Dynamics of protein noise can distinguish between alternate sources of gene‐expression variability
2012; Springer Nature; Volume: 8; Issue: 1 Linguagem: Inglês
10.1038/msb.2012.38
ISSN1744-4292
AutoresAbhyudai Singh, Brandon S. Razooky, Roy D. Dar, Leor S. Weinberger,
Tópico(s)Gene expression and cancer classification
ResumoReport28 August 2012Open Access Dynamics of protein noise can distinguish between alternate sources of gene-expression variability Abhyudai Singh Abhyudai Singh Department of Chemistry and Biochemistry, University of California, San Diego, CA, USA Department of Electrical and Computer Engineering, University of Delaware, Newark, DE, USA Search for more papers by this author Brandon S Razooky Brandon S Razooky Department of Chemistry and Biochemistry, University of California, San Diego, CA, USA Biophysics Graduate Group, University of California, San Francisco, CA, USA The Gladstone Institute of Virology and Immunology, San Francisco, CA, USA Search for more papers by this author Roy D Dar Roy D Dar The Gladstone Institute of Virology and Immunology, San Francisco, CA, USA Center for Systems and Synthetic Biology, University of California, San Francisco, CA, USA Search for more papers by this author Leor S Weinberger Corresponding Author Leor S Weinberger Department of Chemistry and Biochemistry, University of California, San Diego, CA, USA The Gladstone Institute of Virology and Immunology, San Francisco, CA, USA Center for Systems and Synthetic Biology, University of California, San Francisco, CA, USA Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA Search for more papers by this author Abhyudai Singh Abhyudai Singh Department of Chemistry and Biochemistry, University of California, San Diego, CA, USA Department of Electrical and Computer Engineering, University of Delaware, Newark, DE, USA Search for more papers by this author Brandon S Razooky Brandon S Razooky Department of Chemistry and Biochemistry, University of California, San Diego, CA, USA Biophysics Graduate Group, University of California, San Francisco, CA, USA The Gladstone Institute of Virology and Immunology, San Francisco, CA, USA Search for more papers by this author Roy D Dar Roy D Dar The Gladstone Institute of Virology and Immunology, San Francisco, CA, USA Center for Systems and Synthetic Biology, University of California, San Francisco, CA, USA Search for more papers by this author Leor S Weinberger Corresponding Author Leor S Weinberger Department of Chemistry and Biochemistry, University of California, San Diego, CA, USA The Gladstone Institute of Virology and Immunology, San Francisco, CA, USA Center for Systems and Synthetic Biology, University of California, San Francisco, CA, USA Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA Search for more papers by this author Author Information Abhyudai Singh1,2,‡, Brandon S Razooky1,3,4,‡, Roy D Dar4,5 and Leor S Weinberger 1,4,5,6 1Department of Chemistry and Biochemistry, University of California, San Diego, CA, USA 2Department of Electrical and Computer Engineering, University of Delaware, Newark, DE, USA 3Biophysics Graduate Group, University of California, San Francisco, CA, USA 4The Gladstone Institute of Virology and Immunology, San Francisco, CA, USA 5Center for Systems and Synthetic Biology, University of California, San Francisco, CA, USA 6Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA ‡These authors contributed equally to this work *Corresponding author. The Gladstone Institutes, University of California, San Francisco, 1650 Owens Street, San Francisco, CA 94158, USA. Tel.:+1 415 734 4946; Fax:+1 415 355 0855; E-mail: [email protected] Molecular Systems Biology (2012)8:607https://doi.org/10.1038/msb.2012.38 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Within individual cells, two molecular processes have been implicated as sources of noise in gene expression: (i) Poisson fluctuations in mRNA abundance arising from random birth and death of individual mRNA transcripts or (ii) promoter fluctuations arising from stochastic promoter transitions between different transcriptional states. Steady-state measurements of variance in protein levels are insufficient to discriminate between these two mechanisms, and mRNA single-molecule fluorescence in situ hybridization (smFISH) is challenging when cellular mRNA concentrations are high. Here, we present a perturbation method that discriminates mRNA birth/death fluctuations from promoter fluctuations by measuring transient changes in protein variance and that can operate in the regime of high molecular numbers. Conceptually, the method exploits the fact that transcriptional blockage results in more rapid increases in protein variability when mRNA birth/death fluctuations dominate over promoter fluctuations. We experimentally demonstrate the utility of this perturbation approach in the HIV-1 model system. Our results support promoter fluctuations as the primary