Biology subjects

Shi, S.

Publications and source records attributed to Shi, S..

At least 91 records · Page 5Linked to original sources

bioRxiv2021

Crosstalk between myosin II and formin functions in the regulation of force generation and actomyosin dynamics in stress fibers

REF52 fibroblasts have a well-developed contractile machinery, the most prominent elements of which are actomyosin stress fibers with highly ordered organization of actin and myosin IIA filaments. The relationship between contractile activity and turnover dynamics of stress fibers is not sufficiently understood. Here, we simultaneously measured the forces exerted by stress fibers (using traction force microscopy or micropillar array sensors) and the dynamics of actin and myosin (using photoconversion-based monitoring of actin incorporation and high-resolution fluorescence microscopy of myosin II light chain). Our data revealed new features of the crosstalk between myosin II-driven contractility and stress fiber dynamics. During normal stress fiber turnover, actin incorporated all along the stress fibers and not only at focal adhesions. Incorporation of actin into stress fibers/focal adhesions, as well as actin and myosin II filaments flow along stress fibers, strongly depends on myosin II activity. Myosin II-dependent generation of traction forces does not depend on incorporation of actin into stress fibers per se, but still requires formin activity. This previously overlooked function of formins in maintenance of the actin cytoskeleton connectivity could be the main mechanism of formin involvement in traction force generation. Highlights* Cell traction forces are measured together with visualization of actomyosin flow * Actin turnover depends on formin and myosin II activities * Traction forces depend not only on myosin II, but also on formins * Traction force generation may require formin-dependent cytoskeleton connectivity

cell biology↗

bioRxiv2021

Rapid and Visible RPA-Cas12a fluorescence Assay for Accurate Detection of Zoonotic Dermatophytes

BackgroundDermatophytosis is an infectious disease of global significance caused by several fungal species, which affects the hair, nails, or superficial layers of the skin. The most common zoonotic dermatophytes are Microsporum canis, Nannizzia gypsea and Trichophyton mentagrophytes. Woods lamp examination, microscopic identification and fungal culture are the main conventional diagnostic methods used in clinics. Less common methods are dermatophyte PCR and biopsy/histopathology. However, these methods also have limitations for providing both accuracy and timely on-site detection. The recent development of CRISPR-based diagnostic platform provides the possibility of a rapid, accurate, and portable diagnostic tool, which has huge potential for clinical applications. ObjectivesThe purpose of this study is to establish a molecular method for rapid and accurate diagnosis of clinical dermatophytes, which can accelerate clinical diagnostic testing and help timely treatment. MethodsIn this paper, we design a Cas12a-based assay combined with recombinase polymerase amplification (RPA) to differentiate three main zoonotic dermatophytes. The limit of detection (LOD) is determined by using standard strains. A total of 25 clinical samples (hair and scurf) are identified to evaluate the sensitivity and specificity of this assay. ResultsThe RPA-Cas12a method showed high sensitivity and specificity (100% and 100%, respectively). The results could be observed directly by naked-eyes, and all tested samples were consistent with fungal culture and sequencing results. ConclusionsCompared with other methods, the RPA-Cas12a-fluorescence assay requires less time (30 minutes) and less complicated equipment, and visible changes can be clearly observed, which is suitable for on-site clinical diagnosis.

microbiology↗

bioRxiv2021

MLC Seq: De novo sequencing of full-length tRNA isoforms by mass ladder complementation

tRNAs can exist in distinct isoforms because of different chemical modifications, which confounds attempts to accurately sequence individual tRNA species using next generation sequencing approaches or to quantify different RNA modifications at specific sites on a tRNA strand. Herein, we develop a mass spectrometric (MS) ladder complementation sequencing (MLC-Seq), allowing for direct and simultaneous sequencing of full-length tRNA molecules, including those with low abundance. MLC-seq is achieved by improved instrumentation, and advanced algorithms that identify each tRNA species and related isoforms in an RNA mixture, and assemble full MS ladders from partial ladders with missing ladder components. Using MLC-Seq, we successfully obtained the sequence of tRNA-Phe from yeast and tRNA-Glu from mouse hepatocytes, and simultaneously revealed new tRNA isoforms derived from nucleotide modifications. Importantly, MLC-Seq pinpointed the location and stoichiometry changes of RNA modifications in tRNA-Glu upon the treatment of dealkylated enzyme AlkB, which confirmed its known enzymatic activity and suggested previously unidentified effects in RNA editing.

