Function and evolution of Magnaporthe oryzae avirulence gene AvrPib responding to the rice blast resistance gene Pib

功能与稻瘟病菌无毒基因avrpib响应抗稻瘟病Pib基因的进化

Magnaporthe oryzae (Mo) is the causative pathogen of the damaging disease rice blast. The effector gene AvrPib, which confers avirulence to host carrying resistance gene Pib, was isolated via map-based cloning. The gene encodes a 75-residue protein, which includes a signal peptide. Phenotyping and genotyping of 60 isolates from each of five geographically distinct Mo populations revealed that the frequency of virulent isolates, as well as the sequence diversity within the AvrPib gene increased from a low level in the far northeastern region of China to a much higher one in the southern region, indicating a process of host-driven selection. Resequencing of the AvrPiballele harbored by a set of 108 diverse isolates revealed that there were four pathoways, transposable element (TE) insertion (frequency 81.7%), segmental deletion (11.1%), complete absence (6.7%), and point mutation (0.6%), leading to loss of the avirulence function. The lack of any TE insertion in a sample of non-rice infecting Moisolates suggested that it occurred after the host specialization of Mo. Both the deletions and the functional point mutation were confined to the signal peptide. The reconstruction of 16 alleles confirmed seven functional nucleotide polymorphisms for the AvrPiballeles, which generated three distinct expression profiles.

[详细]

  • Scientific Reports 5
  • 5年前
  • Article

Demonstrating quantum contextuality of indistinguishable particles by a single family of noncontextuality inequalities

通过一个家庭的noncontextuality不等式的证明与粒子的量子性

Quantum theory has the intriguing feature that is inconsistent with noncontextual hidden variable models, for which the outcome of a measurement does not depend on which other compatible measurements are being performed concurrently. While various proofs of such contextual behavior of quantum systems have been established, relatively little is known concerning the possibility to demonstrate this intriguing feature for indistinguishable particles. Here, we show in a simple and systematic manner that with projective measurements alone, it is possible to demonstrate quantum contextuality for such systems of arbitrary Hilbert space dimensions, including those corresponding to a qubit. Our demonstration is applicable to a single fermion as well as multiple fermions, and thus also a composite boson formed from an even number of fermions. In addition, our approach gives a clear demonstration of the intimate connection between complementarity and contextuality, two seemingly unrelated aspects of quantum theory.

[详细]

  • Scientific Reports 5
  • 5年前
  • Article

GDC 2: Compression of large collections of genomes

GDC 2的基因组的大量压缩

The fall of prices of the high-throughput genome sequencing changes the landscape of modern genomics. A number of large scale projects aimed at sequencing many human genomes are in progress. Genome sequencing also becomes an important aid in the personalized medicine. One of the significant side effects of this change is a necessity of storage and transfer of huge amounts of genomic data. In this paper we deal with the problem of compression of large collections of complete genomic sequences. We propose an algorithm that is able to compress the collection of 1092 human diploid genomes about 9,500 times. This result is about 4 times better than what is offered by the other existing compressors. Moreover, our algorithm is very fast as it processes the data with speed 200 MB/s on a modern workstation. In a consequence the proposed algorithm allows storing the complete genomic collections at low cost, e.g., the examined collection of 1092 human genomes needs only about 700 MB when compressed, what can be compared to about 6.7 TB of uncompressed FASTA files. The source code is available at http://sun.aei.polsl.pl/REFRESH/index.php?page=projects&project=gdc&subpage=about.

[详细]

  • Scientific Reports 5
  • 5年前
  • Article

Does the passage of seeds through frugivore gut affect their storage: A case study on the endangered plant Euryodendron excelsum

没有种子通过肠道影响其贮存:frugivore对濒危植物猪血木的案例研究

Plant-frugivore mutualism serves an important function in multiple ecological processes. Although previous studies have highlighted the effect of frugivore gut passage on fresh seed germinability, no study has investigated the effect on seed storage after frugivore gut passage. We used the endangered plant, Euryodendron excelsum, to determine the combined effects of frugivore gut passage and storage conditions on the germination percentage and rate of seeds. In particular, three treatments that included storage periods, storage methods, and seed types were designed in the experiment. We hypothesized that seeds that passed through the gut will exhibit enhanced germination capacity and rate during storage. Results showed that the final germination percentage decreased in seeds that passed through the gut, whereas the germination rate increased during seed storage. Germination decreased in most types of seeds under both dry and wet storage after 6 months compared with storage after 1 and 3 months. The results suggest that after frugivore gut passage, E. excelsum seeds cannot form persistent soil seed bank in the field, and were not suitable for species germplasm storage. These finding underscore that seeds that passed through frugivore gut have long-term impact on their viability and germination performance.