noise source in HIV-1 expression. This study illustrates a relatively simple method that complements mRNA smFISH hybridization and can be used with existing GFP-tagged libraries to include or exclude alternate sources of noise in gene expression. Synopsis Intrinsic noise in gene expression stems from two major sources: promoter fluctuations and lowcopy mRNA fluctuations. A new method allows these sources to be distinguished using transient changes in protein variance without the need for involved single molecule mRNA assays, and reveals that promoter fluctuations drive variability in HIV-1 gene expression. This work presents a method that discriminates between two alternate sources of intrinsic noise in gene expression (RNA birth/death processes and promoter fluctuations) even when expression levels are high, thereby complementing mRNA FISH. The method exploits the fact that transcriptional blockage results in more rapid increases in intercellular protein variability when mRNA birth/death processes dominates over promoter fluctuations. Application of the method to the HIV-1 expression system implicates promoter fluctuations, not mRNA birth/death processes, as the primary source of noise in HIV-1 expression. Introduction Clonal cell populations exhibit considerable cell-to-cell variation in the levels of any specific protein (Elowitz et al, 2002; Blake et al, 2003; Kaern et al, 2005; Raser and O'Shea, 2005; Sigal et al, 2006; Raj and van Oudenaarden, 2008). This variation or expression noise is essential for diverse cellular processes, such as the regulation of probabilistic cell-fate decisions and the generation of phenotypic heterogeneity across isogenic cell lines (Suel et al, 2006; Maamar et al, 2007; Chang et al, 2008; Losick and Desplan, 2008; Sureka et al, 2008; Singh and Weinberger, 2009; Razooky and Weinberger, 2011; Munsky et al, 2012). Tight control of expression noise is also vital for optimal functioning of housekeeping proteins, and diverse diseased states have been attributed to an increase in expression noise in particular genes (Cook et al, 1998; Kemkemer et al, 2002; Fraser et al, 2004; Bahar et al, 2006). Collectively, these results suggest that gene-expression noise profoundly affects biological function and underscore the importance of developing methods that pinpoint the source of noise in a given gene circuit. Gene-expression noise is often decomposed into intrinsic and extrinsic noise (Elowitz et al, 2002). While extrinsic noise arises from intercellular differences in the amounts of cellular components (e.g., RNA polymerases and ribosomes), intrinsic noise results from the random timing and discrete nature of biochemical reactions associated with promoter remodeling, transcription, translation, and degradation of mRNA and protein species. Although several techniques are available to separate extrinsic and intrinsic noise (e.g., see Elowitz et al, 2002 and Newman et al, 2006), methods to discriminate between the different sources of intrinsic noise are far less developed. Experimental evidence suggests that transcriptional intrinsic noise originates from two sources: (i) Poisson mRNA fluctuations arising from probabilistic synthesis and decay of individual mRNA transcripts (mRNA birth/death fluctuations) and (ii) promoter switching between different transcriptional states (promoter fluctuations). mRNA birth/death fluctuations constitute a major source of stochasticity in gene expression since many mRNA species are present at very low molecular counts within cells (Bar-Even et al, 2006; Newman et al, 2006; Taniguchi et al, 2010). These mRNA fluctuations are transmitted downstream through translational bursting to generate intercellular variability in protein levels. Alternatively, protein variability results from promoter switching between different transcriptional states. An important consequence of promoter switching is transcriptional bursting, where multiple mRNAs are created per promoter-firing event (Raser and O'Shea, 2004; Golding et al, 2005; Raj et al, 2006; Yunger et al, 2010; Suter et al, 2011; Muramoto et al, 2012). In this study, we sought to develop a method to determine the relative contributions of mRNA birth/death fluctuations and promoter fluctuations to intrinsic gene-expression noise, assuming that protein levels are the only observable state of the system. At a qualitative level, both mRNA birth/death and promoter fluctuations generate similar predictions for steady-state protein noise levels (Bar-Even et al, 2006; Ingram et al, 2008). Thus, steady-state distributions of protein abundances across cells are insufficient to identify the source of intrinsic expression noise. One increasingly popular method to quantify the extent of stochastic promoter switching relies on counting individual mRNA transcripts in single cells by single-molecule fluorescence in situ hybridization (smFISH) (Raj et al, 2006; Zenklusen et al, 2008; Tan and van