small cell lung cancer by upregulating integrinα2β1

Exosomes play critical roles in regulating various physiological and pathological processes,including immune stimulation,immune suppression,cardiovascular diseases,and cancers.Recent studies show that exosomes that transport specific microRNAs(miRNAs)are involved in tumor development.However,the molecular mechanism by which tumor invasion and migration are regulated by exosomes from non-small cell lung cancer(NSCLC)is not well understood.Here,we show that exosomes shuttling low levels of miR-34c-3p are involved in NSCLC progression.Our results showed that exosomes derived from NSCLC cells carrying low levels of miR-34c-3p could be transported into the cytoplasm of NSCLC cells and accelerate NSCLC invasion and migration by upregulating integrinα2β1.A luciferase assay revealed that integrinα2β1 was the direct target of miR-34c-3p,and overexpression of integrinα2β1 could promote the invasion and migration of NSCLC cells.The analysis of exosomes derived from clinical serum samples indicated that the expression of miR-34c-3p was significantly downregulated in exosomes from NSCLC patients compared with that of normal controls.A549-derived exosomes promoted NSCLC cells lung metastases in vivo.Exosomes shuttling low levels of miR-34c-3p were associated with the progression of NSCLC in vitro and in vivo.Our data demonstrate that exosomes shuttling low levels of miR-34c-3p can accelerate the invasion and migration of NSCLC by upregulating integrinα2β1.MiR-34c-3p can be a diagnostic and prognostic marker for NSCLC.High expression of integrinα2β1 is positively related to the migration and metastasis of NSCLC cells.