Challenges and advances in genome mining of ribosomally synthesized and post-translationally modified peptides (RiPPs)

Ribosomally synthesized and post-translationally modified peptides(RiPPs)are a class of cyclic or linear peptidic natural products with remarkable structural and functional diversity.Recent advances in genomics and synthetic biology,are facilitating us to discover a large number of new ribosomal natural products,including lanthipeptides,lasso peptides,sactipeptides,thiopeptides,microviridins,cyanobactins,linear thiazole/oxazole-containing peptides and so on.In this review,we summarize bioinformatic strategies that have been developed to identify and prioritize biosynthetic gene clusters(BGCs)encoding RiPPs,and the genome mining-guided discovery of novel RiPPs.We also prospectively provide a vision of what genomics-guided discovery of RiPPs may look like in the future,especially the discovery of RiPPs from dominant but uncultivated microbes,which will be promoted by the combinational use of synthetic biology and metagenome mining strategies.

Genetic diversity of the S-type small subunit ribosomal RNA gene of Plasmodium knowlesi isolates from Sabah,Malaysian Borneo and Peninsular Malaysia

Objective:To determine the genetic diversity of Plasmodium(P.)knowlesi isolates from Sabah,Malaysian Borneo and Peninsular Malaysia,targeting the S-type SSU rRNA gene and including aspects of natural selection and haplotype.Methods:Thirty-nine blood samples infected with P.knowlesi were collected in Sabah,Malaysian Borneo and Peninsular Malaysia.The S-type SSU rRNA gene was amplified using polymerase chain reaction,cloned into a vector,and sequenced.The natural selection and haplotype of the S-type SSU rRNA gene sequences were determined using DnaSP v6 and illustrated using NETWORK v10.This study's 39 S-type SSU rRNA sequences and eight sequences from the Genbank database were subjected to phylogenetic analysis using MEGA 11.Results:Overall,the phylogenetic analysis showed no evidence of a geographical cluster of P.knowlesi isolates from different areas in Malaysia based on the S-type SSU rRNA gene sequences.The S-type SSU rRNA gene sequences were relatively conserved and with a purifying effect.Haplotype sharing of the S-type SSU rRNA gene was observed between the P.knowlesi isolates in Sabah,Malaysian Borneo,but not between Sabah,Malaysian Borneo and Peninsular Malaysia.Conclusions:This study suggests that the S-type SSU rRNA gene of P.knowlesi isolates in Sabah,Malaysian Borneo,and Peninsular Malaysia has fewer polymorphic sites,representing the conservation of the gene.These features make the S-type SSU rRNA gene suitable for comparative studies,such as determining the evolutionary relationships and common ancestry among P.knowlesi species.

基于3A组装的多拷贝基因策略优化重组水蛭素变体Ⅲ的生产

水蛭素变体Ⅲ(Hirudin varidantⅢ,Hv3)是一种从水蛭中提取的活性成分,在预防和治疗白内障方面具有潜在作用。为了制备足量Hv3用于进一步的临床前应用研究,该研究开发了一种在谷氨酸棒杆菌(Corynebacterium glutamicum)中高效、稳定表达重组水蛭素变体Ⅲ(recombinant Hirudin varidantⅢ,Rhv3)的办法。首先,在C.glutamicum中构建含Ptac启动子和CspA信号肽的Rhv3分泌表达菌株,比较其在不同培养基中的表达情况。结果表明在BHI培养基中Rhv3活性最高,达到3.02×10^(3) ATU/L。为进一步提升Rhv3产量,通过核糖体结合位点(ribosome binding site,RBS)筛选,选用RBS1应用于Rhv3多拷贝菌株构建和表达。然后利用3A组装技术构建含不同信号肽和不同拷贝数的Rhv3分泌表达菌株进一步优化其产量。通过放大发酵培养,Rhv3产量达到1.89 g/L,活性达到10.91×10^(3) ATU/L。最后利用镍柱对Rhv3进行简单纯化,其纯度达到90%。该异源表达策略有效提高Rhv3表达量,为Rhv3的重组生产提供了一种质量可靠、低成本的表达体系。

Testing complete plastomes and nuclear ribosomal DNA sequences for species identification in a taxonomically difficult bamboo genus Fargesia