[详细]

  • Scientific Reports 5
  • 5年前
  • Article

Competition-cooperation relationship networks characterize the competition and cooperation between proteins

竞争合作关系网络的特点,蛋白质之间的竞争和合作

By analyzing protein-protein interaction (PPI) networks, one can find that a protein may have multiple binding partners. However, it is difficult to determine whether the interactions with these partners occur simultaneously from binary PPIs alone. Here, we construct the yeast and human competition-cooperation relationship networks (CCRNs) based on protein structural interactomes to clearly exhibit the relationship (competition or cooperation) between two partners of the same protein. If two partners compete for the same interaction interface, they would be connected by a competitive edge; otherwise, they would be connected by a cooperative edge. The properties of three kinds of hubs (i.e., competitive, modest, and cooperative hubs) are analyzed in the CCRNs. Our results show that competitive hubs have higher clustering coefficients and form clusters in the human CCRN, but these tendencies are not observed in the yeast CCRN. We find that the human-specific proteins contribute significantly to these differences. Subsequently, we conduct a series of computational experiments to investigate the regulatory mechanisms that avoid competition between proteins. Our comprehensive analyses reveal that for most yeast and human protein competitors, transcriptional regulation plays an important role. Moreover, the human-specific proteins have a particular preference for other regulatory mechanisms, such as alternative splicing.

[详细]

  • Scientific Reports 5
  • 5年前
  • Article

Nitrogen limitation as a driver of genome size evolution in a group of karst plants

氮限制作为一个驱动程序的基因组大小进化的一组喀斯特工厂

Genome size is of fundamental biological importance with significance in predicting structural and functional attributes of organisms. Although abundant evidence has shown that the genome size can be largely explained by differential proliferation and removal of non-coding DNA of the genome, the evolutionary and ecological basis of genome size variation remains poorly understood. Nitrogen (N) and phosphorus (P) are essential elements of DNA and protein building blocks, yet often subject to environmental limitation in natural ecosystems. Using phylogenetic comparative methods, we test this hypothesis by determining whether leaf N and P availability affects genome sizes in 99 species of Primulina (Gesneriaceae), a group of soil specialists adapted to limestone karst environment in south China. We find that genome sizes in Primulina are strongly positively correlated with plant N content, but the correlation with plant P content is not significant when phylogeny history was taken into account. This study shows for the first time that N limitation might have been a plausible driver of genome size variation in a group of plants. We propose that competition for nitrogen nutrient between DNA synthesis and cellular functions is a possible mechanism for genome size evolution in Primulina under N-limitation.

[详细]

  • Scientific Reports 5
  • 5年前
  • Article

Molecular and neuronal homology between the olfactory systems of zebrafish and mouse

斑马鱼和小鼠嗅觉系统的分子和神经元的同源性

Studies of the two major olfactory organs of rodents, the olfactory mucosa (OM) and the vomeronasal organ (VNO), unraveled the molecular basis of smell in vertebrates. However, some vertebrates lack a VNO. Here we generated and analyzed the olfactory transcriptome of the zebrafish and compared it to the olfactory transcriptomes of mouse to investigate the evolutionary and molecular relationship between single and dual olfactory systems. Our analyses revealed a high degree of molecular conservation, with orthologs of mouse olfactory cell-specific markers and all but one of their chemosensory receptor classes expressed in the single zebrafish olfactory organ. Zebrafish chemosensory receptor genes are expressed across a large dynamic range and their RNA abundance correlates positively with the number of neurons expressing that RNA. Thus we estimate the relative proportions of neuronal sub-types expressing different chemosensory receptors. Receptor repertoire size drives the absolute abundance of different classes of neurons, but we find similar underlying patterns in both species. Finally, we identified novel marker genes that characterize rare neuronal populations in both mouse and zebrafish. In sum, we find that the molecular and cellular mechanisms underpinning olfaction in teleosts and mammals are similar despite 430 million years of evolutionary divergence.

