Bcs1l, the causative mutation for GRACILE syndrome. The homozygous mice develop mitochondrial hepatopathy with steatosis and fibrosis after weaning. Our aim was to assess cellular mechanisms for disease onset and progression using metabolomics.Liver is a target organ in many mitochondrial disorders, especially if the complex III assembly factor BCS1L is mutated. To reveal disease mechanism due to such mutations, we have produced a transgenic mouse model with c.232A>G mutation in 2O2 production and expression of antioxidants.With mass spectrometry we analyzed metabolite patterns in liver samples obtained from homozygotes and littermate controls of three ages. As oxidative stress might be a mechanism for mitochondrial hepatopathy, we also assessed HHomozygotes had a similar metabolic profile at 14 days of age as controls, with the exception of slightly decreased AMP. At 24 days, when hepatocytes display first histopathological signs, increases in succinate, fumarate and AMP were found associated with impaired glucose turnover and beta-oxidation. At end stage disease after 30 days, these changes were pronounced with decreased carbohydrates, high levels of acylcarnitines and amino acids, and elevated biogenic amines, especially putrescine. Signs of oxidative stress were present in end-stage disease.The findings suggest an early Krebs cycle defect with increases of its intermediates, which might play a role in disease onset. During disease progression, carbohydrate and fatty acid metabolism deteriorate leading to a starvation-like condition. The mouse model is valuable for further investigations on mechanisms in mitochondrial hepatopathy and for interventions. BCS1L mutations have been causative for CIII assembly defects that result in a broad spectrum of symptoms ranging from congenital deafness The cytochrome bc1 complex  is composed of one mitochondrial and ten nuclear encoded proteins that are assembled with the help of two chaperones, BCS1L and TTC19. The recently identified protein TTC19 aids assembly of early subunits of CIII Bcs1l c.232A>G mutation into mice Bcs1lG/G) mice mimick the human disorder and consist of growth failure from the fourth week of live, progressive liver disorder with glycogen depletion and microvesicular steatosis, proximal tubulopathy, and death before 6 weeks Bcs1lG/G mice.To elucidate functions of BCS1L in general, and the pathophysiologic mechanisms in GRACILE syndrome in particular, we introduced the Bcs1lG/G mice to assess changes in liver function at disease onset as well as at the end stage. Targeted metabolite profiles in homogenized liver tissues from the animals was performed in order to get a comprehensive picture of the effect of the homozygous Bcs1l c.232A>G mutation on liver metabolism. Compromised respiratory chain function may impair beta-oxidation resulting in fatty acid accumulation and steatosis Bcs1lG/G mice and therefore assessed proposed The pathophysiologic mechanisms underlying the different BCS1L disorders are unclear. However, in neonatal diseases hepatopathy is a common finding. In this study, our aim was to use the transgenic Comparing the pool of 198 detected metabolites to control animals, significant differences at FDR<0.1 were found both due to the mutation as such (102 compounds) and to the age of the animal (177 compounds). Using the same threshold, 141 metabolites exhibited positive interaction between the presence of the mutation and the age. A summary of the metabolites other than glycerophosphatidylcholines and sphingomyelins is given in a form of a heatmap in p<0.05) whereas sphingomyelins (n = 14) and acylcarnitines (n = 41) were not significantly different . No positive association could be found on day 14 or for any other metabolite class on day 24.In an analysis of changes in sets of metabolites The most marked findings were increased metabolite levels in liver tissue of the animals at the end stage of the disease, aged over 30 days; up to 4-fold changes for most amino acids .In isolated mitochondria from sick  in CIII  was acco in CIII , but no In liver tissue, significant increases for methionine-sulfoxide and all detected prostaglandins (6 out of 15 that were screened for) were found in sick animals aged over 30 days . The antBcs1lG/G mice, with the liver being most affected Bcs1lG/G mice.Since structural and functional CIII deficiencies develop progressively in the organs of In the end stage of the disease, the spectrum of metabolite changes was clearly different, demonstrating a liver deterioration with severe energy deficit, cellular degeneration, oxidative stress, and a starvation-like phenotype. The concomitant up-regulation of larger acylcarnitines, AMP, and a majority of the lipids suggest a decrease of the Krebs cycle activity, and a subsequent failure in anticipated switch in hepatocytes towards beta-oxidation in response to glycogen depletion. Decreased short-chain acylcarnitines indicate a limitation in beta-oxidation. A major deterioration in liver function is reflected in the increase of most amino acids and all detected bile acids.Bcs1lG/G mice Under normal conditions, the liver ensures normoglycemia by glycogenolysis followed by increased gluconeogenesis Bcs1lG/G mice, in which liver and proximal tubules are the first and most affected organs with progressive CIII deficiency Tfam, respiratory chain deficiency seems to be successfully compensated by a switch from fatty acid oxidation to glycolysis, and by increased mitochondrial biogenesis, in myocardium Low lactate content in the liver, despite increased blood levels Bcs1lG/G mice. Phosphatidylcholines are essential for CIII activity since delipidation abolishes its function Bcs1lG/G mice, microvesicular steatosis increases with decreasing CIII activity Energy starvation as well as respiratory chain deficiency may result in liver fat accumulation Bcs1lG/G mice.A recent study in tumour cells with a mitochondrial DNA mutation leading to lack of CIII activity showed that a glutamine-dependent reductive carboxylation, needing isocitrate dehydrogenase 1 and 2, can compensate for impaired mitochondrial oxidative metabolism Bcs1lG/G mice contained elevated levels of the biogenic amino acids spermidine and putrescine, reflecting at least in part a limited capacity of the urea cycle. Both spermidine and putrescine play important roles in mediating cell death. Spermidine induces acetylation and deacetylation of nuclear and cytoplasmic proteins resulting in autophagy, removal of damaged cells and protein aggregates Liver samples at end stage disease in 2O2Impaired respiratory chain function due to CIII deficiency may cause increased ROS production that together with liver steatosis contribute to tissue deterioration Bcs1lG/G mice, ROS modified metabolites such as methionine sulfoxide and prostaglandins were detectable only at end stage disease. Further, antioxidant defence was similar in younger animals and changed only slightly in end stage disease compared to controls. In contrast to findings in fibroblasts harbouring other CIII deficiencies 2O2 levels could be detected in liver mitochondria from Bcs1lG/G mice. Controversy prevails concerning mitochondrial ROS production. Generation of mitochondrial ROS varies depending on tissue and substrate characteristics in vitro studies of CIV and mtDNA deficiencies showing elevated ROS levels, whereas in vivo models do not confirm oxidative stress in tissue In livers of Oxidative stress is believed to be a contributing factor for disease phenotypes in disturbances of oxidative phosphorylation, including in development of steatosis. Cytosolic accumulation of fatty acids activates peroxisomal beta-oxidation and microsomal omega-oxidation resulting in ROS production + dependent deacetylases +/NADH ratios thereby switching on catabolic pathways. AMPK activation in mitochondrial deficiencies is important for regulation of increased mitochondrial biogenesis. This was shown experimentally using an AMP precursor, AICAR, to correct complex IV deficiency in mouse muscle Bcs1lG/G mice suggesting a possible disturbance in Sirtuin 3 activation.Hepatocyte energy production is regulated by the energy sensors AMP kinase (AMPK) and members of the sirtuin family of NADBcs1lG/G mice due to CIII deficiency leads to a starvation-like condition with energy depletion. The findings suggest that the initial disturbances are decreased glycolysis and beta- oxidation concomitant with a decreased ATP production. Impaired mitochondrial metabolism cannot be corrected by eg. the proposed compensatory reductive carboxylation circulus vitiosus occurs with exhausted energy supply and failing compensatory mitochondrial biogenesis to energy deprivation. Oxidative stress is not a central regulatory mechanism for disease onset, but may appear at the end stage of the disease. We envision that our mouse model can be used for further investigations on disease mechanisms in respiratory chain failure and for exploring interventions.In conclusion, using metabolomics we have shown that respiratory chain dysfunction in Bcs1l c.232A>G mutation was introduced into mice using gene targeting Bcs1lG/G mice of three age groups were studied; on postnatal day 14, when the animals are healthy  and on day 24 , when initial histopathological findings appear, and on day 30 or more , when the animals are sick. The same numbers of littermate wild type mice (Bcs1lA/A) or heterozygotes (Bcs1lA/G) at each age were used as controls. Animals were sacrificed by cervical dislocation. Liver tissue samples were either immediately frozen on dry ice, fixed in 4% buffered formalin or used for the isolation of mitochondria All animal experiments were performed with the approval of the Lund regional animal research ethic committee, Sweden . The g at 2°C for 5 min, the resulting supernatants were pipetted into a cryovial  and analyzed immediately to avoid degradation of the analytes.Frozen liver samples were weighed into 2 ml Precellys tubes  equipped with ceramic beads. Tissues were homogenized using ethanol/dichloromethane (1/1) as extraction solvents IDQ kit