HDAC8 drives spindle organization during meiotic maturation of porcine oocytes

ObjectivesHistone deacetylase 8 (HDAC8) is one of the class I HDAC family proteins, which participates in the neuronal disorders, parasitic/viral infections, tumorigenesis and many other biological processes. However, its potential function during female germ cell development has not yet been fully understood.Materials and methodsHDAC8‐targeting siRNA was microinjected into GV oocytes to deplete HDAC8. PCI‐34051 was used to inhibit the enzyme activity of HDAC8. Immunostaining, immunoblotting and fluorescence intensity quantification were applied to assess the effects of HDAC8 depletion or inhibition on the oocyte meiotic maturation, spindle/chromosome structure, γ‐tubulin dynamics and acetylation level of α‐tubulin.ResultsWe observed that HDAC8 was localized in the nucleus at GV stage and then translocated to the spindle apparatus from GVBD to M II stages in porcine oocytes. Depletion of HDAC8 led to the oocyte meiotic failure by showing the reduced polar body extrusion rate. In addition, depletion of HDAC8 resulted in aberrant spindle morphologies and misaligned chromosomes due to the defective recruitment of γ‐tubulin to the spindle poles. Notably, these meiotic defects were photocopied by inhibition of HDAC8 activity using its specific inhibitor PCI‐34051. However, inhibition of HDAC8 did not affect microtubule stability as assessed by the acetylation level of α‐tubulin.ConclusionsCollectively, our findings demonstrate that HDAC8 acts as a regulator of spindle assembly during porcine oocyte meiotic maturation.     

长白山白桦山杨次生林细根形态特征和解剖结构对氮沉降的响应

根系作为植物与土壤物质交换和养分循环的桥梁,长期以来一直是生态学研究的热点。于2017年7月植物生长季,对长白山模拟11年氮(N)沉降控制试验样地的白桦(Betula platyphylla)山杨(Populus davidiana)天然次生林进行了根系采样,并利用根序法研究了根系形态特征和解剖结构对不同梯度N添加处理的响应,旨在探求两物种根系之间潜在生态联系。本试验共设置了三个N添加梯度,分别为对照(CK,0 g N m~(-2 )a~(-1))、低N处理(T_L,2.5 g N m~(-2 )a~(-1))和高N处理(T_H,5.0 g N m~(-2 )a~(-1))。研究结果如下:1)T_L显著抑制白桦和山杨前三级细根皮层厚度的生长。白桦通过增加皮层细胞直径(一级根增加了72.77%,二级根增加了53.22%,三级根增加了39.96%)但减少皮层层数来降低皮层厚度,而山杨主要通过皮层细胞直径的减少(一级根下降了40.80%,二级根下降了28.17%)来降低其皮层厚度。2)T_H显著抑制山杨前三级细根生长。主要通过增加皮层厚度(一级根增加了68.78%,二级根增加了50.81%,三级根增加了88.53%)以及降低导管横截面积来抑制吸收养分,从而达到影响生长的目的。3)白桦T_H相比于T_L细根直径呈抑制生长状态。其主要通过抑制中柱直径(一级根下降了17.61%,二级根下降了16.89%,三级根下降了20.62%)的生长来实现。以上结果表明,在同一立地条件下,白桦和山杨的细根对不同浓度N沉降的响应方式不同。     

Impaired Bone Homeostasis in Amyotrophic Lateral Sclerosis Mice with Muscle Atrophy

There is an intimate relationship between muscle and bone throughout life. However, how alterations in muscle functions in disease impact bone homeostasis is poorly understood. Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by progressive muscle atrophy. In this study we analyzed the effects of ALS on bone using the well established G93A transgenic mouse model, which harbors an ALS-causing mutation in the gene encoding superoxide dismutase 1. We found that 4-month-old G93A mice with severe muscle atrophy had dramatically reduced trabecular and cortical bone mass compared with their sex-matched wild type (WT) control littermates. Mechanically, we found that multiple osteoblast properties, such as the formation of osteoprogenitors, activation of Akt and Erk1/2 pathways, and osteoblast differentiation capacity, were severely impaired in primary cultures and bones from G93A relative to WT mice; this could contribute to reduced bone formation in the mutant mice. Conversely, osteoclast formation and bone resorption were strikingly enhanced in primary bone marrow cultures and bones of G93A mice compared with WT mice. Furthermore, sclerostin and RANKL expression in osteocytes embedded in the bone matrix were greatly up-regulated, and β-catenin was down-regulated in osteoblasts from G93A mice when compared with those of WT mice. Interestingly, calvarial bone that does not load and long bones from 2-month-old G93A mice without muscle atrophy displayed no detectable changes in parameters for osteoblast and osteoclast functions. Thus, for the first time to our knowledge, we have demonstrated that ALS causes abnormal bone remodeling and defined the underlying molecular and cellular mechanisms.     