[详细]

  • Scientific Reports 5
  • 5年前
  • Article

Considerably Unfolded Transthyretin Monomers Preceed and Exchange with Dynamically Structured Amyloid Protofibrils

转甲状腺素蛋白单体数据大大展开与动态结构淀粉样原纤维的交换

Despite numerous studies, a detailed description of the transthyretin (TTR) self-assembly mechanism and fibril structure in TTR amyloidoses remains unresolved. Here, using a combination of primarily small -angle X-ray scattering (SAXS) and hydrogen exchange mass spectrometry (HXMS) analysis, we describe an unexpectedly dynamic TTR protofibril structure which exchanges protomers with highly unfolded monomers in solution. The protofibrils only grow to an approximate final size of 2,900 kDa and a length of 70 nm and a comparative HXMS analysis of native and aggregated samples revealed a much higher average solvent exposure of TTR upon fibrillation. With SAXS, we reveal the continuous presence of a considerably unfolded TTR monomer throughout the fibrillation process, and show that a considerable fraction of the fibrillating protein remains in solution even at a late maturation state. Together, these data reveal that the fibrillar state interchanges with the solution state. Accordingly, we suggest that TTR fibrillation proceeds via addition of considerably unfolded monomers, and the continuous presence of amyloidogenic structures near the protofibril surface offers a plausible explanation for secondary nucleation. We argue that the presence of such dynamic structural equilibria must impact future therapeutic development strategies.

[详细]

  • Scientific Reports 5
  • 5年前
  • Article

The first crop plant genetically engineered to release an insect pheromone for defence

第一种作物基因工程,以释放昆虫信息素为防御

Insect pheromones offer potential for managing pests of crop plants. Volatility and instability are problems for deployment in agriculture but could be solved by expressing genes for the biosynthesis of pheromones in the crop plants. This has now been achieved by genetically engineering a hexaploid variety of wheat to release (E)-β-farnesene (Eβf), the alarm pheromone for many pest aphids, using a synthetic gene based on a sequence from peppermint with a plastid targeting amino acid sequence, with or without a gene for biosynthesis of the precursor farnesyl diphosphate. Pure Eβf was produced in stably transformed wheat lines with no other detectable phenotype but requiring targeting of the gene produced to the plastid. In laboratory behavioural assays, three species of cereal aphids were repelled and foraging was increased for a parasitic natural enemy. Although these studies show considerable potential for aphid control, field trials employing the single and double constructs showed no reduction in aphids or increase in parasitism. Insect numbers were low and climatic conditions erratic suggesting the need for further trials or a closer imitation, in the plant, of alarm pheromone release.

[详细]

  • Scientific Reports 5
  • 5年前
  • Article

Nanopore sensing of individual transcription factors bound to DNA

纳米孔单转录因子结合DNA检测

Transcription factor (TF)-DNA interactions are the primary control point in regulation of gene expression. Characterization of these interactions is essential for understanding genetic regulation of biological systems and developing novel therapies to treat cellular malfunctions. Solid-state nanopores are a highly versatile class of single-molecule sensors that can provide rich information about local properties of long charged biopolymers using the current blockage patterns generated during analyte translocation, and provide a novel platform for characterization of TF-DNA interactions. The DNA-binding domain of the TF Early Growth Response Protein 1 (EGR1), a prototypical zinc finger protein known as zif268, is used as a model system for this study. zif268 adopts two distinct bound conformations corresponding to specific and nonspecific binding, according to the local DNA sequence. Here we implement a solid-state nanopore platform for direct, label- and tether-free single-molecule detection of zif268 bound to DNA. We demonstrate detection of single zif268 TFs bound to DNA according to current blockage sublevels and duration of translocation through the nanopore. We further show that the nanopore can detect and discriminate both specific and nonspecific binding conformations of zif268 on DNA via the distinct current blockage patterns corresponding to each of these two known binding modes.