p150, BIOCRATES Life Sciences AG, Innsbruck, Austria) and LC-MS/MS data were quantified with Analyst 1.4.2 software . All methods are validated for human plasma considering FDA Guidance for industry – Bioanalytical Method Validation. The initial pool of 253 measurements Flow injection analysis (FIA-MS/MS) based platform was employed for the simultaneous quantification of a broad range of endogenous intermediates, namely acylcarnitines, amino acids, sphingomyelins and glycerophospholipids. A chromatographic step (LC-MS/MS) was applied for the additional quantification of a series of amino acids and biogenic amines, small organic acids and other components from the energy metabolism pathway, bile acids, and eicosanoids. Samples were randomized on the plate prior to analysis to avoid potential confounding interaction between concentration and order of injection. Detailed experimental protocols have been published previously http://www.R-project.org/.) for data sorting, statistical analyses and data presentation. All calculations were performed on the log basis 2 transformed concentration data with the non-detect imputed as previously published Measurement data were imported to the environment R  by high resolution respirometry using the substrate- uncoupler- inhibitor protocol Respiratory chain function was analysed in isolated mitochondria incubated in MIR 05  is direct proportional to increased H2O2 release after subsequent substrate and inhibitor addition.Isolated mitochondria were incubated at 37°C in assay medium (MIR 05) supplemented with 4 µl horseradish peroxidase, superoxide dismutase and Amplex red according to the instructions of the manufacturer . Under these conditions, increased fluorescence indicates H, Perkin Elmer) Protein oxidation by ROS was measured as carbonyl group production. Isolated mitochondria were incubated with DNP-hydrazine for 45 min at room temperature. The mixture was diluted with PBS, and 100 µl were used to coat a 96 well plate that was incubated over night at 4°C. The wells were washed three times with 0.9% NaCl+0.05% Tween-20. Then the plate was incubated with primary antibody, anti DNP , diluted 2000 times in incubation buffer  for 1 h incubation at 37°C, washed and incubated with secondary antibody, Swine-Anti-rabbit IgG-HRP,  for 1 h at room temperature. At the end the plate was incubated with substrate solution  according to the recommendations of the manufacturer. The extinction at 490 nm was measured on a spectrophotometer . RNA was extracted from the lysate using the RNeasy kit from Qiagen according to the instructions of the manufacturer and RNA quality was analyzed by agarose gel electrophoresis.For cDNA synthesis 500 ng total RNA were reverse transcribed using the Taqman® reverse transcription reagents from Applied Biosystems and random hexamer primers included in the kit. The resulting cDNA was used as template in quantitative PCR on an Applied Biosystems StepOne cycler using Taqman Gene Expression assays from Applied Biosystems for the specific genes. The variation in replicates is shown as mean ±SD.Figure S1Lipid metabolism. Heatmap depicting the log basis 2 fold changes in lipid (rows) concentration determined in liver tissues between matched pairs (columns) of homozygote  and control  mice. Columns (i.e. matched pairs) are reordered by hierarchical clustering  using the age group  to label the tree leaves. Metabolites are grouped according to the chemical classification employed in the manuscript. To facilitate the reading, fold changes are truncated at +/− 2 and light grey lines are drawn around the main groups highlighted by HCA and chemical classes.(TIF)Click here for additional data file.Table S1Metabolite panel. List of metabolites used in this study.(DOC)Click here for additional data file.


Haliotis spp.), a model system of reproductive protein evolution. We test the evolutionary rates (dN/dS) of abalone sperm lysin and two duplicated egg coat proteins (VERL and VEZP14), and find a signal of co-evolution specific to ZP-N, a putative sperm binding motif previously identified by homology modeling. Positively selected residues in VERL and VEZP14 occur on the same face of the structural model, suggesting a common mode of interaction with sperm lysin. We test this computational prediction biochemically, confirming that the ZP-N motif is sufficient to bind lysin and that the affinities of VERL and VEZP14 are comparable. However, we also find that on phylogenetic lineages where lysin and VERL evolve rapidly, VEZP14 evolves slowly, and vice versa. We describe a model of sexual conflict that can recreate this pattern of anti-correlated evolution by assuming that VEZP14 acts as a VERL mimic, reducing the intensity of sexual conflict and slowing the co-evolution of lysin and VERL.Sperm and egg proteins constitute a remarkable paradigm in evolutionary biology: despite their fundamental role in mediating fertilization (suggesting stasis), some of these molecules are among the most rapidly evolving ones known, and their divergence can lead to reproductive isolation. Because of strong selection to maintain function among interbreeding individuals, interacting fertilization proteins should also exhibit a strong signal of correlated divergence among closely related species. We use evidence of such molecular co-evolution to target biochemical studies of fertilization in North Pacific abalone . We study a discrete functional domain (ZP-N) found in a pair of duplicated egg coat proteins, and we find the ZP-N motif from both proteins bind sperm lysin (a protein important for sperm passage of the egg coat) in a similar fashion. ZP-N is a feature of vertebrate and invertebrate egg coat proteins, as well as yeast mating recognition proteins, demonstrating its broad significance in sexual reproduction. Unexpectedly, we find that the ZP-N motifs of VEZP14 and VERL exhibit inverse patterns of co-evolution with lysin, suggesting that these duplicates may have opposite functions in fertilization. Using computer simulations, we model a novel explanation for this pattern whereby VEZP14 acts as a decoy of VERL in order to decrease the effective amount of sperm lysin and slow the rate of fertilization. Such molecular mimicry could complement other well-established fertilization blocks that females use to control rates of fertilization and limit polyspermy. Reproductive genes are often identified in genome-wide scans as targets of positive selection, and some are among the most rapidly evolving proteins known dN/dS, or ω) has been shown to reflect known protein-protein interactions Haliotis spp. Correlation in the ratio of non-synonymous to synonymous nucleotide substitution (of VE dissolution indicates that two lysin molecules bind each repeat dN/dS) has been shown to be positively correlated across branches of the abalone phylogeny Both lysin and VERL show recurrent adaptive divergence among the 8 abalone species that diverged <18 million years ago in the North Pacific Many of the constituent proteins of the abalone VE have been characterized and are known to also evolve under positive selection Here, we use molecular co-evolutionary analyses in combination with biochemical assays to investigate the molecular interactions between abalone sperm and egg coat proteins during fertilization. A strong signal of co-evolution specifically between lysin and ZP-N motifs focus our biochemical assays that demonstrate the ZP-N motif is sufficient for binding of lysin. Our co-evolutionary analyses also reveal a surprising pattern  that suggests unexpected modes of interaction among these fertilization proteins not evident from binding assays. We develop a population model to explore one such interaction whereby VEZP14 functions as a VERL mimic, binding excess lysin and reducing the intensity of sexual conflict by reducing fertilization rate.dN/dS) are correlated between lysin and ZP-N from both genes across the evolution of North Pacific abalone species – a pattern not seen even with other domains of these genes. Second, affinity assays utilizing surface plasmon resonance (SPR) quantification of association and dissociation rates between green abalone lysin and expressed ZP-N motifs shows binding kinetics are comparable for VERL and VEZP14. While our co-evolutionary analyses are shown to be a useful tool in focusing these more detailed biochemical studies confirming ZP-N is sufficient to bind lysin, they may also offer novel insight into how egg coat proteins mediate sperm passage of the VE during abalone fertilization. We first present results from our co-evolutionary and biochemical studies, after which we discuss implications for models of abalone fertilization.Two distinct but complementary approaches identify ZP-N from duplicated abalone VE proteins, VERL and VEZP14, as sperm lysin receptor motifs. First, evolutionary rates as measured by the ratio of non-synonymous to synonymous nucleotide substitution . We discuss possible explanations for a positive -vs- negative correlation in dN/dS with lysin in detail below in the context of models of abalone fertilization. To summarize: (i) positive selection is a feature of VERL repeats 1 and 2 as well as the ZP-N motif of VEZP14; (ii) the rate of evolution driven by positive selection is somewhat higher for the VEZP14 ZP-N motif; and (iii) whereas the rate of evolution along phylogenetic lineages is correlated between lysin and VERL , lysin and VEZP14's evolutionary rates are anti-correlated (slow with fast).Because cognate sperm and egg proteins must co-evolve to maintain reproductive compatibilities, their rapid divergence among species is predicted to result in correlated evolutionary rates along phylogenetic lineages. We expand upon an earlier study that demonstrated such a positive correlation between abalone lysin and the combined first three full (∼150 AA) repeats of VERL dN/dS with the VEZP14 ZP-N motif . While we were unsuccessful at obtaining protein from VERL repeats 2 and 3, ZP-N expression proteins of high purity from VERL repeat 1 and VEZP14 were obtained  of 5.2×10−7 and 5.8×10−7 