biochemistry↗

bioRxiv2021

Actin polymerization and crosslinking drive left-right asymmetry in single cell and cell collectives

Deviations from mirror symmetry in the development of bilateral organisms are highly stereotypic and genetically predetermined, but their mechanisms are not sufficiently understood. At the cellular level, self-organization of the actin cytoskeleton results in chiral actin swirling, and cells in groups confined to micropatterns demonstrate chiral cell alignment. The relationship between individual and collective cell chirality is unclear, and molecular players involved remain essentially unidentified. Here, by screening major actin-associated proteins and deep-learning-based morphometric analysis of actin patterns, we found that knockdowns of specific actin polymerization regulators, such as mDia1, ARPC2, and cofilins 1&2, abolished chiral actin swirling, while depletion of profilin 1 and CapZ{beta}, reversed its direction in an actin crosslinker -actinin1-dependent manner. Analysis of these and other knockdowns and pharmacological treatments revealed a robust correlation between their effects on the chirality of individual cells and confined cell groups. Thus, actin-driven cell chirality may underlie tissue and organ asymmetry. One Sentence SummaryCell chirality determined by specific regulators of actin polymerization drives left-right asymmetry emergence in cell groups

cell biology↗

bioRxiv2021

The intestinal microbiota-driven dopamine level influences social interaction and innate color preference in zebrafish larvae

Gut microbiota influence neurodevelopment of brain and programing of behaviors. However, the mechanism underlining the relationship between shoals behaviors and intestinal microbiota remain controversial and the roles of neurotransmitters are still unclear. Here we show that, shoaling behavior affected the innate color preference of shoals, indicating that shoals tended to choose a favorable color environment that benefits social contact. Meanwhile, administration of D1-R antagonist disrupted the social interaction which led to the deficits of color preference. More importantly, the altered microbiota caused by an antibiotic OTC decreased the sociability and weakened shoals color preference. When given a supplement of LGG after OTC exposure, fish exhibited an unexpectedly recovery capability in social cohesion and color preference. Our findings show that dopamine level of brain could mediate both social recognition and color preference, and highlight the pathway of microbial metabolites through the microbiota-gut-brain axis that coordinate the production of dopamine.

animal behavior and cognition↗

bioRxiv2021

Apoptotic Extracellular Vesicles (ApoEVs) Safeguard Liver Homeostasis and Regeneration via Assembling an ApoEV-Golgi Organelle

Apoptosis is an integral physiological cell death process that occurs frequently and generates a huge number of apoptotic extracellular vesicles (apoEVs). However, whether apoEVs are necessary for maintaining organ homeostasis remains unclear. Here, we show that circulatory apoEVs engraft in liver and undergo specialized internalization by hepatocytes (HCs) based on surface signature of galactose and N-acetylgalactosamine. Furthermore, apoEVs rescue liver injury in apoptotic-deficient Fas mutant and Caspase-3 knockout mice, which is exerted by restoring the featured hepatic ploidy homeostasis. Surprisingly, apoEVs form a chimeric organelle complex with recipient Golgi apparatus via SNARE-mediated membrane interaction, which consequently facilitates microtubule organization and HC cytokinesis. Notably, through Golgi recovery and ploidy transition, apoEVs contribute to liver regeneration and protect against acute hepatic failure. Collectively, these results identify a previously unrecognized role for apoEVs and the specific mechanisms by which they safeguard liver homeostasis, and suggest the potential of apoEV-based therapy for liver disorders.

cell biology↗

bioRxiv2021

Mutation N501Y in RBD of Spike Protein Strengthens the Interaction between COVID-19 and its Receptor ACE2

