AbstractBackgroundTumor mutational burden (TMB) is a promising biomarker for stratifying patient subpopulation who would benefit from immune checkpoint blockade (ICB) therapies. Although great efforts have been made for standardizing TMB measurement, mutation calling and TMB quantification can be challenging in samples with low tumor content including liquid biopsies. The effect of varying tumor content on TMB estimation by different assay methods has never been systematically investigated.MethodWe established a series of reference standard DNA samples derived from 11 pairs of tumor–normal matched human cell lines across different cancer types. Each tumor cell line was mixed with its matched normal at 0% (control), 1%, 2%, 5%, and 10% mass‐to‐mass ratio to mimic the clinical samples with low tumor content. TMB of these reference standards was evaluated by both ∼1000× whole‐exome sequencing (wesTMB) and targeted panel sequencing (psTMB) at four different vendors. Both regression and classification analyses of TMB were performed for theoretical investigation and clinical practice purposes.ResultsLinear regression model was established that demonstrated in silico psTMB determined by regions of interest (ROI) as a great representative of wesTMB based on TCGA dataset. It was also true in our reference standard samples as the predicted psTMB interval based on the observed wesTMB captured the intended 90% of the in silico psTMB values. Although ∼1000× deep WES was applied, reference standard samples with less than 5% of tumor proportions are below the assay limit of detection (LoD) of wesTMB quantification. However, predicted wesTMB based on observed psTMB accurately classify (>0.97 AUC) for TMB high and low patient stratification even in samples with 2% of tumor content, which is more clinically relevant, as TMB determination should be a qualitative assay for TMB high and low patient classification. One targeted panel sequencing vendor using an optimized blood psTMB pipeline can further classify TMB status accurately (>0.82 AUC) in samples with only 1% of tumor content.ConclusionsWe developed a linear model to establish the quantitative correlation between wesTMB and psTMB. A set of DNA reference standards was produced in aid to standardize TMB measurements in samples with low tumor content across different targeted sequencing panels. This study is a significant contribution aiming to harmonize TMB estimation and extend its future application in clinical samples with low tumor content including liquid biopsy.

Flavone synthase is a key enzyme for flavone biosynthesis and is encoded by two gene families: flavone synthase I (FNSI) and flavone synthase II (FNSII). FNSII is widely distributed in plants, while FNSI has been reported in rice (Oryza sativa) and seven species of Apiaceae. FNSI has likely evolved from the duplication of flavanone 3β-hydroxylase (F3H). In this study, we used multiple bioinformatics tools to identify putative FNSI and F3H genes from 42 publicly available genome and transcriptome datasets. Results showed that rice FNSI does not share a common ancestral sequence with other known FNSI genes and that FNSI is absent from species outside of Apiaceae. Positive selection site identification analysis revealed that four sites within the FNSI tree branches of Apiaceae evolved under significant positive selection. The putative F3H genes identified in this study provide a valuable resource for further function analysis of flavone synthase.

法尼基焦磷酸合成酶(Farnesyl Pyrophosphate Synthase,FPPS,EC:2.5.1.1)是倍半萜合成中的一个关键酶,在植物萜类合成过程中有关键作用.以杧果FPPS基因为研究对象,以拟南芥FPPS基因为参考,通过BLAST方法从番木瓜、金钱橘、甜橙、苹果、小果野蕉、桃子、白梨、葡萄、中华猕猴桃、无油樟基因组数据中获得了FPPS基因,共19条FPPS基因序列,对其进行了系统进化分析和生物信息学分析.系统进化结果显示:双子叶植物纲果树与单子叶植物纲果树关系较远,同是双子叶植物纲的果树关系较近,同科果树亲缘关系更近.生物信息学分析结果显示:氨基酸序列中,酸性蛋白占94.7％,稳定性蛋白占84.2％,有信号肽的蛋白占5.3％,有导肽的蛋白占10.5％,所有蛋白均为有明显跨膜现象的亲水性蛋白;所有FPPS基因亚细胞定位于线粒体中,均有蛋白活性位点.