Oudenaarden, 2010). mRNA smFISH is a powerful and elegant method, but requires that individual mRNA molecules be long enough to accommodate the binding of at least 20 individual probes. This is needed to insure the spot is sufficiently bright to be distinguished in images and counted, and that the specific mRNA species being analyzed are in a concentration low enough that individual diffraction-limited spots do not spatially overlap. Here, we report a method for discriminating mRNA birth/death and promoter fluctuations that can be used with highly expressed mRNAs and that is easily implemented across different cell types. More specifically, we show that the dynamical changes in protein noise levels, in response to perturbations, are sufficient to determine the source of intrinsic expression noise. These perturbations can be generated relatively easily with readily available small-molecule pharmaceutical agents that rapidly and efficiently block transcription and translation. We illustrate the experimental utility of this method by perturbing gene expression from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter. Transient changes in reporter protein noise levels, in response to small-molecule drugs, show that LTR gene-expression noise results primarily (>90%) from promoter fluctuations. Results and Discussion Stochastic gene-expression model To analyze the different sources of intrinsic noise in protein abundance, we considered a stochastic model of gene expression that incorporates both low-copy mRNA fluctuations and transcriptional bursting (Figure 1). In this model, mRNA transcription and degradation are stochastic events that occur at exponentially distributed time intervals. Each transcriptional event creates a burst of B mRNA molecules, where B is a discrete random variable with probability {B=i}=αi, i∊{1,2,3…} and mean 〈B〉, where the chevrons, 〈〉, represent the expected value. Note that B=1 with a probability of one corresponds to Poisson fluctuations in mRNA counts while a large average burst size 〈B〉 implies transcriptional bursting (i.e., promoter switching between active and inactive promoter states). If m(t) denotes the mRNA population count at time t, then the probability Pj(t) of having j mRNA molecules at time t evolves according to the following chemical master equation: Figure 1.Stochastic hybrid model of gene expression with two different sources of noise. Schematic illustrating the two sources of noise: (i) Poisson mRNA fluctuations arising from stochastic production and degradation of individual mRNA molecules, and (ii) promoter fluctuations arising from slow promoter transitions (red arrows) between 'Gene OFF' and 'Gene ON' states. As protein population counts are often very large, stochasticity in protein dynamics is neglected. Download figure Download PowerPoint where km is the frequency of transcription events and γm represents the mRNA degradation rate (Mcquarrie, 1967). Fluctuations in mRNA counts are transmitted downstream to the protein level, which is assumed to be the only observable state of the system. As protein population counts are typically large, we can ignore Poisson noise arising from stochastic birth and death of individual protein molecules. Accordingly, protein dynamics are modeled deterministically as where kp is the mRNA translation rate, γp is the protein degradation rate and p(t) denotes the protein count at time t. Together, Equations (1) and (2) constitute a stochastic hybrid gene-expression model in which mRNA time evolution is discrete and stochastic, while protein levels evolve continuously and deterministically. As in many studies, protein-expression noise is quantified using the coefficient of variation squared, defined as CV2=σ2/〈p〉2, where σ2 is the variance in protein level and 〈p〉 denotes the average protein abundance (Paulsson, 2004, 2005; Pedraza and Paulsson, 2008). For the gene-expression model described above, the steady-state protein noise level is given by where denotes the steady-state mean protein abundance and represents the steady-state Fano factor (also known as 'noise strength') of the mRNA population count (see Supplementary Appendix A in Supplementary information). Equation 3 represents the total intrinsic noise in gene expression and can be decomposed into components representing expression variability originating from mRNA birth/death fluctuations and stochastic promoter switching. When ηm=1, then mRNA population counts have Poisson statistics, and protein noise levels primarily arise from mRNA birth/death fluctuations. In contrast, when ηm=10, then 90% of the protein noise is generated through promoter fluctuations and transcriptional bursting of mRNAs from the promoter. Finally, we point out that ηm is directly related to the mean transcriptional burst size 〈B〉 for many promoters. In particular, analysis shows that for a two-state promoter model, where the ON state is unstable and the promoter