Genetic Analyses of the Internal Transcribed Spacer Sequences Suggest Introgression and Duplication in the Medicinal Mushroom Agaricus subrufescens

The internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster is widely used in fungal taxonomy and phylogeographic studies. The medicinal and edible mushroom Agaricus subrufescens has a worldwide distribution with a high level of polymorphism in the ITS region. A previous analysis suggested notable ITS sequence heterogeneity within the wild French isolate CA487. The objective of this study was to investigate the pattern and potential mechanism of ITS sequence heterogeneity within this strain. Using PCR, cloning, and sequencing, we identified three types of ITS sequences, A, B, and C with a balanced distribution, which differed from each other at 13 polymorphic positions. The phylogenetic comparisons with samples from different continents revealed that the type C sequence was similar to those found in Oceanian and Asian specimens of A. subrufescens while types A and B sequences were close to those found in the Americas or in Europe. We further investigated the inheritance of these three ITS sequence types by analyzing their distribution among single-spore isolates from CA487. In this analysis, three co-dominant markers were used firstly to distinguish the homokaryotic offspring from the heterokaryotic offspring. The homokaryotic offspring were then analyzed for their ITS types. Our genetic analyses revealed that types A and B were two alleles segregating at one locus ITSI, while type C was not allelic with types A and B but was located at another unlinked locus ITSII. Furthermore, type C was present in only one of the two constitutive haploid nuclei (n) of the heterokaryotic (n+n) parent CA487. These data suggest that there was a relatively recent introduction of the type C sequence and a duplication of the ITS locus in this strain. Whether other genes were also transferred and duplicated and their impacts on genome structure and stability remain to be investigated.     

氯化钠密度梯度离心法制备用于显微注射的外源DNA片段

DNA显微注射是生产转基因动物最可靠和最常使用的一种方法,外源DNA的纯度对显微注射的成功起着至关重要的作用。本文介绍用氯化钠密度梯度离心的方法制备用于显微注射的外源DNA片段。与传统的琼脂糖凝胶回收的方法相比较,用此方法制备的外源DNA片段对小鼠受精卵进行显微注射后,受卵体母鼠的胚胎存活率,以及子代小鼠的外源基因整合率均有明显的提高。这一方法可为进一步提高转基因动物的成功率,提供方法学上的参考。 Abstract:DNA microinjection is the most popular and reliable method of producing transgenic animals.The purity of foreign DNA plays an important role for the success of microinjection.In this study,we introduced the use of sodium chloride step gradients in fractionating foreign DNA fragment for microinjection.The data demonstrated that,compared with the conventional agarose gel extraction method,NaCl purification scheme of toreign DNA could improve the treated embryo survival and foreign DNA intergration rate markedly.     

江浙蝮蛇毒溶血毒素晶体培养及初步晶体学研究

从江浙蝮蛇中分离纯化的碱性磷脂酶Ａ２在ｐＨ９．５,０．０５ｍｍｏｌ／ＬＣＨＥＳ缓冲液中,用汽相悬滴扩散的方法,获得了适用于高分辨率Ｘ射线结构分析的单晶．经Ｘ２００Ｂ面探测器分析,表明该晶体属于正交晶系,Ｐ２Ｉ２Ｉ２Ｉ空间群,晶胞参数为ａ＝９７．１３,ｂ＝１０３．６９,ｃ＝２３．２７．并收集了一套衍射数据,独立衍射点数１２００１个,数据完整度为８６．２％,Ｒｍｅｒｇｅ为０．０４５９．最高分辨率达２．０,根据分子量与晶胞体积估算,一个不对称单位含两个分子．     

新疆察布查尔锡伯族体质特征调查

本文调查了新疆察布查尔锡伯族居民220人(男130人、女90人),年龄从20岁到78岁。观察22项,测量65项。调查结果表明,锡伯族居民具有典型的黄种人东亚人种的特征。如头圆宽且高,胡须少,眼裂狭窄、上眼睑褶皱多达睫毛处。耳大、鼻梁较直、鼻高中等,指距长、骨盆宽等。这些特征用等差级数法比较,与达斡尔族、华北地区汉族、蒙古族较接近,与苗族、黎族较远。     

A single dose of recombinant VSV-RABVG vaccine provides full protection against RABV challenge

Highlights:
1. A replication-competent recombinant VSV with RABV-G protein replacement was generated.
2. Single dose of VSV-RABVG immunization induce potent antigen-specific humoral immune response, especially the virus neutralizing antibodies.
3. Mice intranasally immunized with single dose of VSV-RABVG were 100% protected upon RABV challenge.     

Periplaneta Americana extract inhibits osteoclastic differentiation in vitro

ObjectivesPeriplaneta americana extract (PAE) is proven to be promising in treating fever, wound healing, liver fibrosis, and cardiovascular disease. However, the role of PAE in skeletal disorders remains unclear. This study investigated whether PAE regulates osteoclastic differentiation in vitro via the culture using RAW264.7 cells and bone marrow derived macrophages (BMDMs).Materials and MethodsRAW264.7 cells and BMDMs were cultured and induced for osteoclastic differentiation supplementing with different concentrations of PAE (0, 0.1, 1, and 10 mg/mL). Cell counting kit‐8 (CCK‐8) assay was used to detect the cytotoxicity and cell proliferation. TRAP staining, actin ring staining, real‐time quantitative PCR (RT‐qPCR), and bone resorption activity test were performed to detect osteoclastic differentiation. RT‐qPCR and enzyme‐linked immunosorbent assay (ELISA) were conducted to assay the expression and secretion of inflammatory factors. RNA sequencing (RNA‐seq) and western blot analysis were carried out to uncover the underlying mechanism.ResultsCCK‐8 results showed that 10 mg/mL and a lower concentration of PAE did not affect cell proliferation. RT‐qPCR analysis verified that PAE down‐regulated the osteoclastic genes Nfatc1, Ctsk, and Acp5 in macrophages. Moreover, PAE restrained the differentiation, formation, and function of osteoclasts. Besides, RT‐qPCR and ELISA assays showed that PAE decreased inflammatory genes expression and reduced the secretion of inflammatory factors, including IL1β, IL6, and TNFα. Subsequent RNA‐seq analysis identified possible genes and signaling pathways of PAE‐mediated osteoclastogenesis suppression.ConclusionsOur study indicates that PAE has inhibitive effects on osteoclastogenesis and may be a potential therapeutic alternative for bone diseases.