Genes of the LPPT (lipid phosphatase/phosphotransferase) family play important roles in lipid phosphorous transfer and triacylglycerol accumulation in plants. To provide overviews of the plant LPPT family and their overall relationships, here we carried out genome-wide identifications and analyses of plant LPPT family members. A total of 643 putative LPPT genes were identified from 48 sequenced plant genomes, among which 205 genes from 14 plants were chosen for further analyses. Plant LPPT genes belonged to three distinctive groups, namely the LPT (lipid phosphotransfease), LPP (lipid phosphatase), and pLPP (plastidic lipid phosphotransfease) groups. Genes of the LPT group could be further partitioned into three groups, two of which were only identified in terrestrial plants. Genes in the LPP and pLPP groups experienced duplications in early stages of plant evolution. Among 17 Zea mays LPPT genes, divergence of temporal-spatial expression patterns was revealed based on microarray data analysis. Peptide sequences of plant LPPT genes harbored different conserved motifs. A test of Branch Model versus One-ratio Model did not support significant selective pressures acting on different groups of LPPT genes, although quite different nonsynonymous evolutionary rates and selective pressures were observed. The complete picture of the plant LPPT family provided here should facilitate further investigations of plant LPPT genes and offer a better understanding of lipid biosynthesis in plants.

Genes containing an MBOAT (membrane-bound O-acyltransferase) domain form a large gene family in plants whose members play important roles in plant triacylglycerol biosynthesis. Among these genes, most belong to the MBOAT1 subfamily. Here we describe the identification and analysis of MBOAT genes in Solanaceae. Through data mining of four sequenced genomes of Solanaceae, we identified 52 MBOAT members. The MBOAT genes fell into four distinct groups, with MBOAT1 subfamily genes accounting for about half of the total number of genes. Several MBOAT1 genes were present in the genomes of hot pepper, tomato and potato, whereas only one was identified in Nicotiana benthamiana. Most of tomato MBOAT1 genes were localized in chromosomes in the form of clusters, which is the same in potato, indicating that the population of MBOAT1 members was mainly the result of tandem duplication. Some tomato MBOAT1 genes were not expressed, and all MBOAT1 genes were devoid of introns and were significantly shorter than other MBOAT members were. While average pairwise Ka/Ks values were significantly lower within the MBOAT1 subfamily, some MBOAT1 genes showed signs of positive selection.

Praxelis clematidea (Asteraceae), a plant species native to South America, is a noxious weed in southern China. We examined the genetic variation and population structure of 12 populations (76 individuals) of P. clematidea from Fujian, Guangdong, and Hainan Provinces in China using inter-simple sequence repeat (ISSR) analysis. From an initial set of 69 ISSR primers, 10 were selected which yielded 80 reproducible bands. Polymorphic bands (P) were 100%, Shannon's information index (I) was 0.4226, and Nei's gene diversity (H) was 0.2791. We infer that the high levels of genetic diversity exhibited by P. clematidea may have contributed to its invasiveness. Gene flow among populations was 2.4930, which has led to homogenization. The coefficient of population differentiation (Gst = 0.1671) indicated low levels of genetic variation among populations and high levels of genetic polymorphism within populations. There was a negative correlation between population elevation and genetic diversity, while there was a significant positive correlation between genetic distance and geographic distance based on a Mantel test (r = 0.5820, P < 0.01). Some populations from different provinces clustered together in principal coordinate and UPGMA analyses indicating that human-mediated events may have contributed to the dispersal of the species. (C) 2015 Elsevier Ltd. All rights reserved.