M for VERL and VEZP14, respectively  the small quantitative differences in the absolute amount of lysin bound (i) ZP-N motifs from VERL or VEZP14 alone are sufficient to bind abalone lysin, and (ii) the kinetics of binding between lysin and ZP-N from these VE proteins are similar, yielding affinities that differ quantitatively very little (∼10%) based on in vitro binding assays.SPR shows binding between green abalone lysin and expressed ZP-N proteins from VERL and VEZP4 are qualitatively similar. Though slightly more lysin was bound by VERL (as evidenced by a consistently higher absolute refractive index over replicate channels), the kinetics of binding between lysin and both ZP-N motifs over association and dissociation intervals are comparable, yielding equilibrium dissociation constants (Kectively . A negatin bound  likely rdN/dS (ω) for sites predicted to be under positive selection  and low (KD∼10−4 M) affinity between α-Agglutinin and its ligand Aga2p D∼10−7 M) that we predict could be greater for internal repeats based on VE dissolution kinetics. Binding assays using intact VERL isolated from VEs indicate positive cooperativity consistent with VE dissolution assays showing the rate limiting step is lysin's initial binding with VE proteins a) or lower rates of dissociation (kd) between lysin and internal VERL repeats, leading to a net increase in affinity (KD).In contrast, i) also a major component of the abalone VE ii) under strong positive selection consistent with lysin's species-specific VE dissolution iii) can bind lysin at comparable affinity as VERL via the ZP-N receptor motif, VEZP14 is a strong candidate to also be a component of the VE's molecular mechanism mediating sperm entry. One possibility is that VEZP14 is an integral component of the compact VE fibers that decondense upon lysin binding. For example, VEZP14 dimers could form a “molecular zipper” through antiparallel pairing of ZP-N anchored to a scaffold of ∼30 other VE proteins via their ZP polymerization modules per se mediates sperm recognition of mammalian egg coat proteins The raised VE of abalone eggs presents a physical barrier mediating sperm access, commonly viewed as one component of the redundant polyspermy blocks which function to avoid developmental arrest of the embryo due to multiple sperm fusing with the egg nucleus dN/dS between lysin and VERL's ZP-N motifs from repeats 1 and 2  are seminal examples of natural selection i)).We created a model of sexual conflict that can generate the correlations observed among abalone fertilization proteins by assuming that VEZP14 acts as a molecular decoy of VERL. We model the evolutionary dynamics of three diploid genes  in response to sexual conflict over mating rate. In the model, the male (lysin) and female (VERL) proteins co-evolve with one another because of a conflict over optimal mating rate, as expected and previously observed in simulations and natural phylogenetic comparisons opt (ii)). This has two effects (iii)): (i) it stops male-female co-evolution since both now benefit from an increased mating rate; (ii) it provides an impetus for the decoy to evolve traits that optimize the male-female interaction (to tailor mating rate to Popt). For example, the decoy could evolve a different affinity for the male ligand . As differences between the true female receptor and the decoy accumulate, males evolve the ability to preferentially target the true receptor, causing the decoy to become ineffective (iv)). This increases a male's mating rate at the cost of each individual female's fecundity, and co-evolution resumes as per the general model of sexual conflict. As the co-evolutionary chase proceeds, male-female phenotype space may re-encounter the decoy leading to its indefinite maintenance via recurrent positive selection.We imagine that a decoy locus could modify this sexual conflict over mating rate as follows. The female and decoy loci are initially similar, either because the decoy arose as a duplicate of the existing female receptor (as seems to be the case for VEZP14 and VERL) Pi parameter  must take a value that causes female fitness to be greater than at the unmodified mating rate PMF . Second, we aim to model a process that could continue indefinitely, rather than approaching equilibrium. Selection for the decoy to evolve exists because P approaches Popt as the decoy approaches the male (Dopt). The magnitude of female-decoy interference (Pt) increases as the male female distance DMF deviates from Dopt. Third, we model this process in one trait dimension; the decoy is either between the male and female, or not  , otherwise the decoy will begin a co-evolutionary chase with the male. Even at large Pt, the decoy does briefly chase the male in 9 of 20 simulations . Interestingly, among North Pacific abalone lysin and VEZP14 do sometimes take high values on the same branch  . When the branch  despite dN/dS we employ serve to focus genetic or biochemical investigation on the discrete functional domains that mediate molecular interactions. However, because these methods reflect the long-term dynamics of distinct evolutionary lineages, they may also reveal unique modes of interaction not evident in traditional functional assays. We show that the ZP-N motifs of VERL and VEZP14 bind sperm lysin with similar affinity, so the naïve interpretation is that they may also function similarly as duplicate egg coat receptors of lysin. But evidence of anti-correlated co-evolution supports a more complex interpretation: VEZP14 may function as a VERL mimic to bind excess lysin, thereby modifying the dynamics of sexual conflict over fertilization rate. This novel hypothesis, that molecular mimicry functions in addition to other well-established polyspermy blocks, promises new insights and approaches to understanding the extraordinary molecular diversity of reproductive genes and their remarkable patterns of evolution.We present an example of the value of evolutionary analyses in the interpretation of functional phenotypic data. On the simplest level, evolutionary analyses as straightforward as the tests of correlated H. wallalensis, H. sorenseni, H. cracherdii, and H. kamtschatkana) newly obtained from ovary transcripts of the respective taxa using an identical sequencing approach as in Protein coding DNA sequences for sperm and egg genes from 8 North Pacific abalone species dN/dS values. The general approach was developed previously from know cases of protein-protein interaction dN/dS in the absence of genome-wide data necessary for statistical validation of more powerful methods dN/dS for lysin and ZP-N motifs from VERL and VEZP14 were made using the branch model of dN/dS that were either unweighted, or which were weighted by the length of the respective branch as in To test for correlated rates of evolution between lysin and putative binding motifs of VERL and VEZP14, we fit linear regression models to point estimates of branch-specific Mapping of positively selected residues onto structural models was used to identify potential regions of binding between lysin and ZP-N motifs of VERL or VEZP14. Sites models of codon evolution were first used to test for evidence of positive selection on ZP-N motifs, after which posterior probabilities of residues under positive selection were inferred via the Bayes Empirical Bayes algorithm Haliotis fulgens [chosen because of extensive previous biochemical and structural studies of reproductive proteins; see, e.g. in vitro and proteins purified for binding assays with lysin. PCR primers incorporating 5′-BglII and 3′-ApaI restriction sites were designed from H. fulgens sequence to amplify ZP-N motifs from VERL repeats 1, 2, and 3 individually, as well as VEZP14 using standard PCR protocols. PCR products for VERL repeat 1 and VEZP14 ZP-N motifs were verified via Sanger sequencing, but repeated attempts at cloning PCR products from VERL repeats 2 and 3 were unsuccessful. PCR products were digested with BglII and ApaI following manufacturers guidelines , and cloned into vector pMT/BiP/V5His v.A before transfecting S2 cells following manufacturers guidelines for the Drosophila Expression System .ZP-N motifs from 4 cells/ml were grown 24 hours, followed by induction of protein expression using 750 µM CuSO4 for an additional 24 hours. Cells were precipitated from the media and discarded, protease inhibitors  and 1 ml of chelating sepharose  added to the supernatant and stirred overnight at 4°C to allow binding with a C-terminal 6X His tag. Sepharose-bound proteins were washed twice with 50 ml PBS buffer , twice with PBS buffer supplemented to 500 mM NaCl, and bound protein eluted by sequential addition of 10 ml imidazole at increasing concentration . Select fractions were analyzed for expression products in the range of 17.1 and 16.6 kilo Daltons  via SDS PAGE as in ZP-N proteins were purified from transfected S2 cell culture media using immobilized metal affinity chromatography (IMAC). Serum free media (250 mL) containing transfected S2 cell cultures at a density of ∼5×10a) and dissociation (kd) rates from association and dissociation intervals of SPR data by curve fitting using non-linear least-squares regression, implemented via Scrubber2 .Quantitative measurements of binding affinity between lysin and ZP-N motifs from VERL and VEZP14 were made using an in-house surface plasmon resonance (SPR) detection system described previously DMF) between the male and female as determined additively by alleles of sex-limited male (m/m′) and female (f/f′) reproductive loci. Small phenotypic distance values cause high mating rates. The number of offspring per female decreases as her mating rate (P) deviates above or below the optimal female mating rate (Popt). The optimal male mating rate is 1, and males increase mating rate by evolving alleles that are similar to females. Above Popt females are selected to reduce mating rate by evolving away from males and a co-evolutionary chase ensues opt both females and males benefit from an increased mating rate. Pe, Pi, and Pt). Pe (decoy effectiveness) reduces mating rate to