Fargesia,the largest genus within the temperate bamboo tribe Arundinarieae,has more than 90 species mainly distributed in the mountains of Southwest China.The Fargesia bamboos are important components of the subalpine forest ecosystems that provide food and habitat for many endangered animals,including the giant panda.However,species-level identification of Fargesia is difficult.Moreover,the rapid radiation and slow molecular evolutionary rate of Fargesia pose a significant challenge to using DNA barcoding with standard plant barcodes(rbcL,matK,and ITS) in bamboos.With progress in the sequencing technologies,complete plastid genomes(plastomes) and nuclear ribosomal DNA(nrDNA)sequences have been proposed as organelle barcodes for species identification;however,these have not been tested in bamboos.We collected 196 individuals representing 62 species of Fargesia to comprehensively evaluate the discriminatory power of plastomes and nrDNA sequences compared to standard barcodes.Our analysis indicates that complete plastomes have substantially higher discriminatory power(28.6%) than standard barcodes(5.7%),whereas nrDNA sequences show a moderate improvement(65.4%) compared to ITS(47.2%).We also found that nuclear markers performed better than plastid markers,and ITS alone had higher discriminatory power than complete plastomes.The study also demonstrated that plastomes and nrDNA sequences can contribute to intrageneric phylogenetic resolution in Fargesia.However,neither of these sequences were able to discriminate all the sampled species,and therefore,more nuclear markers need to be identified.

Non-conventional peptides in plants:From gene regulation to crop improvement

Conventional peptides(CPs)and non-conventional peptides(NCPs)are generated from small open reading frames,but most CPs are derived from large precursors.NCPs,which are derived from sequences other than conventional open reading frames or annotated coding sequences regions,function in plant development and adaptation to stresses.Ribosome profiling,a technique for studying translational regulation,can be used to identify NCPs.Another new technique,peptidogenomics,which integrates mass spectrometry and genomics,is becoming more widely used for identifying plant NCPs.In recent years,numerous studies have investigated the roles in monocots and dicots of miRNA-derived peptides and upstream open reading frames,which have potential for improving agronomic traits.Investigating the biological functions of NCPs will advance molecular plant breeding by identifying regulators of plant growth and development.We present an overview of NCP identification methods and recent findings about NCP biological functions.

Integrated top-down and bottom-up proteomics mass spectrometry for the characterization of endogenous ribosomal protein heterogeneity

Ribosomes are abundant,large RNA-protein complexes that are the sites of all protein synthesis in cells.Defects in ribosomal proteins(RPs),including proteoforms arising from genetic variations,alternative splicing of RNA transcripts,post-translational modifications and alterations of protein expression level,have been linked to a diverse range of diseases,including cancer and aging.Comprehensive characterization of ribosomal proteoforms is challenging but important for the discovery of potential disease biomarkers or protein targets.In the present work,using E.coli 70S RPs as an example,we first developed a top-down proteomics approach on a Waters Synapt G2 Si mass spectrometry(MS)system,and then applied it to the HeLa 80S ribosome.The results were complemented by a bottom-up approach.In total,50 out of 55 RPs were identified using the top-down approach.Among these,more than 30 RPs were found to have their N-terminal methionine removed.Additional modifications such as methylation,acetylation,and hydroxylation were also observed,and the modification sites were identified by bottomup MS.In a HeLa 80S ribosomal sample,we identified 98 ribosomal proteoforms,among which multiple truncated 80S ribosomal proteoforms were observed,the type of information which is often overlooked by bottom-up experiments.Although their relevance to diseases is not yet known,the integration of topdown and bottom-up proteomics approaches paves the way for the discovery of proteoform-specific disease biomarkers or targets.