SARS-CoV-2 is spreading around the world for the past year. Enormous efforts have been taken to understand its mechanism of transmission. It is well established now that the receptor-binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) as its first step of entry. Being a single-stranded RNA virus, SARS-CoV-2 is evolving rapidly. Recently, several variants such as B.1.1.7, B.1.351, and P.1, with a key mutation N501Y on the RBD, appear to be more infectious to humans. To understand its mechanism, we combined cell surface binding assay, kinetics study, single-molecule technique, and computational method to investigate the interaction between these RBD (mutations) and ACE2. Remarkably, RBD with the N501Y mutation exhibited a considerably stronger interaction characterized from all these methodologies, while the other two mutations from B.1.351 contributed to a less effect. Fluorescence-activated cell scan (FACS) assays found that RBD N501Y mutations are of higher binding affinity to ACE2 than the wild type. Surface plasmon resonance further indicated that N501Y mutation had a faster association rate and slower dissociation rate. Consistent with the kinetics study, atomic force microscopy-based single-molecule force microscopy quantify their strength on living cells, showing a higher binding probability and unbinding force for the mutation. Finally, Steered Molecular Dynamics (SMD) simulations on the dissociation of RBD-ACE2 complexes revealed that the N501Y introduced additional {pi}-{pi} and {pi}-cation interaction for the higher force/interaction. Taken together, we suggested that the reinforced interaction from N501Y mutation in RBD should play an essential role in the higher transmission of COVID-19 variants.

biophysics↗

bioRxiv2021

In vivo structure and dynamics of the RNA genome of SARS-Cov-2

The SARS-CoV-2 coronavirus, which causes the COVID-19 pandemic, is one of the largest positive strand RNA viruses. Here we developed a simplified SPLASH assay and comprehensively mapped the in vivo RNA-RNA interactome of SARS-CoV-2 RNA during the viral life cycle. We observed canonical and alternative structures including 3-UTR and 5-UTR, frameshifting element (FSE) pseudoknot and genome cyclization in cells and in virions. We provide direct evidence of interactions between Transcription Regulating Sequences (TRS-L and TRS-Bs), which facilitate discontinuous transcription. In addition, we reveal alternative short and long distance arches around FSE, forming a "high-order pseudoknot" embedding FSE, which might help ribosome stalling at frameshift sites. More importantly, we found that within virions, while SARS-CoV-2 genome RNA undergoes intensive compaction, genome cyclization is weakened and genome domains remain stable. Our data provides a structural basis for the regulation of replication, discontinuous transcription and translational frameshifting, describes dynamics of RNA structures during life cycle of SARS-CoV-2, and will help to develop antiviral strategies.

genomics↗

bioRxiv2020

A high-quality genome of the mangrove Aegiceras corniculatum aids investigation of molecular adaptation to intertidal environments

Mangroves have colonized extreme intertidal environments characterized by high salinity, hypoxia, and other abiotic stresses. During millions of years of evolution, mangroves have adapted to these habitats, evolving a series of highly specialized traits. Aegiceras corniculatum, a pioneer mangrove species that evolved salt secretion and crypto-vivipary, is an attractive ecological model to investigate molecular mechanisms underlying adaptation to intertidal environments. Here we report a high-quality reference genome of A. corniculatum using the PacBio SMRT sequencing technology, comprising 827 Megabases (Mb) and containing 32,092 protein-coding genes. The longest scaffold and N50 for the assembled genome are 13.76 Mb and 3.87 Mb. Comparative and evolutionary analyses revealed that A. corniculatum experienced a whole-genome duplication (WGD) event around 35 million years ago after the divergence between Aegiceras and Primula. We inferred that maintenance of cellular environmental homeostasis is an important adaptive process in A. corniculatum. The 14-3-3 protein-coding genes were retained after the recent WGD event, decoding a calcium signal to regulate Na+ homeostasis. A. corniculatum has more H+-ATPase coding genes, essential for the maintenance of low Na+ concentration in the cells, than its relatives. Photosynthesis and oxidative-phosphorylation pathways are overrepresented among significantly expanded gene families and might supply the energy needed for salt secretion. Genes involved in natural antioxidant biosynthesis, contributing to scavenging reactive oxygen species against high salinity, have also increased in copy number. We also found that all homologs of DELAY OF GERMINATION1 (DOG1), a pivotal regulator of seed dormancy, lost their heme-binding ability in A. corniculatum. This loss may contribute to crypto-vivipary. Our study provides a valuable resource to investigate molecular adaptation to extreme environments in mangroves.

genomics↗

bioRxiv2020

ILRUN downregulates ACE2 expression and blocks infection of human cells by SARS-CoV-2