Popt. Pi  further reduces mating rate when the decoy is similar to the female. Interference decreases as the decoy approaches the male, and the mating rate thus approaches Popt. Pt  depends on the male-female distance. Dopt is the optimal male-female distance for decoy function; when DMF deviates from Dopt, the decoy interferes with the female and reduces mating rate (Pt). Equations and parameter values describing these relationships are in We extended the diploid two-locus sexual conflict models of −5 per locus per generation. Other simulation parameters and equations are shown in the graph detailing the model . Linear regressions were fit to point estimates as was done with the lysin and ZP-N data, with correlated rates inferred as a significant slope parameter (p>0.05).Simulations are run on populations of N = 10,000 individuals for 15,000 generations . Femaleshe model . As in [Figure S1dN/dS or ω) for reproductive proteins  or non-reproductive  genes were estimated using the branch model of codeml dN/dS values are seen, even when comparing among functionally distinct regions of the same gene , and the correlation in rates observed between lysin and the target ZP-N motif Click here for additional data file.Figure S2Drosophila S2 expressed ZP-N motifs. The ZP-N motif from VEZP14 the first repeat of green abalone VERL were cloned, expressed in Drosophila S2 cells , and successively eluted from immobilized metal affinity columns (IMAC) using three concentrations of imidazole . (A) IMAC elutions employing 50–250 mM imidazole contain a large quantity of 20–24 kDa protein (dark green box); deglycosylation using PNGaseF  resulted in a single protein band of approx. 17 kDa (expected size of VERL repeat 1 ZP-N is 17.1 kDa). (B) IMAC elutions employing 250 mM imidazole contain a single band of approx. 22 kDa . Western blots confirmed the identified bands as expressed proteins from the presence of a V5 epitope tag, and shotgun proteomic analyses match mass spectra from multiple peptides of both to the respective ZP-N motifs from VERL or VEZP14. Retarded migration and results from PNGaseF experiments are consistent with N-linked glycosylation of the expressed ZP-N proteins used in binding assays (Affinity purification of g assays .(TIF)Click here for additional data file.Supplement S1decoy.pl, the main perl script; decoy.ctl, the control file where simulation and model parameters can be set; and sub_*.pm, various subroutines called by the main perl script. For additional detail please contact the authors.Perl scripts for running population simulations of sexual conflict with a female decoy. The files are: (ZIP)Click here for additional data file.


Deoxyribonucleic acid (DNA) of the human immunodeficiency virus (HIV) provides the most sensitive measurement of residual infection in patients on effective combination antiretroviral therapy (cART). Droplet digital PCR (ddPCR) has recently been shown to provide highly accurate quantification of DNA copy number, but its application to quantification of HIV DNA, or other equally rare targets, has not been reported. This paper demonstrates and analyzes the application of ddPCR to measure the frequency of total HIV DNA (pol copies per million cells), and episomal 2-LTR  circles in cells isolated from infected patients. Analysis of over 300 clinical samples, including over 150 clinical samples assayed in triplicate by ddPCR and by real-time PCR (qPCR), demonstrates a significant increase in precision, with an average 5-fold decrease in the coefficient of variation of pol copy numbers and a >20-fold accuracy improvement for 2-LTR circles. Additional benefits of the ddPCR assay over qPCR include absolute quantification without reliance on an external standard and relative insensitivity to mismatches in primer and probe sequences. These features make digital PCR an attractive alternative for measurement of HIV DNA in clinical specimens. The improved sensitivity and precision of measurement of these rare events should facilitate measurements to characterize the latent HIV reservoir and interventions to eradicate it. Exponential amplification of nucleic acids by the polymerase chain reaction (PCR) is the cornerstone of modern molecular biology. Numerous methods have been developed to obtain quantitative information from PCR about the concentration of deoxyribonucleic acid (DNA) before amplification. The most widely used form of quantitative PCR (qPCR) is “real-time” PCR (qPCR), in which initial concentrations are extrapolated from sequential measurements during the cycling reaction Few fields have been more profoundly impacted by quantitative PCR than virology. The quantification and dynamics of HIV burden in infected patient were originally elucidated using quantitative PCR to measure viral ribonucleic acid (RNA) in blood plasma Measurement of HIV DNA in translational research studies has relied upon a variety of home-brew assays This paper describes an assay for HIV pol and 2-LTR circles in human peripheral blood samples based on droplet digital PCR. The assay relies on well-established biochemistry, using primer sets and TaqMan hydrolysis probes published previously “Clinical samples” of HIV-1 seropositive patients analyzed had been previously collected in ongoing research studies approved by the applicable Institutional Review Boards at Brigham and Women's Hospital, Johns Hopkins University, the University of North Carolina, Chapel Hill, and the University of California, San Francisco. The authors were blinded to patient data for these samples. The cohorts from which these samples had been drawn were known. Most of these samples (>95%) were peripheral blood mononuclear cells, and the remainder consisted of purified CD4+ T cells. Purification of these CD4+ T cells had been performed at the collection site, and thus purification methods were not known.“PBMC samples” were extracted from the blood of HIV-negative healthy donors at the University of California San Diego (UCSD). All subjects gave written, informed consent and blood collection protocols were approved by the UCSD Institutional Review Board. Peripheral blood mononuclear cells (PBMC) were purified from whole blood with Lymphoprep, following the manufacturer's recommended protocol.“Infected CD4+ T cells” were purified from blood collected identically using a RosetteSep (StemCell Technologies) CD4+ T Negative Isolation Kit. Purified CD4 cells were incubated overnight prior to activation with surface-bound anti-CD3/anti-CD28 and infected with NL4-3 at a multiplicity of infection of 0.2. Two days after infection, cells were washed and cryo-preserved until DNA extraction.260/A280 absorptivity ratio using a NanoDrop 2000 spectrophotometer (Thermo Scientific). When the DNA concentration was below the desired concentration for emulsification, the concentration was increased by ethanol precipitation and re-suspension. Where specified, templates were thoroughly mixed with background human genomic DNA obtained by identical extraction methods from HIV seronegative donors (“PBMC DNA”) or with sonicated salmon sperm DNA (Agilent Technologies).Cellular DNA was extracted using a Qiagen DNA Blood Midi Kit, following the manufacturer's protocol. In all cases, DNA was ethanol precipitated following elution to increase concentration. The DNA concentration was estimated from the AExtracted DNA was heated to 95°C for 10 minutes then quenched on ice prior to digest with the restriction enzyme BSAJ-I (New England Biolabs) at 60°C for 1 hour.Plasmids encoding the entire HIV genome  or a 2-LTR junction  were used as standards for qPCR.Both real-time and digital PCR reactions used published primers to conserved regions of HIV pol (Hxb2 positions 2536–2662) Real-time PCR reactions were performed in a 50 µL solution containing 25 µL PerfeCTa Multiplex Master Mix containing ROX (Quanta Biosciences), 900 nM primers, 250 nM probe, and 1 µg of template DNA. Reactions were conducted in triplicate. Cycling proceeded in an Applied Biosystems 7900HT Sequence Detection System with the following parameters: 50°C for 2 minutes, 95°C for 10 minutes, followed by 50 cycles of 95°C for 15 seconds and 60°C for 1 minute. A standard curve of pNL4-3 plasmid DNA in triplicate, 10-fold serial dilutions was included on each plate. PCR reactions used FAM-labeled pol probes multiplexed with VIC-labeled RNAse P for genomic DNA quantification, together with the corresponding forward and reverse primers. A FAM-labeled 2-LTR probe and 2-LTR primers were used in a separate reaction for each sample.The PCR reaction mixture was loaded into the Bio-Rad QX-100 emulsification device and droplets were formed following the manufacturer's instructions. The contents were transferred to a 96-well reaction plate and sealed with a pre-heated Eppendorf 96-well heat sealer for 2 seconds, as recommended by Bio-Rad.Total DNA was amplified separately in an Applied Biosystems GeneAmp 9700 thermal cycler. Each reaction consisted of a 20 µL solution containing 10 µL ddPCR Probe Supermix, 900 nM primers, 250 nM probe, and template DNA with the following cycling conditions: 10 minutes at 95°C, 40 cycles each consisting of a 30 second denaturation at 94°C followed by a 58°C extension for 60 seconds, and a final 10 minutes at 98°C. After cycling droplets were analyzed immediately or stored at 4°C overnight and until analysis.Raw fluorescence data for each well were exported from the manufacturer's software (Bio-Rad QuantaSoft v. 1.2) for analysis. Analysis of individual event data was performed using custom software written in Mathematica 8.0 (Wolfram Research). Droplets were classified as positive, negative or ambiguous following a custom algorithm that filters out potentially spurious events.  