核糖蛋白替代鱼粉对大菱鲆幼鱼生长性能的影响

该试验旨在研究核糖蛋白替代饵料中鱼粉对大菱鲆(Scophthalmus maximus)幼鱼生长性能的影响,探讨核糖蛋白(Ribosomal protein)替代鱼粉的可行性,并确定最适的替代比例。试验选择体质健壮、体重约为25g的大菱鲆幼鱼400尾,随机分成4组,每组5个重复,每个重复20尾大菱鲆幼鱼。对照组大菱鲆幼鱼投喂基础饵料,试验各组大菱鲆幼鱼分别投喂使用核糖蛋白替代1%、1.5%和2%鱼粉的试验饵料。饲喂8周后,检测各组大菱鲆幼鱼的生长性能。结果表明,在大菱鲆幼鱼饵料中使用核糖蛋白替代1.5%的鱼粉可以显著提高试验末重和增重率,分别比对照组显著提高了5.36%和10.53%(P<0.05);使用核糖蛋白替代1%、1.5%和2%的鱼粉可以使特定生长率分别显著提高7.14%、11.90%、8.73%(P<0.05);使用核糖蛋白替代1.5%的鱼粉可以显著降低大菱鲆幼鱼的饵料系数,显著降低了13.41%(P<0.05)。综上所述,在大菱鲆幼鱼养殖中使用核糖蛋白替代饵料中1.5%的鱼粉对其生长性能效果最优。

益气活血中药复方影响CVB_3病毒性心肌炎模型乳鼠心肌细胞MCP-1、Rps4基因表达的实验研究

目的:观察益气活血中药复方对CVB3病毒性心肌炎模型大鼠心肌细胞small inducible cytokine A2(MCP-1/CCL2)、ribosomal protein S4基因表达的影响,以期从基因水平揭示益气活血中药复方治疗CVB3心肌炎的作用机制,进一步证实益气活血中药复方是治疗CVB3病毒性心肌炎的有效方剂。方法:通过改良的抑制性消减杂交技术(SSH),克隆了受CVB3攻击的宿主细胞中被中药调控的基因,并通过荧光rt-PCR对上述结果进行验证。结果:益气活血中药复方能够上调CVB3病毒性心肌炎模型大鼠心肌细胞small inducible cytokine A2(MCP-1/CCL2)、ribosomal protein S4基因的表达。结论:益气活血中药复方可能通过调节small inducible cytokine A2(MCP-1/CCL2)、ribosomal protein S4基因的表达而实现治疗病毒性心肌炎的目的。

南极冰藻Chlamydomonas sp.ICE-L的cDNA文库构建及品质检测

为构建南极冰藻Chlamydomonas sp．ICE-L的cDNA文库，提取对数生长期南极冰藻ICE-L的总RNA，以此为模板，通过PowerScript逆转录酶逆转录合成第一链cDNA；再以第一链cDNA产物为模板，用LD-PCR合成第二链cDNA。该cDNA产物经分级分离转入大肠杆菌中，即获得南极冰藻IcBL的cDNA原始文库，其滴度为1．6×10^6 cfu／mL。扩增后的cDNA文库的滴度为1．0×10^10 cfu／mL。用PCR方法测得文库的重组率大干97％，插入cDNA的长度为0．5～1．8kb，0．9kb以上插入片段占50％以上。取适量扩增文库稀释并铺平板，挑取72个独立菌落，对其中22个独立菌落所插入的cDNA进行测序，克隆到了一个具有5’和3’非编码区的40S ribosomal protein S5全长基因序列，GenBank收录号为AM167929。

负载核糖体蛋白L8的树突状细胞对黑色素瘤的抑制作用

背景与目的:研究发现核糖体蛋白L8(ribosomal protein L8,RPL8)在黑色素瘤中表达能激活黑色素瘤患者外周血单个核细胞增殖并产生白细胞介素2,提示RPL8可能参与抗肿瘤免疫应答,有望成为抗肿瘤治疗的靶点。本研究通过RPL8蛋白负载树突状细胞(dentritic cell,DC),探讨负载RPL8蛋白的DC对黑色素瘤的免疫效应。方法:原核表达RPL8蛋白,纯化后致敏小鼠骨髓来源DC,流式细胞仪检测DC表面标志,MTT法检测细胞毒性T淋巴细胞对小鼠黑色素瘤细胞的杀伤作用;负载RPL8蛋白DC免疫治疗小鼠后,观察肿瘤体积变化及小鼠生存时间。结果:纯化蛋白经蛋白质印迹法(Western blot)分析见约28×103大小的特异性条带;DC经RPL8及细菌脂多糖(Lipoplysaccharide,LPS)诱导成熟后细胞表面CD11c、CD80、MHC-Ⅰ类、MHC-Ⅱ类分子表达增高,能激活T淋巴细胞,对B16细胞有抑制作用,RPL8-DC组的抑制率在效靶比为30∶1时高达70%,较PBS组和DC组明显增高;负载RPL8蛋白DC免疫治疗小鼠后,肿瘤体积缩小,小鼠的生存期明显延长。结论:负载RPL8的DC对黑色素瘤有生长抑制作用。