The human protein-coding gene ILRUN (inflammation and lipid regulator with UBA-like and NBR1-like domain, previously C6orf106) is a recently-characterised inhibitor of the transcription regulators p300 and CREB-binding protein (CBP). Here we have utilised RNA-seq to define cellular pathways regulated by ILRUN in the context of severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2) infection. We find that inhibition of ILRUN expression increases cellular expression of several members of the renin-angiotensin aldosterone system (RAAS), including the SARS-CoV-2 entry receptor angiotensin converting enzyme 2 (ACE2). Furthermore, inhibition of ILRUN results in increased SARS-CoV-2 replication. These data identify ILRUN as a novel inhibitor of SARS-CoV-2 replication and represents, to our knowledge, the first report of ILRUN as a regulator of the RAAS. SIGNIFICANCE STATEMENTThere is no doubt that the current rapid global spread of COVID-19 has had significant and far-reaching impacts on our health and economy and will continue to do so. Research in emerging infectious diseases, such as severe acute respiratory syndrome-associated coronavirus (SARS-CoV-2), is growing rapidly, with new breakthroughs in the understanding of host-virus interactions and the development of innovative and exciting therapeutic strategies and new knowledge and tools to better protect against the impacts of disease. The human protein-coding gene ILRUN is a recently-characterised inhibitor of the transcription regulators p300 and CREB-binding protein (CBP). Here we present the first evidence that ILRUN modulation has implications for SARS-CoV-2 infections. Virus infectivity assays confirmed that gene silencing of ILRUN had a proviral effect and increased SARS-CoV-2 replication, whilst over-expression of ILRUN inhibited SARS-CoV-2 production. Additionally, we observed that ILRUN also regulates the expression of key elements of the RAAS. These data have important implications for the development of antiviral strategies to deal with the current SARS-CoV-2 pandemic.

cell biology↗

bioRxiv2020

Germline Selection by Meiosis Defends the Transmission of defective Mitochondria with mtDNA variants

Germline selection of mtDNA is vital in maternal inheritance of mtDNA, as it can eliminate severe mtDNA mutations. However, current evidence concerning germline selection at meiosis level comes from incomplete mtDNA sequencing in human first polar body (PB1), which lacks persuasion. Here, we found various variants, including pathogenic mutation sites, present on whole genome of mtDNA in human PB1 compared with its oocyte. And that PB1 mitochondria with mtDNA variants were defective. Afterwards, to further explore how mitochondria enter PB, the defective mitochondria transfer in mouse germline, including cumulus-oocyte-complexes at germinal vesicle and matured oocytes stage. It confirmed that in the first and second meiosis, active purification selected defective mitochondria into PB1 and PB2. Thus, twice meiosis is the last defense system for purifying selection of mtDNA mutations during oogenesis, which also demonstrated that PB1 and PB2 would be final destination of deleterious mtDNA mutations in germline selection.

cell biology↗

bioRxiv2020

Dincta: Data INtegration and Cell Type Annotation of Single Cell Transcriptomes

AO_SCPLOWBSTRACTC_SCPLOWWe proposed a method for data integration and cell type annotation (Dincta) of single cell transcriptomes in a unify framework. The Dincta can handle three cases. In the first case, the data has been annotated the cell type for all cells, Dincta can integrate the the data into a common low dimension embedding space such that cells with different cell types separate while cells from the different batches but in the same cell type cluster together. In the second case, the data was only annotated for part of cells, such as one sample, Dincta can integrate the data into a common low dimension embedding space such that cells with different cell types separate while cells from the different batches but in the same cell type cluster together. Moreover, it can infer the known or novel cell type of the cells with unknown cell type initially. In the third case, there are no cell type information of cells, we can run Dincta in an unsupervised way. It can infer the number of new cell types and annotate the cells into its correspond cell type, and do data integration keeping cells from different cell type separate while removing the batch effects to mix cells in the same cell type. Dincta is simple, accurate and efficient to integrate data, which keeps the cell type information preserved while removes the batch effects, and infers the known or novel cell types of cells.

bioinformatics↗

bioRxiv2020

CUBIC-Cloud: An Integrative Computational Framework Towards Community-Driven Whole-Mouse-Brain Mapping