Events tμ was estimated from the number of positive events n detected in the corresponding channel and the number of total accepted droplets N by maximum likelihood. The distribution of templates within a drop was assumed to follow a Poisson distribution, and the number of positive droplets was assumed to follow a binomial distribution. Confidence intervals were estimated under the same assumptions. The droplet size was assumed to be 0.91 nl, consistent with the instrument manufacturer's software.The number of template copies per unit volume Template copies per sample were computed by averaging over all available replicate wells. Total cellular DNA input was measured by halving the estimated number of RPP30 copies, and copy numbers per diploid cell equivalent were computed as the ratio of template (pol or 2-LTR) copies per diploid cell.Statistical analyses were performed using Graph-Pad Prism 5.0 software  and Mathematica 8.0.It was desirable to load as much DNA as possible into each well to maximize assay sensitivity, because the HIV targets of interest are extremely rare amidst a large background of host cellular DNA. To determine the maximum amount of cellular DNA that could be loaded into a single well, 10, 100 or 1000 copies of pNL4-3 or 2-LTR plasmid were spiked into a background of salmon sperm DNA prior to droplet formation . MeasureThe Bio-Rad digital PCR system measures only endpoint droplet fluorescence after all thermal cycling is completed. Thus measured copy numbers might vary with the number of amplification cycles used. For perfectly efficient amplification, a single starting template would be amplified to the probe concentration (250 nM) in 25 cycles, resulting in signal saturation. To test the sensitivity of ddPCR to variation in cycle number and to determine the optimal cycle number, test samples consisting of plasmid  or dilutN = 156) were analyzed by both qPCR and ddPCR in triplicate. Samples were collected from 24 patients at varying times during the first 6 months following initiation of combination antiretroviral therapy (cART). Measurements made by the two methods were linearly correlated (Pearson R2 = 0.64), with a slope statistically indistinguishable from unity  was computed for each bin, and these averages were fitted by linear regression  of 32 or fewer, the coefficient of variation was low and independent of template concentration. In this regime, the error distribution was approximately log-normal with a standard deviation of 0.07 log10 copies.To determine the distribution of assay noise in the qPCR assay, plasmid or infected cell templates were serially diluted and evaluated by qPCR with 10–12 replicates per sample . For temAt low template concentrations, random sampling of templates occurs in qPCR, suggesting that the noise distribution should become Poissonian in this regime. However, the ratio between the copy number estimated per well and number of events N in the Poisson distribution might not be the expected value, primarily because the absolute copy number estimates rely on a  standard curve. The coefficient of variation was therefore fitted to a model that assumed log-normal errors at high copy number and Poisson distribution at low copy number . The inc6 cells.Next, the limit of detection of the ddPCR assay was evaluated. The assay used in previous experiments loaded DNA from approximately 500,000 cells into each triplicate, making detection of templates present at fewer than 2 copies per million theoretically impossible. To differentiate the intrinsic detection limit for the ddPCR assay from this loading limit, a serial dilution of DNA isolated from infected CD4 cells into uninfected PBMC DNA was tested with a number of replicate wells that increased as the reciprocal of the template number to a maximum of 36 wells . This alAll experiments described above used the same conserved primer-probe set. Because of the high diversity of HIV genomes in vivo, even regions that are relatively well conserved may vary in some patients. Mismatches in the primers or the probes might reduce the efficiency of amplification or probe hydrolysis without eliminating the signal completely. In this scenario, qPCR may substantially underestimate the true template copy number. This problem can be circumvented by using patient-specific primers and probes if HIV DNA sequence data are available, but this approach is cumbersome 10 pol c/106 cells) was reduced in all cases, with an average 57% reduction.To determine how ddPCR measurements would be affected by sequence variation, samples were selected from a large cohort with pol sequence data available. Among these 84 patients, 21 had variations in the sequence complementary to the probe and over half had variations at one or both primer sites. Four patients with the most extreme mismatches between pol sequence and the consensus primer/probe set were evaluated by both ddPCR and qPCR using both patient-specific and consensus primers and probes . With qPSeveral applications of digital PCR reported recently have demonstrated that the high accuracy possible in theory can be practically realized with a variety of clinical specimens To maximize assay sensitivity, the maximum input DNA concentration was established first. Inhibition of the PCR reaction occurred abruptly when over 1.5 μg of extracted DNA was loaded into the 20 μl reaction input.  AlthoughThe number of amplification cycles used had no discernible effect on measured values of either pol or 2-LTR, provided that at least 30 cycles were used.  Twenty PR2 = 0.64), and there was no discernible bias between the two methods. Therefore both assays indeed provide similar measurements, and the principal difference between them is the structure and magnitude of noise.Because identical primers and probes were used for the two assays, we expected both assays to provide similar information. To determine the correlation and bias of the two methods, 156 clinical samples were tested by both assays. Values measured by the two techniques were strongly correlated . This procedure, which has been speculated to remove secondary structure, was used consistently here. Samples were not digested with a restriction enzyme prior to real-time analyses, so it is possible that the restriction digest used prior to digital PCR had the inadvertent benefit of removing inhibitory secondary structure in episomal DNA.The term “limit of detection” is used in various ways depending on the context. The definition that seems most applicable in typical HIV research applications is simply the template concentration below which detection will usually not occur at levels distinguishable from background noise. In the triplicate format used predominantly here, this limit is between 3 and 4 copies per million cells, depending on the number of droplets formed. A far more mathematically demanding definition used in clinical chemistry requires that the 95% confidence intervals of an undetected sample not overlap with the 95% confidence interval of a sample at the limit of detection As shown in 10One important limitation of the Bio-Rad QX-100 system is the dynamic range. Because the system works by counting positive reactions among approximately 15,000 droplets per sample well, the dynamic range is limited to between 4 and 5 logThe ddPCR work described here used the Bio-Rad QX-100 droplet digital PCR system exclusively, and our results may not apply to alternative implementations of digital PCR. The current QX-100 system suffers from two limitations that significantly impact the HIV DNA assay reported here. The first is the unexplained false positive events mentioned above. In addition, categorization of events as positive or negative based on measured fluorescence values is not trivial. The default thresholds set by Bio-Rad's QuantaSoft analysis software result in some obviously incorrect calls. Re-analysis of raw fluorescence data using custom software corrected most of these errors, but false positive could not be completely eliminated.  and S7 WDespite these limitations, droplet digital PCR was found to be a practical method for highly accurate measurement of HIV DNA targets in clinical specimens. Existing qPCR assays for pol and 2-LTR HIV DNA were readily adapted to digital PCR without modification of primer/probe sets, re-optimization of thermal cycling parameters or changes in clinical sample processing. Unexpectedly, ddPCR also proved more robust to target sequence variation. Most importantly, the improved accuracy and precision resulted in reliable quantification of proviral and episomal HIV DNA targets well below the limit of quantification of qPCR. These features make ddPCR particularly well suited to measurement of the size of the HIV latent reservoir and suggest that this assay could prove useful for clinical studies aimed at eradication of HIV from infected patients.Figure S1Values measured by digital PCR do not vary with cycle number. DNA isolated from CD4+ T cells infected in vitro was serially diluted, emulsified into droplets and thermally cycled for 30 to 50 cycles before analysis. No significant differences in measured pol or 2-LTR copy numbers were observed over this range of cycling times. No positive events were observed after 20 cycles (not shown). False-positive events in no-template control wells were also unaffected by cycle number (data not shown).(PDF)Click here for additional data file.Figure S2Poissonian noise among ddPCR replicates. (a) Copy numbers estimated from each well are plotted versus the average over triplicate wells for 370 clinical samples. In addition to the 156 samples described All patients were on cART and were enrolled in studies requiring at least 6 months of suppressed plasma viremia (<50 HIV RNA copies/ml). Wells with no events detected are excluded from the plot in (a), but not from the analysis in (b). Red dashed lines show the expected value plus or minus one standard deviation, assuming the data are Poisson distributed. (b) To more rigorously assess whether the data in (a) are consistent with a Poisson distribution, the coefficient of variation was computed for each well (black dots), and errors were averaged over bins of width 0.2 log10 (blue squares). Observed average errors (solid black regression line) were smaller than the Poissonian prediction (dashed violet line) by 0.15±0.10 log10, but this difference was not statistically significant.