采用酵母双杂交法筛选PSRPK相关蛋白基因

本课题组在前期研究中,从多头绒泡菌中分离到一个SR蛋白激酶基因psrpk,并将其编码蛋白命名为PSRPK(SR protein kinase ofPhysarum Polycephalum).为分离PSRPK相关蛋白基因以了解PSRPK的功能,构建了转化率为2×106转化子/3μg pGADT7-Rec、密度为5.35×108cells/mL、滴度为2.34×109cfu/mL的多头绒泡菌酵母双杂交AD库.以PSRPK为饵蛋白筛选该文库得到接合率为41.18%的杂交酵母,在SD/-Leu/-Trp/-Ade/-H is培养板上筛选获得1476个杂交克隆,其中,X-gal滤纸显色呈强蓝色的克隆有342个.对大于500 bp的67个克隆测序获得35个cDNA片段,其中编码Plasm in C、branched-chain am inoacid am inotransferase(BCAT)类似蛋白、MSF1类似蛋白、m ixed-linked glucanase precursor类似蛋白、FCY1p类似蛋白和40S ribosomal protein S2类似蛋白等7个cDNA片段在阳性杂交酵母克隆中出现多次,其余仅出现一次.在35个cDNA片段的编码序列中有15个具有同源蛋白,31个编码序列的丝氨酸含量大于或等于6%,符合激酶底物的组成特征.

Effect of moxibustion on mTOR-mediated autophagy in rotenone-induced Parkinson's disease model rats

Defects in autophagy-mediated clearance of α-synuclein may be one of the key factors leading to progressive loss of dopaminergic neurons in the substantia nigra. Moxibustion therapy for Parkinson’s disease has been shown to have a positive effect, but the underlying mechanism remains unknown. Based on this, we explored whether moxibustion could protect dopaminergic neurons by promoting autophagy mediated by mammalian target of rapamycin （mTOR）, with subsequent elimination of α-syn. A Parkinson’s disease model was induced in rats by subcutaneous injection of rotenone at the back of their necks, and they received moxibustion at Zusanli （ST36）, Guanyuan （CV4）and Fengfu （GV16）, for 10 minutes at every point, once per day, for 14 consecutive days. Model rats without any treatment were used as a sham control. Compared with the Parkinson’s disease group, the moxibustion group showed significantly greater tyrosine hydroxylase immunoreactivity and expression of light chain 3-II protein in the substantia nigra, and their behavioral score, α-synuclein immunoreactivity,the expression of phosphorylated mTOR and phosphorylated ribosomal protein S6 kinase （p-p70S6K） in the substantia nigra were significantly lower. These results suggest that moxibustion can promote the autophagic clearance of α-syn and improve behavioral performance in Parkinson’s disease model rats. The protective mechanism may be associated with suppression of the mTOR/p70S6K pathway.

Application of PCR-DGGE in Research of Bacterial Diversity in Drinking Water

Objective To analyze the structure of bacteria in drinking water by molecular biological techniques, Methods DNA of bacteria in drinking water was directly extracted without culture. 16S ribosomal DNA fragments, including V-6, -7, and -8 regions, were amplified with universal primers （EUBf933CJC and EUBr1387） and analyzed by DGGE. Results DGGE indicated that amplification products could be separated, The results showed that DGGE could be used in the separation of different microbial 16SrRNA genes extracted from drinkng water. Though there were special bacteria in different water samples, the predominant bacteria were essentially the same. Three sequences of the reclaimed specific bands were obtained, and phylogenetic tree of these bands was made. Conclusion Bacterial diversity in drinking water is identified by molecular biological techniques.