[详细]

  • Scientific Reports 5
  • 5年前
  • Article

Differential gene retention as an evolutionary mechanism to generate biodiversity and adaptation in yeasts

差分基因保留在酵母中的生物多样性和适应的进化机制

The evolutionary history of the characters underlying the adaptation of microorganisms to food and biotechnological uses is poorly understood. We undertook comparative genomics to investigate evolutionary relationships of the dairy yeast Geotrichum candidum within Saccharomycotina. Surprisingly, a remarkable proportion of genes showed discordant phylogenies, clustering with the filamentous fungus subphylum (Pezizomycotina), rather than the yeast subphylum (Saccharomycotina), of the Ascomycota. These genes appear not to be the result of Horizontal Gene Transfer (HGT), but to have been specifically retained by G. candidum after the filamentous fungi–yeasts split concomitant with the yeasts’ genome contraction. We refer to these genes as SRAGs (Specifically Retained Ancestral Genes), having been lost by all or nearly all other yeasts, and thus contributing to the phenotypic specificity of lineages. SRAG functions include lipases consistent with a role in cheese making and novel endoglucanases associated with degradation of plant material. Similar gene retention was observed in three other distantly related yeasts representative of this ecologically diverse subphylum. The phenomenon thus appears to be widespread in the Saccharomycotina and argues that, alongside neo-functionalization following gene duplication and HGT, specific gene retention must be recognized as an important mechanism for generation of biodiversity and adaptation in yeasts.

[详细]

  • Scientific Reports 5
  • 5年前
  • Article

Analysis of resistance and tolerance to virus infection in Drosophila

果蝇对病毒感染的耐受性及耐受性分析

Drosophila is a useful model for identifying and characterizing the genetic components involved in host resistance and tolerance to virus infection. The van Rij laboratory explains how to conduct such experiments, controlling for common confounding factors.

[详细]

  • Nature Protocols 10, 1084 (2015)
  • 5年前
  • Protocol

Acute TNF-induced repression of cell identity genes is mediated by NF{kappa}B-directed redistribution of cofactors from super-enhancers [RESEARCH]

急性TNF诱导的细胞特征的基因抑制NFκ} {介导的b-directed再分配的辅因子超促进剂[研究]

The proinflammatory cytokine tumor necrosis factor (TNF) plays a central role in low-grade adipose tissue inflammation and development of insulin resistance during obesity. In this context, nuclear factor kappa-light-chain-enhancer of activated B cells (NFB), is directly involved and required for the acute activation of the inflammatory gene program. Here we show that the major transactivating subunit of NFB, v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), is also required for acute TNF-induced suppression of adipocyte genes. Notably, this repression does not involve RELA binding to the associated enhancers but rather loss of cofactors and enhancer RNA (eRNA) selectively from high occupancy sites within super-enhancers. Based on these data we have developed models that with high accuracy predict which enhancers and genes are repressed by TNF in adipocytes. We show that these models are applicable to other cell types where TNF represses genes associated with super-enhancers in a highly cell type-specific manner. Our results propose a novel paradigm for NFB-mediated repression, whereby NFB selectively redistributes cofactors from high occupancy enhancers, thereby specifically repressing super-enhancer-associated cell identity genes.

[详细]

  • Genome Research
  • 5年前
  • RESEARCH

Real-time multi-view deconvolution

实时多视图反褶积

Summary: In light-sheet microscopy, overall image content and resolution are improved by acquiring and fusing multiple views of the sample from different directions. State-of-the-art multi-view (MV) deconvolution simultaneously fuses and deconvolves the images in 3D, but processing takes a multiple of the acquisition time and constitutes the bottleneck in the imaging pipeline. Here we show that MV deconvolution in 3D can finally be achieved in real-time by processing cross-sectional planes individually on the massively parallel architecture of a graphics processing unit (GPU). Our approximation is valid in the typical case where the rotation axis lies in the imaging plane.

Availability: Source code and binaries are available on github (https://github.com/bene51/), native code under the repository ’gpu_deconvolution’, Java wrappers implementing Fiji plugins under ’SPIM_Reconstruction_Cuda’

Contact: bschmid@mpi-cbg.de

Supplementary Information: Supplementary data are available at Bioinformatics online.

[详细]

  • Bioinformatics
  • 5年前
  • APPLICATIONS NOTE

damidseq pipeline: an automated pipeline for processing DamID sequencing datasets

damidseq管道:一种用于处理DamID测序数据自动化的流水线

Summary: DamID is a powerful technique for identifying regions of the genome bound by a DNA-binding (or DNA-associated) protein. Currently no method exists for automatically processing next-generation sequencing DamID (DamID-seq) data, and the use of DamID-seq datasets with normalisation based on read-counts alone can lead to high background and the loss of bound signal. DamID-seq thus presents novel challenges in terms of normalisation and background minimisation. We describe here damidseq_pipeline, a software pipeline that performs automatic normalisation and background reduction on multiple DamID-seq FASTQ datasets.