spends most of the time in the OFF state, ηm=1+〈B〉 (see Supplementary Appendix B). Thus, ηm is representative of the mean burst size for many promoters including HIV LTR, which have been shown to reside mostly in the OFF state (Raj et al, 2006; Singh et al, 2010; Suter et al, 2011). We next explore methods to determine ηm for a specific promoter or gene. Equations 3 and 4 show that increasing the frequency of transcription events, km, increases the steady-state mean protein abundance, but decreases the steady-state noise level such that the product remains fixed. Thus, intrinsic noise scales inversely with mean protein abundance, consistent with experimental observations in both prokaryotes and eukaryotes (Kepler and Elston, 2001; Thattai and van Oudenaarden, 2001; Ozbudak et al, 2002; Simpson et al, 2004; Bar-Even et al, 2006; Newman et al, 2006). One method to determine ηm would be through the scaling of versus , as the scaling factor is proportional to it (Equation 3). However, the scaling factor also depends on the mRNA translation rate kp and protein and mRNA half-lives, and thus, this method requires a priori knowledge of these parameters. Moreover, for this versus method, one needs to quantify protein abundances in absolute molecule counts rather than fluorescence intensities, as is typically the case. Thus, steady-state measurements of protein noise magnitude are insufficient to determine ηm and, hence, insufficient to discriminate between the different components of intrinsic noise. Below, we predict analytically and demonstrate experimentally that ηm can be inferred from transient changes of protein population mean and noise magnitude in response to transcriptional and translational perturbations to gene expression. Analytical results: dynamics of protein noise magnitude in response to perturbations Small-molecule drugs provide a convenient method to perturb genetic circuits and realize transient changes in protein levels across different cell types. In this section, we theoretically analyze the effects of small-molecule drugs that specifically block transcription and translation. These drugs are routinely used to determine the stability of mRNAs and proteins (Zhou, 2004; Yen et al, 2008; Hao and Baltimore, 2009). Such experiments typically involve bulk assays that track transient changes in mean molecular abundances across a population of cells. In response to a complete block of translation or transcription, the mean protein levels 〈p(t)〉 decay as, respectively, where t represents the time since drug addiction. Thus, both mRNA and protein half-life can be quantified by monitoring 〈p(t)〉 in response to blocks in transcription and translation. Next, we investigate how cell-to-cell variation in protein levels (coefficient of variation squared) changes in response to these small-molecule drugs and whether this change provides information about unknown system parameters, such as ηm. We first consider a translational block to gene expression. Recall that, in this gene-expression model, protein production and degradation are modeled as differential equations. A translational block would result in a deterministic exponential decay in protein levels in each individual cell, which would shift the distribution of protein level across the cell population to lower levels without changing its coefficient of variation. Thus, in case of a translational block, this model predicts that no additional information can be gained from the higher order statistical moments of p(t). We next consider a transcriptional block to gene expression by setting the frequency of transcriptional events km=0 at time t=0. Assuming the system was at steady state before the block, the protein-noise level (measured by the coefficient of variation squared, CV2(t)) changes as where is the initial protein-noise level given by (3), and function is a monotonically increasing function of t (see Supplementary Appendix C in Supplementary information). With transcription blocked, mRNA levels exponentially decay, leading to an increase in cell-to-cell variability, as would be expected from the inverse scaling of noise and mean levels. This increase in mRNA noise is transmitted to encoded proteins, causing CV2(t) to monotonically increase over time (Figure 2). Interestingly, this analysis indicates that the rate of increase of CV2(t) is dependent on the source of intrinsic noise. This point is illustrated in Figure 2, which plots the function for different values of ηm, and hence different relative contributions of mRNA birth/death and promoter fluctuations to expression noise. More specifically, as we increase ηm, the initial rise in the protein-noise level becomes more and more gradual. Intuitively, this occurs because, when expression noise results primarily from promoter fluctuations, then mRNA counts are sufficiently high, and mRNA degradation is essentially a deterministic process. Thus, when transcription is