(PDF)Click here for additional data file.Figure S3Noise in qPCR assay. The cycle threshold (CT), which measures the number of PCR cycles required for template amplification, varies only slightly (median 0.07 cycles) at low CT values (higher target amounts). Templates consisting of either plasmid or samples from an HIV-infected patient were diluted into HIV- cellular DNA background to determine the effect of input copy number on assay variability. (a) As the number of input template copies decreases below about 300 copies/106 cells, the standard deviation of the CT value among replicate wells increases rapidly. Poissonian sampling noise (dotted line) imposes a theoretical minimum on the assay noise (dotted line), but the observed noise (dashed line) is much larger (least-squares fit  = 23× larger). (b) The corresponding number of pol copies N estimated based on the best fit to a standard curve ((TIF)Click here for additional data file.Figure S4The ddPCR assay is more precise, particularly for 2-LTR circles. Samples isolated from the PBMC of infected patients were assayed by both methods. To compare the precision throughout the tested range, the C.V. was averaged over bins as in Fig. S2 (b). All 370 clinical samples analyzed by ddPCR in a) For both methods and both targets, the C.V. increases with a slope statistically that is not significantly different from ½, the expected value for Poisson-distributed noise (dashed line). Trend lines computed independently for the four assays did not have distinguishable slopes. Therefore, in order to estimate the average relative precision, maximum likelihood fits shown assume the ½ exponent. The offset between the trend lines indicates the relative precision. (b) For the pol target, ddPCR is 4-fold more precise. (c) The precision improvement is much greater for the 2-LTR target, with an average 20-fold improvement over qPCR.(PDF)Click here for additional data file.Figure S5Triage classification of events. Raw fluorescence data were first filtered to eliminate events consistent with irregular droplet size (“rain” and “hail”). The remaining events were analyzed according to the algorithm shown. First, the largest droplet clusters were identified. In most cases, positive events were rare and nearly all events were associated with droplets that were negative in both fluorescence channels. Significant clusters were then approximated with a binormal distribution, and the probability of each droplet was determined for each of these distributions. Events that were highly unlikely within any of the binormal distributions were classified as ambiguous. Finally, independent assortment of the duplexed targets was used to eliminate events with an unlikely combination of fluorescence amplitudes. Restriction enzymes used in this study were always expected to cut between the duplexed amplicons, so the number of positive events in each channel was assumed independent. This was used to identify spurious double-positive events.(PDF)Click here for additional data file.Figure S6Sample dot-plots illustrate threshold ambiguities in ddPCR. Raw fluorescence values from a single well are shown. Default thresholds set by Bio-Rad QuantaSoft analysis software (version 1.1) are shown as colored rectangles, and the corresponding event counts in each quadrant are shown. (a) “Rain” and “hail” extend outward from the central peak of dual-negative events, but these are easily distinguished from true positive events by cluster analysis. (b) The pattern is similar to (a), but the sparseness of hail complicates its discrimination from true positive events. The one dual-positive event called by QuantaSoft can be eliminated by the assumption of independent assortment.(PDF)Click here for additional data file.Figure S7Sample false positives in ddPCR. Positive events called in no-template control wells may reflect erroneous calls by the default thresholding algorithm (a), but most false-positive events are well separated from true negative events and thus cannot be identified mathematically (b).(PDF)Click here for additional data file.Table S1PCR Primers and Probes.(DOCX)Click here for additional data file.Table S2Summary of Clinical Characteristics for Unblinded Patients. The 156 samples analyzed by both qPCR and ddPCR were drawn from ACTG5248, during the first 6 months of treatment.(DOCX)Click here for additional data file.


Standard (static) CT angiography is used to identify the intracerebral hemorrhage (ICH) spot sign. We used dynamic CT-angiography to describe spot sign characteristics and measurement parameters over 60-seconds of image acquisition.We prospectively identified consecutive patients presenting with acute ICH within 4.5 hours of symptom onset, and collected whole brain dynamic CT-angiography (dCTA). Spot parameters , time to maximum HU, time to spot diagnostic definition, spot volume and hematoma volumes) were measured using volumetric analysis software.We enrolled 34 patients: three were excluded due to secondary causes of ICH. Of the remaining 31 patients there were 18 females (58%) with median age 70 (range 47–86) and baseline hematoma volume 33 ml (range 0.7–103 ml). Positive dCTA spot sign was present in 13 patients (42%) visualized as an expanding 3-dimensional structure temporally evolving its morphology over the scan period. Median time to spot appearance was 21 s (range 15–35 seconds). This method allowed tracking of spots evolution until the end of venous phase (active extravasation) with median duration of 39 s (range 25–45 seconds). The average density and time to maximum density was 204HU and 30.8 s (range 23–31 s) respectively. Median time to spot diagnosis was 20.8 s using either 100 or 120HU definitions.Dynamic CTA allows a 3-dimensional assessment of spot sign formation during acute ICH, and captured higher spot sign prevalence than previously reported. This is the first study to describe and quantify spot sign characteristics using dCTA; these can be used in ongoing and upcoming ICH studies. Computed Tomographic angiography (CTA) is non-invasive test that visualizes the transit of radio-opaque contrast through the vasculature, and is commonly used in acute cerebrovascular disease to identify sites of vessel occlusion or rupture. The CTA “spot sign” is a site of active contrast extravasation A recent evaluation of spot signs on delayed angiographic phases suggests its prevalence has been underestimated and confirmed the potential for delayed imaging to detect late phase spot signs Dynamic CTA (dCTA) is a non-invasive low-radiation dose technique that acquires a time series of bone subtracted CTA images of the whole brain, which avoids the timing restrictions inherent to routine static CTA acquisition. The temporal wash-in and washout of intravascular contrast material over the scan duration provides the opportunity to assess for spot sign formation over the entire image acquisition protocol , and to characterize its temporal evolution in a quantitative manner. We have previously used dCTA to explore the underlying pathology of the spot sign Ottawa Health Science Network Research Ethics Board (OHSN-REB) approved this study with a waiver of consent for data collection. The Ottawa Hospital is a tertiary care stroke centre with a “code stroke” imaging protocol including non-contrast computerized tomography (NCCT), CTA of the neck arteries, and whole brain CT-perfusion. All patients presenting less than 4.5 hours with symptoms consistent with stroke or ICH and no known contraindications to iodinated contrast undergo the full imaging protocol as part of clinical care. We prospectively identified consecutive patients presenting with acute non-traumatic ICH, and collected imaging data performed as part of the “code stroke” protocol.TM) as per our institutional stroke protocol. This system utilizes 320 ultra-high resolution detector rows (0.5 mm in width) to image the entire brain in a single gantry rotation, resulting in ultrafast scan times and reduced contrast and radiation exposures. We injected 40 ml of intravenous contrast at 5 ml/s through a power injector (Medrad® Stellant® CT Injection Systems) followed by a saline bolus. We acquired multiple low-dose (80 kVp) volume scans of the entire brain (skull base to vertex) in sequential order during contrast infusion to obtain whole brain perfusion and dynamic vascular analysis in one examination We obtained baseline head CT, CTP and dynamic CTA images using Toshiba volume CT scanner  An experienced neuroradiologist (S.C. 9 years' experience) reconstructed dynamic CTA data from the whole brain perfusion data into non-subtracted orthogonal multiplanar reformats, bone subtracted 3D MIP and volume rendered images using a VitreaBetween December 2008 and June 2013, 34 consecutive patients presented with acute ICH were recruited who underwent dCTA as part of a code stroke protocol. Three patients with secondary ICH  were excluded. Thirty-one patients with spontaneous ICH were included: 18 female (58%); median age 70 (range 47–86). The overall baseline hematoma volume was 33 ml (range 0.7–103 ml). Thirteen patients (42%) were dCTA spot sign (dspot) positive. Median baseline total hematoma volume in dspot positive patients was 48.6 ml (range 4–103) and dspot negative patients was 22 ml (range 0.7–94.1 ml) (p = 0.007). The median rate of contrast extravasation was 0.06 ml/s (range 0.04–1.1 ml/s). All dspots appeared in the arterial phase. In one case, dCTA showed a posteriorly located spot that appeared 3 seconds after an anterior one within the same hematoma  & S2. OnAll spot signs continued through both arterial and venous phases (holophasic) until the end of image acquisition at 60 seconds. This continuity of spot sign presence over the whole scanning period allowed the assessment of contrast leakage between start and end points. The dspot was best appreciated on orthogonal non-subtracted multiplanar reformats when displayed as a continuous loop  [12], [1This study provides further insight into the dynamic evolution of the spot sign during acute primary ICH. In our series, dspot was present in 42% of patients, and contrast