Periplaneta americana extract (PAE), the animal medicine material extracted from the insects Periplaneta americana, is proven to possess a variety of pharmacological functions. However, the role of PAE in skeletal disorders remains unclear. In this study, we found that PAE decreased osteoclast genes expression Nfatc1, Ctsk, and Acp5 in macrophages. Besides, PAE restrained the differentiation, formation, and function of osteoclasts. Moreover, PAE suppressed the LPS‐induced inflammation. Subsequent RNA‐seq analysis identified the signaling pathways of PAE‐mediated osteoclastogenesis suppression. Our study indicated that PAE has inhibitive effects on osteoclastic differentiation and may be a potential therapeutic Chinese medicine for bone diseases.     

High-sensitive electrochemical detection of point mutation based on polymerization-induced enzymatic amplification

Here a highly sensitive electrochemical method is described for the detection of point mutation in DNA. Polymerization extension reaction is applied to specifically initiate enzymatic electrochemical amplification to improve the sensitivity and enhance the performance of point mutation detection. In this work, 5'-thiolated DNA probe sequences complementary to the wild target DNA are assembled on the gold electrode. In the presence of wild target DNA, the probe is extended by DNA polymerase over the free segment of target as the template. After washing with NaOH solution, the target DNA is removed while the elongated probe sequence remains on the sensing surface. Via hybridizing to the designed biotin-labeled detection probe, the extended sequence is capable of capturing detection probe. After introducing streptavidin-conjugated alkaline phosphatase (SA-ALP), the specific binding between streptavidin and biotin mediates a catalytic reaction of ascorbic acid 2-phosphate (AA-P) substrate to produce a reducing agent ascorbic acid (AA). Then the silver ions in solution are reduced by AA, leading to the deposition of silver metal onto the electrode surface. The amount of deposited silver which is determined by the amount of wild target can be quantified by the linear sweep voltammetry (LSV). The present approach proved to be capable of detecting the wild target DNA down to a detection limit of 1.0×10(-14) M in a wide target concentration range and identifying -28 site (A to G) of the β-thalassemia gene, demonstrating that this scheme offers a highly sensitive and specific approach for point mutation detection.     

Cloning, expression, purification, and characterization of AmphiTip30, a member of short-chain dehydrogenases/reductases family from the amphioxus Branchiostoma belcheri tsingtauense

In this paper we describe the cloning, expression and identification study of the TIP30 gene from amphioxus (Branchiostoma belcheri). The amphioxus TIP30 cDNA is comprised of 1499 bp and is translated in one open-reading frame to give a protein of 237 amino acids, with a predicted 23 amino acids signal peptide, a 147 bp 5'-UTR and a 638 bp 3'-UTR. A multiple alignment of TIP30 from amphioxus with other known TIP30 sequences shows the conservation of most amino acid residues involved in the peculiar structural domains found within TIP30's. Phylogenetic analysis places AmphiTIP30 at the base of the phylogenetic tree, suggesting that AmphiTIP30 is the archetype of the vertebrate TIP30 genes. We express the amphioxus TIP30 gene in Escherichia coli. driven by T7 promoter. The recombinant amphioxus TIP30 protein was purified by HisTrap affinity column. Subsequently, the binding constant and enzyme activity was mensurated. Western blot and immunohistochemistry analysis confirmed that amphioxus has a native molecular mass of approximately 26 kDa, and TIP30 was strongly expressed in ovary. Finally, the initial function of TIP30 is discussed.     

Comparative Proteomics of Milk Fat Globule Membrane Proteins from Transgenic Cloned Cattle

The use of transgenic livestock is providing new methods for obtaining pharmaceutically useful proteins. However, the protein expression profiles of the transgenic animals, including expression of milk fat globule membrane (MFGM) proteins, have not been well characterized. In this study, we compared the MFGM protein expression profile of the colostrum and mature milk from three lines of transgenic cloned (TC) cattle, i.e., expressing recombinant human α-lactalbumin (TC-LA), lactoferrin (TC-LF) or lysozyme (TC-LZ) in the mammary gland, with those from cloned non-transgenic (C) and conventionally bred normal animals (N). We identified 1, 225 proteins in milk MFGM, 166 of which were specifically expressed only in the TC-LA group, 265 only in the TC-LF group, and 184 only in the TC-LZ group. There were 43 proteins expressed only in the transgenic cloned animals, but the concentrations of these proteins were below the detection limit of silver staining. Functional analysis also showed that the 43 proteins had no obvious influence on the bovine mammary gland. Quantitative comparison revealed that MFGM proteins were up- or down-regulated more than twofold in the TC and C groups compared to N group: 126 in colostrum and 77 in mature milk of the TC-LA group; 157 in colostrum and 222 in mature milk of the TC-LF group; 49 in colostrum and 98 in mature milk of the TC-LZ group; 98 in colostrum and 132 in mature milk in the C group. These up- and down-regulated proteins in the transgenic animals were not associated with a particular biological function or pathway, which appears that expression of certain exogenous proteins has no general deleterious effects on the cattle mammary gland.