blocked, mRNAs degrade approximately deterministically across single cells, which reduces protein levels and generates a slow increase in CV2(t). On the other hand, when expression noise originates from mRNA birth/death fluctuations, then a transcriptional block results in stochastic degradation of mRNAs across the cells, which rapidly increases the protein-noise levels (Figure 2). In summary, the increase in protein cell-to-cell variability after transcription is blocked is dependent on ηm, and the dynamics of CV2(t) can discriminate between alternative sources of intrinsic stochasticity in gene expression. Finally, we point out that although the above results were derived assuming deterministic protein dynamics (i.e., Equation 2), modeling protein synthesis and decay stochastically does not significantly alter the predictions in Figure 2 (see Supplementary Appendix D). Figure 2.Transient changes in gene-expression noise can discriminate between mRNA birth/death and promoter fluctuations. Predictions for changes in gene-expression noise after transcription are blocked when protein half-life⩽2 × mRNA half-life (γm⩽2γp, left, note that this is expressed in terms of degradation rate) and protein half-life>2 × mRNA half-life (γm>2γp, right, note that this is expressed in terms of degradation rate), for different contributions of promoter fluctuations to gene-expression noise. If variability in protein levels mainly results from stochastic birth and death of individual mRNA molecules, then CV2(t) is predicted to change much more rapidly than when protein variability results from promoter fluctuations. The y axis is normalized by the CV2(t) at t=0. These simulations were preformed assuming a 2.5-h protein half-life with a 3-h (left) and 1-h (right) mRNA half-life. Download figure Download PowerPoint Equation 7 shows that the rise in protein-noise levels after a transcription block is dependent on ηm, γm, and γp. Assuming protein and mRNA degradation rates are known or have been determined from the kinetics of protein decay (Equation 6), ηm can be directly inferred from CV2(t). Since this procedure to quantify ηm depends on examining relative changes in CV2(t), it will work irrespective of whether protein levels are quantified in terms of fluorescence intensity or molecular counts. However, if data on absolute protein molecular count are available, then the frequency of transcriptional events km and the mRNA translation rate kp can also be determined from the steady-state protein mean and noise level (Equations 3 and 4). Hence, by combining transient data on the statistical moments of p(t) (Equations 6 and 7) together with their steady-state values (Equations 3 and 4), one can infer all the parameters of the gene-expression model. One limitation of this method is that it may not be applicable in cases where the mRNA half-life is much shorter than the protein half-life. This limitation arises because when mRNA half-life is short, mRNA transcripts quickly decay to zero once transcription in blocked. With no available mRNA, protein decay will be dominated by protein degradation alone and will not contain any information about the underlying mRNA dynamics. For example, when γm is much larger than γp, then decay in mean protein levels is always given by p(t)=exp(−γpt), irrespective of whether transcription or translation is blocked (Equation 6), hence providing no information on the mRNA half-life. Thus, for this method to work, reporter systems need to be used where both mRNA and protein degradation reactions are first-order processes that occur with known comparable rates. Moreover, small-molecule drugs used for perturbing transcription/translation should not alter these decay rates. Importantly, the change in protein expression variability after a transcription block becomes insensitive to ηm, when ηm is large. For example, model predictions for ηm equal to 1 and 10 are well separated in Figure 2, but predictions for ηm equal to 10 and 80 are close to each other. Thus, it may not be possible to get precise estimates of ηm by this method, especially when ηm is large (i.e., high levels of transcriptional bursting from the promoter). Experimental results: HIV-1 LTR gene-expression noise results primarily from promoter fluctuations In this section, we illustrate the experimental utility of this method by quantifying the relative contributions of mRNA birth/death and promoter fluctuations to HIV-1 expression noise. Stochastic expression of viral proteins from the HIV-1 LTR promoter critically influences the viral-fate decision between active replication and post-integration latency (a dormant state of the virus analogous to phage lysogeny) (Weinberger et al, 2005; Weinberger and Shenk, 2007; Weinberger et al, 2008). Thus, this is an important system to identify the source of intrinsic noise in gene expression. To discriminate between the different sources of intrinsic noise using the