Microbiota transplantation: concept,methodology and strategy for its modernization

Fecal microbiota transplantation (FMT) has become a research focus of biomedicine and clinical medicine in recent years. The clinical response from FMT for different diseases provided evidence for microbiota-host interactions associated with various disorders, including Clostridium difficile infection, inflammatory bowel disease, diabetes mellitus, cancer, liver cirrhosis, gutbrain disease and others. To discuss the experiences of using microbes to treat human diseases from ancient China to current era should be important in moving standardized FMT forward and achieving a better future. Here, we review the changing concept of microbiota transplantation from FMT to selective microbiota transplantation, methodology development of FMT and stepup FMT strategy based on literature and state experts’ perspectives.     

An appropriate loading control for western blot analysis in animal models of myocardial ischemic infarction

An appropriate loading control is critical for Western blot analysis. Housekeeping proteins (HKPs), such as β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin, are commonly used to normalize protein expression. But HKP expression can be impacted by certain experimental conditions, such as ischemic myocardial infarction. This study was undertaken to look for an appropriate loading control for western blot analysis of ischemic myocardium. Myocardial ischemic infarction was induced by left anterior descending coronary artery (LAD) ligation in Rhesus monkeys and C57BL/6 mice. The heart tissue samples from different areas and time points after surgery were subjected to western blot or gel staining. The level of β-actin, GAPDH, β-tubulin, and total protein were tested. The total protein level was consistent in all groups, whereas the protein level of β-tubulin and β-actin were different in all groups. However, the protein level of GAPDH was stable in the Rhesus monkey model. We concluded that total protein was the most appropriate internal control in different stages of myocardial ischemic disease of various animal models. GAPDH is a reliable internal control only for ischemic myocardium of Rhesus monkey.     

镰刀菌Q7-31T内切葡聚糖酶Egn21的分离纯化及酶学性质

【目的】为进一步研究镰刀菌Q7-31T产生的植物细胞壁降解酶的酶系信息。【方法】以1%(W/V)蛋白胨为氮源,0.5%(W/V)燕麦秸秆为碳源,20°C、120 r/min振荡培养3 d,诱导发酵培养菌株,获得的粗酶液经过Sephacry S-100凝胶柱层析和DEAE琼脂糖弱阴离子交换柱层析,最终得到纯化的内切葡聚糖酶,并对其进行酶学性质分析及串联质谱鉴定。【结果】研究表明:Egn21的分子质量为44.25 kDa,等电点为4.91;酶学特性研究显示:Egn21降解羧甲基纤维素的最适反应温度为40°C,在45°C以下比较稳定。该酶最适pH为6.0,在pH为5.0–8.0条件之间比较稳定。Co~(2+)、Zn~(2+)和Mg~(2+)对其没有明显作用,而Fe~(2+)、Ca~(2+)、K~(+)、Na~(+)和Mn~(2+)对酶活性有抑制作用,Hg~(2+)会使酶失去活性。【结论】从Q7-31T中分离纯化得到的内切葡聚糖酶Egn21,经过酶学特性与串联质谱鉴定结果显示其属于GH5家族。     

Phylogenetic relationships and divergence dates of softshell turtles (Testudines: Trionychidae) inferred from complete mitochondrial genomes

The softshell turtles (Trionychidae) are one of the most widely distributed reptile groups in the world, and fossils have been found on all continents except Antarctica. The phylogenetic relationships among members of this group have been previously studied; however, disagreements regarding its taxonomy, its phylogeography and divergence times are still poorly understood as well. Here, we present a comprehensive mitogenomic study of softshell turtles. We sequenced the complete mitochondrial genomes of 10 softshell turtles, in addition to the GenBank sequence of Dogania subplana, Lissemys punctata, Trionyx triunguis, which cover all extant genera within Trionychidae except for Cyclanorbis and Cycloderma. These data were combined with other mitogenomes of turtles for phylogenetic analyses. Divergence time calibration and ancestral reconstruction were calculated using BEAST and RASP software, respectively. Our phylogenetic analyses indicate that Trionychidae is the sister taxon of Carettochelyidae, and support the monophyly of Trionychinae and Cyclanorbinae, which is consistent with morphological data and molecular analysis. Our phylogenetic analyses have established a sister taxon relationship between the Asian Rafetus and the Asian Palea + Pelodiscus + Dogania + Nilssonia + Amyda, whereas a previous study grouped the Asian Rafetus with the American Apalone. The results of divergence time estimates and area ancestral reconstruction show that extant Trionychidae originated in Asia at around 108 million years ago (MA), and radiations mainly occurred during two warm periods, namely Late Cretaceous–Early Eocene and Oligocene. By combining the estimated divergence time and the reconstructed ancestral area of softshell turtles, we determined that the dispersal of softshell turtles out of Asia may have taken three routes. Furthermore, the times of dispersal seem to be in agreement with the time of the India–Asia collision and opening of the Bering Strait, which provide evidence for the accuracy of our estimation of divergence time. Overall, the mitogenomes of this group were used to explore the origin and dispersal route of Trionychidae and have provided new insights on the evolution of this group.     