Fecal microbial dysbiosis in Chinese patients with inflammatory bowel disease

AIM To analyze the alterations of fecal microbiota in Chinese patients with inflammatory bowel disease(IBD).METHODS Fecal samples from 15 patients with Crohn's disease(CD)(11 active CD, 4 inactive CD), 14 patients with active ulcerative colitis(UC) and 13 healthy individuals were collected and subjected to 16 S ribosomal DNA(rDNA) gene sequencing. The V4 hypervariable regions of 16 S rDNA gene were amplified from all samples and sequenced by the Illumina Mi Seq platform. Quality control and operational taxonomic units classification of reads were calculated with QIIME software. Alpha diversity and beta diversity were displayed with R software.RESULTS Community richness(chao) and microbial structure in both CD and UC were significantly different from those in normal controls. At the phyla level, analysis of the microbial compositions revealed a significantly greater abundance of Proteobacteria in IBD as compared to that in controls. At the genera level, 8 genera in CD and 23 genera in UC(in particular, the Escherichia genus) showed significantly greater abundance as compared to that in normal controls. The relative abundance of Bacteroidetes in the active CD group was markedly lower than that in the inactive CD group. The abundance of Proteobacteria in patients with active CD was nominally higher than that in patients with inactive CD; however, the difference was not statistically significant after correction. Furthermore, the relative abundance of Bacteroidetes showed a negative correlation with the CD activity index scores.CONCLUSION Our study profiles specific characteristics and microbial dysbiosis in the gut of Chinese patients with IBD. Bacteroidetes may have a negative impact on inflammatory development.

Exploration of the mechanism by which icariin modulates hippocampal neurogenesis in a rat model of depression

Icariin(ICA) has a significant capacity to protect against depression and hippocampal injury,but it cannot effectively cross the bloodbrain barrier and accumulate in the brain.Therefore,the mechanism by which ICA protects against hippocampal injury in depression remains unclear.In this study,we performed proteomics analysis of cerebrospinal fluid to investigate the mechanism by which ICA prevents dysfunctional hippocampal neurogenesis in depression.A rat model of depression was established through exposure to chronic unpredictable mild stress for 6 weeks,after which 120 mg/kg ICA was administered subcutaneously every day.The results showed that ICA alleviated depressive symptoms,learning and memory dysfunction,dysfunctional neurogenesis,and neuronal loss in the dentate gyrus of rats with depression.Neural stem cells from rat embryonic hippocampi were cultured in media containing 20% cerebrospinal fluid from each group of rats and then treated with 100 μM corticosterone.The addition of cerebrospinal fluid from rats treated with ICA largely prevented the corticosterone-mediated inhibition of neuronal proliferation and differentiation.Fifty-two differentially expressed proteins regulated by chronic unpredictable mild stress and ICA were identified through proteomics analysis of cerebrospinal fluid.These proteins were mainly involved in the ribosome,PI3 K-Akt signaling,and interleukin-17 signaling pathways.Parallel reaction monitoring mass spectrometry showed that Rps4 x,Rps12,Rps14,Rps19,Hsp90 b1,and Hsp90 aa1 were up-regulated by chronic unpredictable mild stress and down-regulated by ICA.In contrast,Htr A1 was down-regulated by chronic unpredictable mild stress and up-regulated by ICA.These findings suggest that ICA can prevent depression and dysfunctional hippocampal neurogenesis through regulating the expression of certain proteins found in the cerebrospinal fluid.The study was approved by the Experimental Animal Ethics Committee of Guangzhou University of Chinese Medicine of China in March 2017.

AMPK-associated signaling to bridge the gap between fuel metabolism and hepatocyte viability

The adenosine monophosphate-activated protein kinase (AMPK) and p70 ribosomal S6 kinase-1 pathway may serve as a key signaling flow that regulates energy metabolism; thus, this pathway becomes an attractive target for the treatment of liver diseases that result from metabolic derangements. In addition, AMPK emerges as a kinase that controls the redox-state and mitochondrial function, whose activity may be modulated by antioxidants. A close link exists between fuel metabolism and mitochondrial biogenesis. The relationship between fuel metabolism and cell survival strongly implies the existence of a shared signaling network, by which hepatocytes respond to challenges of external stimuli. The AMPK pathway may belong to this network. A series of drugs and therapeutic candidates enable hepatocytes to protect mitochondria from radical stress and increase cell viability, which may be associated with the activation of AMPK, liver kinase B1, and other molecules or components. Consequently, the components downstream of AMPK may contribute to stabilizing mitochondrial membrane potential for hepatocyte survival. In this review, we discuss the role of the AMPK pathway in hepatic energy metabolism and hepatocyte viability. This information may help identify ways to prevent and/or treat hepatic diseases caused by the metabolic syndrome. Moreover, clinical drugs and experimental therapeutic candidates that directly or indirectly modulate the AMPK pathway in distinct manners are discussed here with particular emphasis on their effects on fuel metabolism and mitochondrial function.