Availability and implementation: Open-source and freely available from http://owenjm.github.io/damidseq_pipeline. The damidseq_pipeline is implemented in Perl and is compatible with any Unix-based operating system (e.g. Linux, Mac OSX).

Contact: o.marshall@gurdon.cam.ac.uk

[详细]

  • Bioinformatics
  • 5年前
  • APPLICATIONS NOTE

biRte: Bayesian Inference of Context Specific Regulator Activities and Transcriptional Networks

Birte:贝叶斯推断上下文特定的调节活性和转录网络

In the last years there has been an increasing effort to computationally model and predict the influence of regulators (transcription factors, miRNAs) on gene expression. Here we introduce biRte as a computationally attractive approach combining Bayesian inference of regulator activities with network reverse engineering. biRte integrates target gene predictions with different omics data entities (e.g. miRNA and mRNA data) into a joint probabilistic framework. The utility of our method is tested in extensive simulation studies and demonstrated with applications from prostate cancer and E. coli growth control. The resulting regulatory networks generally show a good agreement with the biological literature.

Further information: See supplemental material.

Availability: biRte is available on Bioconductor (http://bioconductor.org)

Contact: frohlich@bit.uni-bonn.de

[详细]

  • Bioinformatics
  • 5年前
  • ORIGINAL PAPER

Predicting tumor purity from methylation microarray data

预测肿瘤纯度的甲基化基因芯片数据

Motivation: In cancer genomics research, one important problem is that the solid tissue sample obtained from clinical settings is always a mixture of cancer and normal cells. The sample mixture brings complication in data analysis and results in biased findings if not correctly accounted for. Estimating tumor purity is of great interest, and a number of methods have been developed using gene expression, copy number variation or point mutation data.

Results: We discover that in cancer samples, the distributions of data from Illumina Infinium 450k methylation microarray are highly correlated with tumor purities. We develop a simple but effective method to estimate purities from the microarray data. Analyses of the TCGA lung cancer data demonstrate favorable performance of the proposed method.

Availability: The method is implemented in InfiniumPurify, which is freely available at https://bitbucket.org/zhengxiaoqi/infiniumpurify.

Contact: xqzheng@shnu.edu.cn; hao.wu@emory.edu

[详细]

  • Bioinformatics
  • 5年前
  • 基因组 DISCOVERY NOTE

Data Safe Havens in health research and healthcare

健康研究和医疗保健的数据安全港

Motivation: The data that put the ‘evidence’ into ‘evidence-based medicine’ are central to developments in public health, primary and hospital care. A fundamental challenge is to site such data in repositories that can easily be accessed under appropriate technical and governance controls which are effectively audited and are viewed as trustworthy by diverse stakeholders. This demands socio-technical solutions that may easily become enmeshed in protracted debate and controversy as they encounter the norms, values, expectations and concerns of diverse stakeholders. In this context, the development of what are called ‘Data Safe Havens’ has been crucial. Unfortunately, the origins and evolution of the term have led to a range of different definitions being assumed by different groups. There is, however, an intuitively meaningful interpretation that is often assumed by those who have not previously encountered the term: a repository in which useful but potentially sensitive data may be kept securely under governance and informatics systems that are fit-for-purpose and appropriately tailored to the nature of the data being maintained, and may be accessed and utilized by legitimate users undertaking work and research contributing to biomedicine, health and/or to ongoing development of healthcare systems.

Results: This review explores a fundamental question: ‘what are the specific criteria that ought reasonably to be met by a data repository if it is to be seen as consistent with this interpretation and viewed as worthy of being accorded the status of ‘Data Safe Haven’ by key stakeholders’? We propose 12 such criteria.