苯丙氨酸解氨酶(PAL,phenylalanine ammonia-lyase [EC:4.3.1.24])是植物次生代谢尤其是苯丙烷途径的关键酶,与植物抵抗病原菌入侵密切相关,具有重要的植物生理学意义;其催化产物是辣椒素等植物天然产物的前体.采用BLASTP方法,依托全基因组数据库,获得了番茄、马铃薯、本氏烟草、辣椒等4种茄科植物及杨树、拟南芥的PAL基因家族成员共27条序列,并对其进行初步的生物信息学分析、理化性质分析及结构分析.结果表明:在进化过程中,茄科植物烟草、番茄、马铃薯和辣椒的亲缘关系较近,拟南芥、杨树与茄科植物的亲缘关系较远;酸性蛋白质占96.3％,所有蛋白均为亲水性稳定蛋白、有明显跨膜现象、无信号肽;所有PAL亚细胞定位于细胞质中,具有活性位点的蛋白占96.3％.本研究结果为进一步研究茄科植物中PAL代谢机理提供理论支持.

二酰甘油酰基转移酶2(Diacylglycerol O-acyltransferase 2,DGAT2)是植物中三羧酸甘油酯(TAG)合成途径的限速酶,其编码基因属于酰基转移酶基因超家族.本研究依托植物全基因组数据库Phytozome,通过BLAST搜索获得了蓖麻(Ricinus communis L.)、拟南芥(Arabidopsis thaliana Heynh.)、毛果杨(Populus trichocarpa Torr.＆A.Gray.)和木薯(Manihot esculenta Crantz.)4种双子叶植物酰基转移酶基因超家族所编码的73条多肽序列,并从中鉴定出5条DGAT2序列.理化性质和跨膜结构域分析表明,5条DGAT2序列均为疏水性跨膜蛋白,其中木薯DGAT2为一次跨膜蛋白且在叶绿体膜中大量分布,这与其他植物的DGAT2序列存在差异;木薯DGAT2蛋白在进化过程中发生了功能分化且可能与木薯的抗逆作用有关.

In this study, we download ESTs form dbEST database and first designed 27 primer pairs for Praxelis clematidea after EST pretreatment and SSR locus identification. The sample were collected from Wenchang City in Hainan province as DNA template, and the optimization and establishment of EST-derived SSR-PCR reaction system for Praxelis clematidea was carried out by single factor test and orthogonal experiment design of L16(45). The result was indicated that the optimal EST-SSRs PCR reaction system (20μL) was contained 2.75 mmol/L Mg2+, 35 ng DNA template, 2.25 U Tag DNA polymerase, 0.3 mmol/L dNTPs and 0.6 μmol/L primer. This system was tested by 14 Praxelis clematidea from different places, which stable and clear polymorphic bands were acquired. Accordingly, the development of EST-SSRs can be used for the genetic diversity research of Praxelis clematidea.

Polysaccharides fromSedum aizoonL. were extracted by hot water, ultrasonic wave and enzyme, respectively. Then crude polysaccharides were de-proteined by sevag method, and the process was optimized taking advantages of single-factor and L9(3)4-orthogonal array method. The results revealed hot water method was the most efficient extraction method among the three methods, which the yield of polysaccharides was (2.48±0.7000)%. The optimized de-proteinization process was that the sample was added one of two volumes Sevag reagent (CHCl3-C4H10O (V/V), 3:1), shook for 25 min once, then centrifuged to remove denatured protein and organic reagent. This process was repeated for three times. The protein removal rate and polysaccharide loss rate were (61.28±0.25)% and (29.16±0.19)%, separately. And the process was stable according to verification test, which was significantly higher than reported values in the literature.

对来自海南、广东和福建的12个假臭草居群54个样本的内转录间隔区Internal transcribed spacer (ITS)序列变异进行检测.结果共发现12个单倍型,且所有居群均共享一个单倍型.分子差异性分析(AMOVA)显示,居群内的遗传变异小(-4.57％),遗传分化低(GST=-0.061,FST=-0.04569,NST=-0.031),无明显的谱系结构(NST＞GST,p＞0.01),且居群间平均基因流(Nm=1.18＞1)高,说明假臭草居群内基因交流频繁.ITS序列的失配曲线表明假臭草近期发生过多次入侵扩张,对假臭草的防治应遵循检疫与治理并重的原则,降低其对入侵地的生态环境危害.