Effects of electroacupuncture on the expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in the hippocampus of rats with vascular dementia

This study investigated the mechanism underlying electroacupuncture therapy for vascular dementia through electroacupuncture at the acupoints of Baihui （DU20）, Dazhui （DU14）, and bilateral Shenshu （BL23） in a rat model of vascular dementia produced by bilateral middle cerebral artery occlusion. Morris water maze test showed that electroacupuncture improved the learning ability of vascular dementia rats. Western blot assay revealed that the expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in vascular dementia rats was significantly increased after electroacupuncture, compared with the model group that was not treated with acupuncture. The average escape latency was also shortened after electroacupuncture, and escape strategies in the spatial probe test improved from edge and random searches, to linear and trending swim pathways. The experimental findings indicate that electroacupuncture improves learning and memory ability by up-regulating expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in the hippocampus of vascular dementia rats.

Molecular Replacement Studies of Cucurmosin from Cucurbita Moschata:Structure Homology with Trichosanthin

High diffraction quality crystals of cucurmosin, a type I ribosome inactivating protein isolated from the sarcocarp of Cucurbita moschata (pumpkin), have been grown under newly optimised conditions. With in-house rotating anode X-ray source, these crystals diffract to 1.65 ?resolution which is much higher than that of the previously reported crystals that diffracted only to 3 ?resolution. The crystals belong to space group P212121 with cell parameters a = 41.5, b = 58.4 and c = 99.3 . Molecular replacement studies indicate that the cucurmosin structure is homologous to trichosanthin. The initial structural model has been obtained and the model fitting/ refinement is in progress.

Alisol B 23-acetate-induced HepG2 hepatoma cell death through mTOR signaling-initiated G_1 cell cycle arrest and apoptosis: A quantitative proteomic study

Objective: The present study aimed to investigate the molecular events in alisol B 23-acetate(ABA) cytotoxic activity against a liver cancer cell line.Methods: First, we employed a quantitative proteomics approach based on stable isotope labeling by amino acids in cell culture(SILAC) to identify the different proteins expressed in HepG2 liver cancer cells upon exposure to ABA. Next, bioinformatics analyses through DAVID and STRING on-line tools were used to predict the pathways involved. Finally, we applied functional validation including cell cycle analysis and Western blotting for apoptosis and mTOR pathway-related proteins to confirm the bioinformatics predictions.Results: We identified 330 different proteins with the SILAC-based quantitative proteomics approach. The bioinformatics analysis and the functional validation revealed that the mTOR pathway, ribosome biogenesis, cell cycle, and apoptosis pathways were differentially regulated by ABA. G1 cell cycle arrest, apoptosis and mTOR inhibition were confirmed.Conclusions: ABA, a potential mTOR inhibitor, induces the disruption of ribosomal biogenesis. It also affects the mTOR-MRP axis to cause G1 cell cycle arrest and finally leads to cancer cell apoptosis.

Changes of microbial community structures and functional genes during biodegradation of phenolic compounds under high salt condition

The changes of microbial community structures and functional genes during the biodegradation of single phenol and phenol plus p-cresol under high salt condition were explored.It was found that the phenol-fed system（PFS） exhibited stronger degrading abilities and more stable biomass than that of the phenol plus p-cresol-fed system（PCFS）.The microbial community structures were revealed by a modern DNA fingerprint technique,ribosomal intergenic spacer analysis（RISA）.The results indicated that the microbial community of PFS changed obviously when gradually increased phenol concentration,while PCFS showed a little change.16S rRNA sequence analysis of the major bands showed that Alcanivorax sp.genus was predominant species during phenolic compounds degradation.Furthermore,amplified functional DNA restriction analysis（AFDRA） on phenol hydroxylase genes showed that the fingerprints were substantially different in the two systems,and the fingerprints were not the same during the different operational periods.