Contact: paul.burton@bristol.ac.uk

[详细]

  • Bioinformatics
  • 5年前
  • REVIEW

How Many Genes Are Expressed in a Transcriptome? Estimation and Results for RNA-Seq

有多少基因在转录组表达?对于RNA序列估计和结果

by Luis Fernando García-Ortega, Octavio Martínez

RNA-seq experiments estimate the number of genes expressed in a transcriptome as well as their relative frequencies. However, an undetermined number of genes can remain undetected due to their low expression relative to the sample size (sequence depth). Estimation of the true number of genes expressed in a transcriptome is essential in order to determine which genes are exclusively expressed in specific tissues or under particular conditions. A reliable estimate of the true number of expressed genes is also required to accurately measure transcriptome changes and to predict the sequencing depth needed to increase the proportion of detected genes. This problem is analogous to ecological sampling problems such as estimating the number of species at a given site. Here we present a non-parametric estimator for the number of undetected genes as well as for the extra sample size needed to detect a given proportion of the undetected genes. Our estimators are superior to ones already published by having smaller standard errors and biases. We applied our method to a set of 32 publicly available RNA-seq experiments, including the evaluation of 311 individually sequenced libraries. We found that in the majority of the cases more than one thousand genes are undetected, and that on average approximately 6% of the expressed genes per accession remain undetected. This figure increases to approximately 10% if individual sequencing libraries are analyzed. Our method is also applicable to metagenomic experiments. Using our method, the number of undetected genes as well as the sample size needed to detect them can be calculated, leading to more accurate and complete gene expression studies.

[详细]

  • PloS one
  • 5年前

Emerging Object Representations in the Visual System Predict Reaction Times for Categorization

新兴的可视化系统中的对象表示预测反应时间分类

by J. Brendan Ritchie, David A. Tovar, Thomas A. Carlson

Recognizing an object takes just a fraction of a second, less than the blink of an eye. Applying multivariate pattern analysis, or “brain decoding”, methods to magnetoencephalography (MEG) data has allowed researchers to characterize, in high temporal resolution, the emerging representation of object categories that underlie our capacity for rapid recognition. Shortly after stimulus onset, object exemplars cluster by category in a high-dimensional activation space in the brain. In this emerging activation space, the decodability of exemplar category varies over time, reflecting the brain’s transformation of visual inputs into coherent category representations. How do these emerging representations relate to categorization behavior? Recently it has been proposed that the distance of an exemplar representation from a categorical boundary in an activation space is critical for perceptual decision-making, and that reaction times should therefore correlate with distance from the boundary. The predictions of this distance hypothesis have been born out in human inferior temporal cortex (IT), an area of the brain crucial for the representation of object categories. When viewed in the context of a time varying neural signal, the optimal time to “read out” category information is when category representations in the brain are most decodable. Here, we show that the distance from a decision boundary through activation space, as measured using MEG decoding methods, correlates with reaction times for visual categorization during the period of peak decodability. Our results suggest that the brain begins to read out information about exemplar category at the optimal time for use in choice behaviour, and support the hypothesis that the structure of the representation for objects in the visual system is partially constitutive of the decision process in recognition.

[详细]

  • PLOS Computational Biology
  • 5年前

BASiCS: Bayesian Analysis of Single-Cell Sequencing Data

基础知识:单细胞测序数据的贝叶斯分析

by Catalina A. Vallejos, John C. Marioni, Sylvia Richardson

Single-cell mRNA sequencing can uncover novel cell-to-cell heterogeneity in gene expression levels in seemingly homogeneous populations of cells. However, these experiments are prone to high levels of unexplained technical noise, creating new challenges for identifying genes that show genuine heterogeneous expression within the population of cells under study. BASiCS (Bayesian Analysis of Single-Cell Sequencing data) is an integrated Bayesian hierarchical model where: (i) cell-specific normalisation constants are estimated as part of the model parameters, (ii) technical variability is quantified based on spike-in genes that are artificially introduced to each analysed cell’s lysate and (iii) the total variability of the expression counts is decomposed into technical and biological components. BASiCS also provides an intuitive detection criterion for highly (or lowly) variable genes within the population of cells under study. This is formalised by means of tail posterior probabilities associated to high (or low) biological cell-to-cell variance contributions, quantities that can be easily interpreted by users. We demonstrate our method using gene expression measurements from mouse Embryonic Stem Cells. Cross-validation and meaningful enrichment of gene ontology categories within genes classified as highly (or lowly) variable supports the efficacy of our approach.

[详细]

  • PLOS Computational Biology
  • 5年前