Recent advancements in tissue clearing technologies have offered unparalleled opportunities for researchers to explore the whole mouse brain at cellular resolution. With the expansion of this experimental technique, however, a scalable and easy-to-use computational tool is in demand to effectively analyze and integrate whole-brain mapping datasets. To that end, here we present CUBIC-Cloud, a cloud-based framework to quantify, visualize and integrate whole mouse brain data. CUBIC-Cloud is a fully automated system where users can upload their whole-brain data, run analysis and publish the results. We demonstrate the generality of CUBIC-Cloud by a variety of applications. First, we investigated brain-wide distribution of PV, Sst, ChAT, Th and Iba1 expressing cells. Second, A{beta} plaque deposition in AD model mouse brains were quantified. Third, we reconstructed neuronal activity profile under LPS-induced inflammation by c-Fos immunostaining. Last, we show brain-wide connectivity mapping by pseudo-typed Rabies virus. Together, CUBIC-Cloud provides an integrative platform to advance scalable and collaborative whole-brain mapping.

neuroscience↗

bioRxiv2020

The Formin Inhibitor, SMIFH2, Inhibits Members of the Myosin Superfamily

The small molecular inhibitor of formin FH2 domains, SMIFH2, is widely used in cell biological studies. It was selected in a chemical screen as a compound inhibiting formin-driven actin polymerization in vitro, but not polymerization of pure actin, and found to be active against several types of formins from different species (Rizvi et al., 2009). Here, in experiments with cultured fibroblasts, we found that SMIFH2 inhibits retrograde flow of myosin 2 filaments and contraction of stress fibers. We further checked the effect of SMIFH2 on non-muscle myosin 2A and skeletal muscle myosin 2 in vitro and found that SMIFH2 inhibits myosin ATPase activity and ability to translocate actin filaments in the in vitro motility assay. While inhibition of myosin 2A in vitro required somewhat higher concentration of SMIFH2 than inhibition of retrograde flow and stress fiber contraction in cells, inhibition of several other non-muscle myosin types, e.g. mammalian myosin 10, Drosophila myosin 7a and Drosophila myosin 5 by SMIFH2, was equally or more efficient than inhibition of formins. Since actin polymerization and myosin contractility are linked in many cytoskeleton processes, additional careful analysis is needed in each case when function of formins was proposed solely on the basis of experiment with SMIFH2.

cell biology↗

bioRxiv2020

Stable isotope informed genome-resolved metagenomics uncovers potential trophic interactions in rhizosphere soil

The functioning, health, and productivity of soil is intimately tied to a complex network of interactions, particularly in plant root-associated rhizosphere soil. We conducted a stable isotope-informed, genome-resolved metagenomic study to trace carbon from Avena fatua grown in a 13CO2 atmosphere into soil. We collected paired rhizosphere and non-rhizosphere soil at six and nine weeks of plant growth and extracted DNA that was then separated by density using gradient centrifugation. Thirty-two fractions from each sample were grouped by density, sequenced, assembled, and binned to generate 55 unique microbial genomes that were >70% complete. The complete 18S rRNA sequences of several micro-eukaryotic bacterivores and fungi were enriched in 13C. We generated several circularized bacteriophage (phage) genomes, some of which were the most labelled entities in the rhizosphere. CRISPR locus targeting connected one of these phage to a Burkholderiales host predicted to be a plant pathogen. Another highly labeled phage is predicted to replicate in a Catenulispora sp., a possible plant growth-promoting bacterium. We searched the genomes for traits known to be used in interactions involving bacteria, micro-eukaryotes and plant roots and found that heavily isotopically-labeled bacteria have the ability to modulate plant signaling hormones, possess numerous plant pathogenicity factors, and produce toxins targeting micro-eukaryotes. Overall, 13C stable isotope-informed genome-resolved metagenomics revealed that very active bacteria often have the potential for strong interactions with plants and directly established that phage can be important agents of turnover of plant-derived carbon in soil.

microbiology↗

bioRxiv2020

Adult newborn granule cells confer emotional-state-dependent plasticity in memory retrieval

