A Zernike-moment-based non-local denoising filter for cryo-EM images

Cryo-electron microscopy (cryo-EM) plays an important role in determining the structure of proteins, viruses, and even the whole cell. It can capture dynamic structural changes of large protein complexes, which other methods such as X-ray crystallography and nuclear magnetic resonance analysis find difficult. The signal-to-noise ratio of cryo-EM images is low and the contrast is very weak, and therefore, the images are very noisy and require filtering. In this paper, a filtering method based on non-local means and Zernike moments is proposed. The method takes into account the rotational symmetry of some biological molecules to enhance the signal-to-noise ratio of cryo-EM images. The method may be useful in cryo-EM image processing such as the automatic selection of particles, orientation determination, and the building of initial models.     

栓菌漆酶在毕赤酵母中高效表达及重组酶的性质

栓菌420（Trametes sp. 420）漆酶基因lacD以两种方式在巴斯德毕赤酵母（Pichia pastoris）进行异源表达,产生两种重组漆酶：rLacDx（具有天然N-末端）和rLacDe（N-末端带有8个额外的氨基酸残基）。摇瓶发酵18d,rLacDx和rLacDe的产量分别为1.21×10⁵u/L、7.38×10⁴u/L ［以2,2′-连氮-3-乙苯-二噻唑-6磺酸（ABTS）为底物］。在高密度发酵条件下,rLacDx的产量增加到2.39×10⁵u/L,同时其生产周期降至7.5 d。两种重组酶对愈创木酚底物的氧化特性相似,且在50℃和pH3～10的范围内均稳定。然而,rLacDx对底物ABTS的比活力（1761u/mg）高于rLacDe （1122u/mg）,其表观K_m值(427μmol/L)低于rLacDe (604μmol/L)。     

多重实时荧光PCR相对定量法快速诊断唐氏综合征

为了建立一种基于多重实时荧光相对定量PCR技术并应用之于唐氏综合征分子诊断, 选择21号染色体上唐氏综合征特异区域基因片段(DSCR3)为目的基因, 以12号染色体上的磷酸甘油醛脱氢酶基因(GAPDH)为参照基因, 设计合成两对引物以及分别以不同荧光标记的TaqMan探针, 在同一个反应管中进行扩增。以相对定量指标△CT值区分唐氏综合征患者与正常人。采用EB 病毒转化技术, 把唐氏综合征患者外周血B 淋巴细胞转化成永生淋巴母细胞系作为标准品。通过优化反应条件, 使得目的基因和参照基因的扩增效率基本一致, 接近100%, 模板浓度在3～300 ng/μL范围内, △CT值的变异系数小于15%, 浓度在30 ng/μL时, 变异系数最小(＜10%), 以该浓度的DNA作为模板进行批内和批间实验的△CT值重复性好, 变异系数分别为9.8%和13.3%。运用建立的方法检测20例唐氏综合征患者的血标本和30例正常人的血标本, 正常人△CT值范围是-1.90～-1.30, 患者的△CT值范围是-2.95～-2.15, 两组之间无交叉重叠, 有明显差异(P＜0.001)。唐氏综合征患者永生细胞系建系成功 ,染色体核型和DNA 分析表明建系前后遗传是稳定的。因此, 实时荧光定量PCR比较△CT值的相对定量法快速诊断唐氏综合征是可行的。     

敲除trim47基因斑马鱼脑和脾的比较转录组学分析

用CRISPR/Cas9技术敲除斑马鱼(Danio Rerio)的trim47, 收集TRIM47–/–(trim47基因敲除)和WT(野生型)斑马鱼的大脑和脾脏进行RNA-seq分析, 以鉴定差异表达基因(DEGs)。总共确定了271个DEGs, 经过简要分析, 将这些DEGs注释为KEGG途径和GO富集分析, 与WT组相比, TRIM47-/-组的脑中DEGs集中在细胞黏附和谷氨酸能突触信号传导途径中。在脾脏中, 与野生型组相比, TRIM47-/-组的DEGs在补体和凝血级联信号通路中发生了变化。使用qRT-PCR验证了脑和脾中与补体途径相关的基因, 与转录组数据一致。这些结果表明, Trim47在脾脏和大脑中起着重要的生物学作用, 尤其是通过补体途径参与先天免疫功能。体内感染实验表明, trim47基因敲除可提高斑马鱼中鲤春病毒血症病毒(SVCV)的感染率。总之, 这些发现为TRIM成员在先天免疫中的功能提供了新线索。     

Spontaneous modulation of a dynamic balance between bacterial genomic stability and mutability: roles and molecular mechanisms of the genetic switch

Bacteria need a high degree of genetic stability to maintain their species identities over long evolutionary times while retaining some mutability to adapt to the changing environment.It is a long unanswered question that how bacteria reconcile these seemingly contradictory biological properties.We hypothesized that certain mechanisms must maintain a dynamic balance between genetic stability and mutability for the survival and evolution of bacterial species.To identify such mechanisms,we analyzed bacterial genomes,focusing on the Salmonella mismatch repair(MMR)system.We found that the MMR gene mutL functions as a genetic switch through a slipped-strand mispairing mechanism,modulating and maintaining a dynamic balance between genetic stability and mutability during bacterial evolution.This mechanism allows bacteria to maintain their phylogenetic status,while also adapting to changing environments by acquiring novel traits.In this review,we outline the history of research into this genetic switch,from its discovery to the latest findings,and discuss its potential roles in the genomic evolution of bacteria.     