extravasation appeared to persist throughout the scanning period, with a median peak density of 204 HU at a median time-to-maximal density of 30.8 seconds. Moreover, dCTA allowed the assessment of rate of contrast extravasation and the measurement of several time and density parameters that may prove to be useful radiological surrogates of hematoma expansion in future ICH studies. Finally, dCTA facilitated the identification of spot signs by allowing us to easily visualize their contrast pooling in later images. Had our study relied on static first-pass arterial weighted CTA, our spot sign rate would have been 35% at 25 seconds post-contrast bolus, and only 19% at 20 seconds post-contrast bolus.Choosing the optimal timing parameters for standard (static) CTA acquisition for detection of contrast leak and assessment of the spot sign is challenging Spot sign is not simply a dot in a hematoma as captured in standard CTA snapshot images; rather, it is a temporally evolving phenomenon Our observed variability of extravasation rate, volumes, density and duration among our spot cases may reflect internal and external factors influencing dspot formation or “rate of leak”. Such factors are not yet well defined, but could relate to point-to-point physiological changes happening intracranially and systemically , hematoma characteristics  and radiographic factors .The dCTA series gives us the opportunity not only to visualize the evolution and spreading of the spot over time but also the luxury of looking backwards. An obvious area of contrast pooling in the later images could be tracked back to its point of origin in earlier phases, which helped us to define confidently the presence, number and location of the spot in all cases. Our experience is consistent with studies that showed increased sensitivity and specificity of detecting spot sign and predicting of hematoma expansion by adding delayed imaging techniques including CTP source images In contrast to abnormal permeability (e.g. tumor perfusion) where contrast leaks from abnormal vasculature in every voxel throughout the tumor, we found dspots all had a point source and expanded 3-dimensionally through the hematoma. This may be compared to an “oil spill” effect where the leakage has a point source followed by multidirectional expansion. In this scenario, the use of color perfusion maps that average out the contrast flow data may not be appropriate to estimate the rate of contrast extravasation in acute ICH.Our study has several limitations. To limit the level of radiation exposure, we acquired low dose (80 kV) images which decreased the signal to noise ratio and potentially introduced more HU measurement errors as compared to standard 120 kV images; HU measurements were not corrected for this decreased dose. For similar reasons, we limited the temporal resolution to 2 seconds during the arterial phase and 5 seconds during the venous phase, which limited the assessment of spot sign flow dynamics. In addition, the patients within our study presented with large baseline hematoma volumes which may have contributed to the relatively high dynamic spot positive rate. Finally, this is a descriptive study describing dynamic spot sign characteristics and proposing measurable spot sign parameters, and was not intended to predict hematoma growth or clinical outcomes. The utility of our proposed spot sign parameters must be tested in a larger sample size, and a prospective study is in progress.In this pilot study, we describe dspot formation and propose quantifiable dspot parameters for future study. We further suggest that dCTA can facilitate the detection of spot signs using comparable contrast and radiation exposures to standard CTA. This technique can potentially improve imaging-based patient selection for emerging ICH therapies.Video S1Sagittal reformatted images show temporal evolution of anterior and posterior spots in the same hematoma.(AVI)Click here for additional data file.Video S2Dynamic 3D MIP bone-subtracted images show temporal evolution of anterior and posterior spots in the same hematoma in a cut out volume.(AVI)Click here for additional data file.


Pattern formation by segregation of cell types is an important process during embryonic development. We show that an experimentally yet unexplored mechanism based on collective motility of segregating cells enhances the effects of known pattern formation mechanisms such as differential adhesion, mechanochemical interactions or cell migration directed by morphogens. To study in vitro cell segregation we use time-lapse videomicroscopy and quantitative analysis of the main features of the motion of individual cells or groups. Our observations have been extensive, typically involving the investigation of the development of patterns containing up to 200,000 cells. By either comparing keratocyte types with different collective motility characteristics or increasing cells' directional persistence by the inhibition of Rac1 GTP-ase we demonstrate that enhanced collective cell motility results in faster cell segregation leading to the formation of more extensive patterns. The growth of the characteristic scale of patterns generally follows an algebraic scaling law with exponent values up to 0.74 in the presence of collective motion, compared to significantly smaller exponents in case of diffusive motion. Understanding pattern formation in various fields of life and especially in the morphogenetic events taking place in developing embryos has always been a subject of interest in biology. Several models have been proposed to explain these events. The Turing theory argues that diffusible morphogens are produced and diffuse forming a heterogeneous steady-state spatial prepattern and thus different local morphogen concentrations react with cells to differentiate them The Murray-Oster mechanochemical model of pattern formation is based on the view that local cellular forces act on the viscoelastic extracellular matrix and other cells thus creating spatial heterogeneity leading to changes in pattern and form Pattern formation in embryonic morphogenesis is a fast process in several species, however the reason for such speed is a long unsolved problem suggesting that several already known but not fully explored mechanisms should act in a synergistic way to accelerate such processes.We demonstrate the existence of a mechanism for pattern formation based on segregation  in which cell migration is a key component in addition to differential adhesion. Cell migration is often seen as persistent random-walk in 2-dimensional cell culture. It is characterized by cell velocity and directional persistence, i.e. how long the cells tend to move linearly before changing direction. If directional persistence is high, groups of cells are more likely to move in a correlated fashion. This correlated or collective migration  is further enhanced by adhesive bonds between individual cells. In computer simulations where cell adhesion strength, cell velocity and correlation between cell velocities are adjustable parameters it has been shown that a population of two initially mixed cell types gradually segregates in homogeneous clusters in a self-propelled manner We argue that collective migration resulting from high directional persistence of individual cells facilitates and accelerates the formation of segregated clusters from a mix of cell types. To support this notion we present in vitro experiments and analyses with mixed cell types where cell velocity and directional persistence are varied or altered by drugs. These experimental variations result in characteristic differences in both the speed and extent of cluster formation by self-propelled segregation. Increased collective cell migration, characterized by larger directional persistence, leads to faster and more extensive cell segregation yielding larger segregated clusters. We demonstrate that the growth of the characteristic scale of the segregated patterns follows an algebraic scaling law with high exponent values showing the accelerating effect of collective cell motion on the segregation process, compared to the lower exponent values resulting from simulations in the absence of collective motion Analogies can also be found between in vitro experiments with mixed 2D cell cultures and cell migration related pattern formation during embryonic development. Exploring the impact of collective migration on pattern formation of cells in 2D culture may also provide new ways for pattern formation in tissue engineering.Collective cell migration is characterized by lateral association of individual cells migrating in a correlated way with high directional persistence. To study the impact of collective migration on cell segregation phenomena we used mixed monolayer co-cultures of keratocyte types from various species and video-imaged the formation of segregated cell clusters. We used primary keratocytes from goldfish, EPC fish keratocyte line  and HaCaT, a human keratocyte line. These keratocyte types can form clusters in a density dependent way and are able to migrate collectively with inherently different parameters of correlated motion. For comparison we also tested C2C12, a mouse myoblast cell line, as these cells tend to migrate as single cells without clustering even at high cell density .The cell types studied have different inherent motility characteristics: they differ in velocity and directional persistence of cell migration. Directional persistence arises from the cell's ability to maintain front-rear polarity during migration. Migrating cells' directional persistence is characterized by their persistence length and persistence time, calculated on the basis of the Orstein-Uhlenbeck model Collective cell migration in 2-dimensional cultures is a density-dependent phenomenon. Beyond a critical density We utilized the inherent motility characteristics of the cell types studied to test the impact of collective migration, characterized by cells' directional persistence, on the segregation of cell types in co-cultures where two cell types were