人降钙素基因相关肽转基因马铃薯的RT-PCR分析

报道经过农杆菌介导将人降钙素基因相关肽(calcitoningenerelatedpeptide,CGRP)基因由马铃薯块茎专一表达classIpatatin基因5′侧翼区和CaMV35S启动子驱动构建的马铃薯表达载体导入马铃薯,PCR鉴定获得了转基因植株。RTPCR分析证实classIpatatin基因5′侧翼区驱动的CGRPmRNA在转基因马铃薯中的表达。研究结果在开发转基因马铃薯生物反应器表达医用多肽中具有重要意义。     

Zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) mediates multiple intracellular signaling pathways

Insect PGRPs can function as bacterial recognition molecules triggering proteolytic and/or signal transduction pathways, with the resultant production of antimicrobial peptides. To explore if zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) has such effects, RNA interference (siRNA) and high-density oligonucleotide microarray analysis were used to identify differentially expressed genes regulated by zfPGRP-SC. The mRNA levels for a set of genes involved in Toll-like receptor signaling pathway, such as TLRs, SARM, MyD88, TRAF6 and nuclear factor (NF)-kappa B2 (p100/p52), were examined by quantitative RT-PCR (QT-PCR). The results from the arrays and QT-PCR showed that the expression of 133 genes was involved in signal transduction pathways, which included Toll-like receptor signaling, Wnt signaling, BMP signaling, insulin receptor signaling, TGF-beta signaling, GPCR signaling, small GTPase signaling, second-messenger-mediated signaling, MAPK signaling, JAK/STAT signaling, apoptosis and anti-apoptosis signaling and other signaling cascades. These signaling pathways may connect with each other to form a complex network to regulate not just immune responses but also other processes such as development and apoptosis. When transiently over-expressed in HEK293T cells, zfPGRP-SC inhibited NF-κB activity with and without lipopolysacharide (LPS) stimulation.     

Development of an industrial medium for economical 2,3-butanediol production through co-fermentation of glucose and xylose by Klebsiella oxytoca

An industrial medium containing urea as a sole nitrogen source, low levels of corn steep liquor and mineral salts as nutrition factors to retain high 2,3-butanediol production through co-fermentation of glucose and xylose (2:1, wt/wt) by Klebsiella oxytoca was developed. Urea and corn steep liquor were identified as the most significant factors by the two-level Plackett–Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors and a central composite design was employed to determine their optimal levels. Under the optimal medium, the yield of 2,3-butanediol plus acetoin relative to glucose and xylose was up to 0.428 g/g, which was 85.6% of theoretical value. The cheap nitrogen source and nutrition factors combining the co-fermentation process using lignocellulose derived glucose and xylose as the carbon source in the developed medium would be a potential solution to improve the economics of microbial 2,3-butanediol production.     

Analysis of the monosaccharide composition of purified polysaccharides in Ganoderma atrum by capillary gas chromatography

Introduction – Ganoderma, one of the best‐known traditional Chinese medicines, has attracted considerable attention owing to the fact that dozens of polysaccharides isolated from it have shown diverse and potentially significant pharmacological activities. However, no work has been reported on the analysis of monosaccharide composition of polysaccharide isolated from the aqueous extract of Ganoderma atrum yet. Objective – To develop a simple and sensitive GC‐based method for the analysis of monosaccharide composition of purified polysaccharides in Ganoderma atrum. Methodology – The polysaccharide was first hydrolysed to give the constituent monosaccharides, which were subsequently derived into acetylated aldononitriles and analysed by gas chromatography using a capillary column packed with a (5%phenyl) methylpolysiloxane stationary phase with the addition of acetyl inositol as the inner standard. High‐performance liquid chromatography was also used for comparison. Results – The stable derivatives of the most common monosaccharides could be separated and reproducibly determined with high sensitivity. The limits of detection and quantification were 0.013 and 0.043 mg/mL, respectively. The intermediary precision values (expressed as the RSD) were less than 10%. The mean recovery of the method was 100 ± 3%, with RSD values of less than 5%. The results obtained from GC and HPLC methods were found to be close to each other within acceptable error ranges. Conclusion – This study demonstrated that the developed method could be applied as an accurate method for the compositional analysis of monosaccharides in the field of biological and biochemical study. Copyright © 2009 John Wiley & Sons, Ltd.