Structural basis of the ultrasensitive calcium indicator GCaMP6

GCaMP is one of the most widely used calcium indicators in neuronal imaging and calcium cell biology.The newly developed GCaMP6 shows superior brightness and ultrasensitivity to calcium concentration change.In this study,we determined crystal structures of Ca2+-bound GCaMP6 monomer and dimer and presented detailed structural analyses in comparison with its parent version GCaMP5G.Our analyses reveal the structural basis for the outperformance of this newly developed Ca2+indicator.Three substitution mutations and the resulting changes of local structure and interaction explain the ultrasensitivity and increased fluorescence intensity common to all three versions of GCaMP6.Each particular substitution in the three GCaMP6 is also structurally consistent with their differential sensitivity and intensity,maximizing the potential of using GCaMP6 in solving diverse problems in neuronal research and calcium signaling.Our studies shall also be beneficial to further structure-guided optimization of GCaMP and facilitate the design of novel calcium indicators.     

Description of a New Species of the Genus Brachytarsophrys Tian and Hu, 1983 （Amphibia： Anura： Megophryidae） from Southern China Based on Molecular and Morphological Data

A new species, Brachytarsophrys popei sp. nov., is described based on a series of specimens collected from Mount Jinggang, Jiangxi Province, Taoyuandong Nature Reserve, Hunan Province and Nanling Nature Reserve, Guangdong Province, China. The new species can be easily distinguished from other known congeners by morphology, morphometrics and molecular data of the mitochondrial 16S rRNA gene. It is characterized by its relatively small size with 86.2 mm in snout-vent length in adult female and 70.7 mm-83.5 mm in males; vomerine teeth bearing on two markedly elevated ridges, which projecting behind far beyond the posterior level of the choanae, widely separated by a distance nearly 1.5 times length of one; margin of tongue de~ply notched behind; toes about one-third to two-thirds webbed in males, at most one-third webbed in female; the webs extending as a wide fringes along either side of toes; upper eyelid with tubercles, one of which is enlarged and becoming a remarkably prominent, bluntly conical light- yellow horn; black tiny nuptial spines on the dorsal surface of the first finger and second finger base, single vocal sac in males; gravid females bear pure yellowish oocytes; tadpoles with a transverse white stripe on ventral surface and two longitudinal white stripes along the sides of body. The new species represents the fifth known Brachytarsophrys species.     

Na+ permeation and block of hERG potassium channels

The inactivation gating of hERG channels is important for the channel function and drug-channel interaction. Whereas hERG channels are highly selective for K+, we have found that inactivated hERG channels allow Na+ to permeate in the absence of K+. This provides a new way to directly monitor and investigate hERG inactivation. By using whole cell patch clamp method with an internal solution containing 135 mM Na+ and an external solution containing 135 mM NMG+, we recorded a robust Na+ current through hERG channels expressed in HEK 293 cells. Kinetic analyses of the hERG Na+ and K+ currents indicate that the channel experiences at least two states during the inactivation process, an initial fast, less stable state followed by a slow, more stable state. The Na+ current reflects Na+ ions permeating through the fast inactivated state but not through the slow inactivated state or open state. Thus the hERG Na+ current displayed a slow inactivation as the channels travel from the less stable, fast inactivated state into the more stable, slow inactivated state. Removal of fast inactivation by the S631A mutation abolished the Na+ current. Moreover, acceleration of fast inactivation by mutations T623A, F627Y, and S641A did not affect the hERG Na+ current, but greatly diminished the hERG K+ current. We also found that external Na+ potently blocked the hERG outward Na+ current with an IC50 of 3.5 mM. Mutations in the channel pore and S6 regions, such as S624A, F627Y, and S641A, abolished the inhibitory effects of external Na+ on the hERG Na+ current. Na+ permeation and blockade of hERG channels provide novel ways to extend our understanding of the hERG gating mechanisms.     

Diagnostic cytologic features of an epididymal melanotic neuroectodermal tumor of infancy present in scrotal fluid: a case report

BACKGROUND: The cytologic features of melanotic neuroectodermal tumor of infancy (MNTI) have been described in rare cases only, and these reports have been based solely on direct smears obtained from aspirated tumor material or from touch preparations of resected tumors. CASE: We report, to our knowledge, the first documented case of an epididymal MNTI diagnosed from aspirated hydrocele fluid processed using the ThinPrep system (Cytyc Corp., Boxborough, Massachusetts, U.S.A.). CONCLUSION: Based on a 15 year retrospective review of scrotal fluids submitted for cytologic evaluation to our laboratory, MNTI represented the sole neoplasm that was represented in the fluid and could be specifically diagnosed. Compared to the direct smear evaluation in our case and other published reports, the ThinPrep method enables the recognition of both neuroblast-like cells and larger melanin-containing epithelial cells in scrotal fluid, thereby aiding in the specific diagnosis of MNTI and minimizing the risk of misdiagnosing this tumor as a more aggressive neoplasm.