Sensing of mycobacterial arabinogalactan by galectin‐9 exacerbates mycobacterial infection

Mycobacterial arabinogalactan (AG) is an essential cell wall component of mycobacteria and a frequent structural and bio‐synthetical target for anti‐tuberculosis (TB) drug development. Here, we report that mycobacterial AG is recognized by galectin‐9 and exacerbates mycobacterial infection. Administration of AG‐specific aptamers inhibits cellular infiltration caused by Mycobacterium tuberculosis (Mtb) or Mycobacterium bovis BCG, and moderately increases survival of Mtb‐infected mice or Mycobacterium marinum‐infected zebrafish. AG interacts with carbohydrate recognition domain (CRD) 2 of galectin‐9 with high affinity, and galectin‐9 associates with transforming growth factor β‐activated kinase 1 (TAK1) via CRD2 to trigger subsequent activation of extracellular signal‐regulated kinase (ERK) as well as induction of the expression of matrix metalloproteinases (MMPs). Moreover, deletion of galectin‐9 or inhibition of MMPs blocks AG‐induced pathological impairments in the lung, and the AG‐galectin‐9 axis aggravates the process of Mtb infection in mice. These results demonstrate that AG is an important virulence factor of mycobacteria and galectin‐9 is a novel receptor for Mtb and other mycobacteria, paving the way for the development of novel effective TB immune modulators.     

枝条覆盖对平茬花棒生长特征和浅层土壤水分的影响

为了综合评价枝条覆盖对不同立地(平地、丘顶、丘间低地)平茬花棒(Hedysarum scoparium)生长特征和浅层土壤水分的影响,以吉兰泰荒漠-绿洲过渡区平茬花棒为研究对象,于2017年4—10月对覆盖与无覆盖平茬花棒物候期、土壤容积含水量、土壤储水量和生长指标等相关指标进行测定。实验结果表明:(1)枝条覆盖有利于平茬花棒提前萌芽,较无覆盖提前5 d以上。(2)枝条覆盖使不同立地平茬花棒生长初期和生长末期0—100 cm土层平均土壤容积含水量增加20.12%以上,且土壤含水量变异系数随土层深度增加变化减缓;土壤储水量在生长初期和生长末期枝条覆盖较无覆盖增加27%以上。(3)枝条覆盖使平茬花棒株高、新生枝数、基径、冠幅较无覆盖增加10.91%以上,同时其生物量鲜重和干重较无覆盖分别增加4.45%—24.17%和3.78%—18.93%。尤其丘间低地平茬花棒生物量增加效果更加明显(P>0.05)。但枝条覆盖不能改变平茬花棒生物量分配格局;(4)枝条覆盖和平茬对花棒生长均具有积极作用,枝条覆盖对丘间低地平茬花棒生长促进作用最好。     

Hh-induced Smoothened conformational switch is mediated by differential phosphorylation at its C-terminal tail in a dose- and position-dependent manner

The activation of Smoothened (Smo) requires phosphorylation at three clusters of Serine residues in Drosophila Hedgehog (Hh) signaling. However, the mechanism by which phosphorylation promotes Smo conformational change and subsequently activates Smo in response to Hh gradient remains unclear. Here, we show that the conformational states of Smo are determined by not only the amount but also the position of the negative charges provided by phosphorylation. By using a Smo phospho-specific antibody, we demonstrate that Smo is differentially phosphorylated at three clusters of serine residues in response to levels of Hh activity. Mutating the first cluster, compared to mutating the other clusters, impairs Smo activity more severely, whereas mutating the last cluster prohibits C-terminus dimerization. In addition, phosphorylation of the membrane proximal cluster promotes phosphorylation of the distal cluster. We propose a zipper-lock model in which the gradual phosphorylation at these clusters induces a gradual conformational change in the Smo cytoplasmic tail, which promotes the interaction between Smo and Costal2 (Cos2). Moreover, we show that Hh regulates both PKA and CK1 phosphorylation of Smo. Thus, the differential phosphorylation of Smo mediates the thresholds of Hh activity.     

Construction and expression of humanized chimeric T cell receptor specific for chronic myeloid leukemia cells

Chimeric T cell receptors (chTCRs), composed of the single-chain variable fragments (scFv) of murine antibodies and human signaling molecules, are used to redirect the specificity of autologous or allogeneic T lymphocytes. To develop novel therapeutic agents for treatment of chronic myeloid leukemia (CML), we engineered a scFv from the hybridoma cell line CMA1 which produces monoclonal antibody specific against CML. The genes encoding the heavy and light chain variable regions were amplified from CMA1 cDNA and a humanized chTCR was constructed. Expression of the novel hchTCR was verified in NIH3T3 cells transduced with retroviral vectors. The results demonstrated that hchTCR can be expressed and presented on cell surface normally. These results suggest that retroviral vectors expressing hchTCR specific for CML cells may be used to redirect human T lymphocytes.