dynamics of protein noise magnitude, it is essential to use a reporter system in which the mRNA half-life is not very short compared to the protein half-life. Toward that end, we used flow cytometry measurements of a destabilized version of GFP (d2GFP) that exhibits a 2.5-h half-life. Previous measurements quantified a 3-h half-life for the d2GFP mRNA (Raj et al, 2006), so γm≈γp for d2GFP, making it an ideal reporter system for this study. To quantify HIV-1 LTR expression noise, we use our previously described library of isoclonal populations (Singh et al, 2010), where each isoclonal population carries a single copy of a minimal vector encoding HIV-1 LTR driving d2GFP, integrated at a unique location in the human genome. To focus on intrinsic noise and remove extrinsic contributions to noise measurements, gating of flow cytometry data was performed as described (Newman et al, 2006; Singh et al, 2010). To filter extrinsic noise, a two-color fluorescent reporter system (Elowitz et al, 2002) was implemented where each cell contains a single integrated copy of the LTR driving d2GFP and a second copy of the LTR driving mCherry at a different integration site (see Supplementary Figure S7). After filtering of extrinsic noise, isoclonal populations exhibit an inverse scaling of steady-state noise level with mean protein abundance (Singh et al, 2010), as predicted by the model (Equation 3). This inverse scaling of versus is consistent with both mRNA birth/death and promoter fluctuations (Equation 3), and as mentioned above steady-state protein noise levels are insufficient to discriminate between the different sources of intrinsic noise. To discriminate between mRNA birth/death and promoter fluctuations, we analyzed dynamic changes in HIV-1 LTR expression noise after perturbation with two transcriptional inhibitors, actinomycin D, a small-molecule drug that rapidly and efficiently blocks transcription by preventing RNA polymerase II (RNAp II) elongation (Sobell, 1985), and Flavopiridol, a small-molecule drug that blocks transcription by inhibiting the interaction of P-TEFb with RNAp II (Chao and Price, 2001). Introducing either transcriptional inhibitor to any of the isoclonal populations resulted in a decrease in d2GFP fluorescence intensity levels that was consistent with the reported 2.5-h d2GFP protein and 3-h d2GFP mRNA half-lives (see Supplementary information and Supplementary Figure S3). The fact that the decay in d2GFP fluorescence after actinomycin D treatment is consistent with the reported 3-h mRNA half-life (Raj et al, 2006) suggests that the dynamics of the reporter decay are not affected by the presence of the drug. The data also show that d2GFP fluorescence decays, in the presence of cycloheximide, at ∼3.5 h both in the presence and absence of actinomycin D (Supplementary Figure S2; Supplementary Appendix E). In response to both transcriptional inhibitors, the cell-to-cell variability in fluorescence intensity levels across all the isoclonal populations (CV2(t)) gradually increases over time (Figure 3). Fitting the data to model predictions (see Supplementary Appendix F for details) shows that this increase in CV2(t) is inconsistent, for all clones, with a model where intercellular variability in protein levels originates only from mRNA birth/death fluctuations (Figure 3). Instead, the data indicate that promoter fluctuations act as the dominant source of gene-expression noise in the HIV-1 LTR promoter with a mRNA Fano factor (ηm) of at least 10 across four different integration sites (Supplementary Appendix F). Direct measurements of mRNA population statistics by mRNA smFISH also show high levels of transcriptional bursting from the HIV-1 LTR (ηm≈80; see Supplementary Appendix G) and provide an independent experimental verification of the proposed method. Figure 3.Transcriptional bursting is a significant source of variability in HIV-1 LTR gene-expression across different integration sites. Time courses of CV2(t) of GFP expression, as measured by flow cytometry, for four isoclonal Jurkat T lymphocyte populations (F32, G95, LL44, and LL8) after perturbation with transcriptional blocking drugs actinomycin D (left column) or flavopiridol (right column). The transient increases in gene-expression noise after transcriptional blocking were consistent with a model in which HIV-1 LTR expression noise is primarily due to transcriptional bursting of mRNAs from the viral promoter (red lines; ηm=15). In contrast, a model in which mRNA birth/death fluctuations act as the dominant source of gene-expression noise (black dashed lines; ηm=1), overestimates the observed transient increase in CV2(t) for all clones. All quantities are normalized by their corresponding values of CV2(t) at t=0; error bars show 95% confidence interval for the CV2(t). Source data is available for this figure in the Supplementary Information. Download figure
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