Achieving optimal behavior requires animals to flexibly retrieve prior knowledge. Here we show that adult newborn granule cells (anbGCs) mediate emotional-state-dependent plasticity of memory retrieval. We find that acute social reward (aSR) enhances memory retrieval by increasing the reactivation of engram cells, while acute social stress (aSS) weakens retrieval and reduces the reactivation. Such bidirectional regulation relies on the activation of distinct populations of anbGCs by aSR and aSS, triggering opposing modifications of dDG activity, which is sufficient to regulate and predict the performance of memory retrieval. Concordantly, in emotional disorder models, aSR-dependent memory plasticity is impaired, while the effect of aSS remains intact. Together, our data revealed that anbGCs mediate plasticity of memory retrieval, allowing animals to flexibly retrieve memory according to the current emotional state, and suggested the essential roles of anbGCs in translating emotional information to the regulation of memory expression.

neuroscience↗

bioRxiv2020

PIWIL1 Promotes Gastric Cancer via a piRNA-Independent Mechanism

Targeted cancer therapy aims to achieve specific elimination of cancerous but not normal cells. Recently, PIWI proteins, a subfamily of the PAZ-PIWI Domain (PPD) protein family, have emerged as promising candidates for targeted cancer therapy. PPD proteins are essential for small non-coding RNA pathways. The Argonaute subfamily partners with microRNA and small interfering RNA, whereas the PIWI subfamily partners with PIWI-interacting RNA (piRNA). Both PIWI proteins and piRNA are mostly expressed in the germline and best known for their function in transposon silencing, with no detectable function in mammalian somatic tissues. However, PIWI proteins become aberrantly expressed in multiple types of somatic cancers, thus gaining interest in targeted therapy. Despite this, little is known about the regulatory mechanism of PIWI proteins in cancer. Here we report that one of the four PIWI proteins in humans, PIWIL1, is highly expressed in gastric cancer tissues and cell lines. Knocking out PIWIL1 expression (PIWIL1-KO) drastically reduces gastric cancer cell proliferation, migration, metastasis, and tumorigenesis. RNA deep sequencing of gastric cancer cell line SNU-1 reveals that PIWIL1-KO significantly changes the transcriptome, causing the up-regulation of most of its associated transcripts. Surprisingly, few bona fide piRNAs exist in gastric cancer cells. Furthermore, abolishing the piRNA-binding activity of PIWIL1 does not affect its oncogenic function. Thus, PIWIL1 function in gastric cancer cells is independent of piRNA. This piRNA-independent regulation involves interaction with the UPF1-mediated nonsense-mediated mRNA decay (NMD) mechanism. Altogether, our findings reveal a novel and piRNA-independent function of PIWIL1 in promoting gastric cancer. SIGNIFICANCEPrecision medicine aims to cure cancer without affecting normal tissues. PIWI proteins provide a promising opportunity for precision medicine because they are normally expressed only in the testis for male fertility but gain expression in diverse types of cancers. Thus, inhibiting PIWI expression may stop cancer development (and spermatogenesis) without affecting normal body function. To establish causality between PIWI and cancer, we show here that the expression of PIWIL1, a human PIWI protein, promotes gastric cancer. Surprisingly, this oncogenic function does not require piRNA, the expected partner of PIWI proteins, but involves the nonsense-mediated mRNA decay mechanism. These findings reveal a new function and action mechanism of PIWI proteins in oncogenesis, guiding the identification of PIWI inhibitors to cure cancer.

cancer biology↗

bioRxiv2020

Two decades of suspect evidence for adaptive DNA-sequence evolution - Less negative selection misconstrued as positive selection

Evidence for biological adaptation is often obtained by studying DNA sequence evolution. Since the analyses are affected by both positive and negative selection, studies usually assume constant negative selection in the time span of interest. For this reason, hundreds of studies that conclude adaptive evolution might have reported false signals caused by relaxed negative selection. We test this suspicion two ways. First, we analyze the fluctuation in population size, N, during evolution. For example, the evolutionary rate in the primate phylogeny could vary by as much as 2000 fold due to the variation in N alone. Second, we measure the variation in negative selection directly by analyzing the polymorphism data from four taxa (Drosophila, Arabidopsis, primates, and birds, with 64 species in total). The strength of negative selection, as measured by the ratio of nonsynonymous/synonymous polymorphisms, fluctuates strongly and at multiple time scales. The two approaches suggest that the variation in the strength of negative selection may be responsible for the bulk of the reported adaptive genome evolution in the last two decades. This study corroborates the recent report1 on the inconsistent patterns of adaptive genome evolution. Finally, we discuss the path forward in detecting adaptive sequence evolution.

evolutionary biology↗