initially mixed. The basic motive of spontaneous cell segregation in such a system is differential adhesion of the participating cell types. If homotypic adhesion strength (i.e. adhesion between cells of the same cell type) is larger than heterotypic adhesion strength (i.e. adhesion between two different cell types) this difference can be the adhesion-based driving force for spontaneous cell segregation here.Although we did not quantitatively study cell adhesion between pairs of the studied cell types it is observed that none of these cell types tends to spontaneously mix with or attach to the other cell type, instead they tend to segregate . TherefoWe thoroughly mixed pairs of cell types after labeling each cell type either red or green using fluorescent cell tracking dyes as indicated for the specific case. Confluent mixed co-cultures were made from keratocyte types: EPC+primary goldfish keratocytes  and EPC+Cluster formation is strongly influenced by the motility characteristics of the participating cell types: their relative speed and persistence length. To further investigate the impact of directional persistence on segregation and cluster formation we changed the directional persistence of cells using NSC-23766: a specific inhibitor of Rac1 activity We tested various concentrations of this Rac1 inhibitor on all cell types studied and found that primary goldfish keratocytes responded to low concentrations (30 µM) of NSC-23766 by altering their motility whereas other cell types were responsive to much higher concentrations (over 150 µM) of the drug (data not shown). Therefore we used primary goldfish keratocytes and EPC keratocytes for tests with Rac1 inhibitor. Higher Rac1 inhibitor concentration (60 µM) in mixed cultures caused prompt rounding up and eventual death for primary goldfish keratocytes but did not affect the motility or morphology of EPC keratocytes.Primary goldfish keratocytes at various densities responded to low concentrations (30 µM) of NSC-23766 by increasing their persistence length while there seemed no change in their morphology or adhesion. As the persistence increasing effect depended on cell density, we measured persistence lengths both in low density cultures of solitary goldfish keratocytes and in high density mixed cultures of goldfish keratocytes and EPC cells. In both settings partial Rac1 inhibition resulted in considerable increase in persistence length . VelocitWe used the persistence length increasing effect of the Rac1 inhibitor to increase the collective characteristic of the migration of primary goldfish keratocytes. We simultaneously monitored cell cluster formation in several untreated or NSC-treated confluent mixed cultures of EPC and primary goldfish keratocytes and compared cluster diameters and areas . Fig. 8 Several untreated (n = 4) and NSC-treated (n = 4) co-cultures of approx. 200 000 cells covering an area were imaged after cluster formation reached maximum . These iTo compare experimental results on the segregation of different cell types with the predicted data of computer simulations d of clusters formed by primary fish keratocytes in mixtures with EPC keratocytes and compared with cluster diameters of HaCaT keratocytes in co-culture with EPC keratocytes. These two cell types were chosen as they tend to form isolated clusters (islands) when mixed with EPC keratocytes that tend to form a network.To compare our 2-dimensional in vitro segregation data with earlier 3-dimensional in vitro data d as segregation evolves. The relationship between 1/γ and d is expected to be linear if the forming clusters have identical shape and size at any given time point throughout the segregation process. This, however, does not hold for our in vitro system where cluster diameters are never uniform but have a non-Gaussian, wide distribution with fat tail (data not shown). This can be the reason why the decay of the segregation index and the increase in cluster diameters are characterized by different exponent values.The calculated segregation index γ is inversely proportional to average cluster diameter Pattern formation in various ontogenetic events such as embryonic development involves several driving mechanisms like differential cell adhesion, cell migration and differentiation instructed by diffusible morphogens or mechanochemical interaction of cells with other cells or the extracellular matrix. These mechanisms must act in a concerted or synergistic way in order to provide for robustness of pattern formation, which is considerably resistant to environmental variations.We demonstrate the existence of an additional patterning mechanism which can speed up and further enhance the robustness of existing and already known mechanisms. This patterning mechanism is based on the different motility characteristics of the pattern forming cell types: cell velocity and directional persistence. It does not require pre-patterned environment and only assumes that adhesion forces between the segregating cell types (heterotypic adhesion) are weak and adhesion forces among cells of their own cell type (homotypic) are strong. If these criteria are met, the speed and extent of cell segregation, measured by the size of segregated homotypic cell clusters, will be strongly influenced by the velocity and ‘collectiveness’ of cell migration, characterized by the directional persistence of migrating cells of each segregating cell type.Our results from experiments comparing cell types with inherently different collective motility characteristics  as well Simulations of cell segregation phenomena use cell adhesion, the velocity and correlation of cells' motion and noise as adjustable parameters having considerable impact on the outcome of segregation both in terms of speed and extent Cells' directional migration is based on their inherent ability to maintain their front-rear polarity during migration. Directional migration is influenced by regulators of actin polymerization such as Rac GTP-ase which promotes the network-type polymerization of actin required for both the advance of the cell's lamellopodium at the front and the formation of a new peripheral lamellopodium when the cell changes direction Embryonic development is very fast in several species including the zebrafish where it takes less than 72 hours to complete Heisenberg and coworkers have recently shown in the developing zebrafish that during epibolic gastrulation the directional persistence and the resultant net speed of mesendodermal cells is regulated by controlling the proportion of different protrusion types: lamellipodium or fillopodium or bleb, formed during migration. Directional persistence is decreased along with an increase in bleb formation, enhanced either by decreasing the membrane to cortex attachment or by increasing the cortical tension of mesendodermal cells. Directional persistence in these cells is regulated independently of instantaneous cell speed Computer simulations coupled with and based on experimental findings with live cells could be a very useful tool for advancing our understanding of cell segregation and pattern formation phenomena.Cell cultures and co-cultures were made of either freshly isolated primary cells or commercially available cell lines. Cells were kept in Dulbecco's Modified Eagle Medium  supplemented with either 10% or 15% fetal calf serum , 4 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 0.25 µg/ml amphotericin B (Sigma) in a humidified incubator with 5% CO2 atmosphere in tissue culture grade Petri dish or 6-well plate (Greiner).EPC fish keratocyte cell line was grown in DMEM 10% FCS at 25°C, HaCaT human keratocyte cell line and C2C12 mouse myoblast cell line were kept at 37°C in DMEM supplemented with 10% or 15% FCS, respectively. The cell lines were passed after brief incubation with 0.5 mg/ml trypsin and 0.2 mg/ml EDTA (Sigma) in PBS .Primary keratocytes were isolated from scales of goldfish (Carassius auratus). Briefly, without sacrificing the goldfish few of its scales were removed then halved and covered with glass coverslip in DMEM 10% FCS in a tissue culture grade Petri dish at 25°C in a 5% CO2 incubator. Keratocytes migrated out of the scales within one day and subsequently they were passed and seeded after incubating in Ca-free PBS (GIBCO) for 4 minutes then suspending in DMEM 10% FCS.EPC cell line and primary fish keratocytes were grown at 25°C and imaged at 33°C whereas other cell lines were kept at 37°C.Time-lapse recordings were performed on either a Zeiss or a Leica system.Time-lapse recordings with phase contrast and double fluorescence applications were performed on a Zeiss Axio Observer Z1 inverted microscope with 10× EC-Plan Neofluar objective coupled to a Zeiss Axiocam MRM CCD camera and equipped with a Marzhauser SCAN-IM powered stage. For epifluorescent excitation Zeiss Colibri LED illumination system was used. Power stage positioning, focusing, illumination, primary image collection and stitching of primary images into mosaics (2×2 or 5×7) were controlled by Zeiss Axiovision 4.8 software and a custom-made experiment manager software module on a PC.For time-lapse recordings with phase contrast and single fluorescence applications a computer-controlled Leica DM IRB inverted microscope equipped with a Marzhauser SCAN-IM powered stage was used with a 10× N-PLAN objective . This microscope was coupled to an Olympus DP70 color CCD camera. Zeiss HXP 120 metal-halide illumination system was used for fluorescent excitation. A time-lapse experiment manager software (CellMovie) controlled the field of view and plane positioning, illumination and image acquisition on a PC.Cell cultures were kept at 37°C or 33°C in humidified 5% CO2 atmosphere in tissue culture grade Petri dishes (Greiner) in computer-controlled wide-screen microscope stage incubators (CellMovie) mounted on the powered stage of both microscopes.Phase contrast and epifluorescent images of cells were collected consecutively every 10 minutes from each of the microscopic fields (n = max. 6) for various durations ranging from 12 to 48 hours. Images were edited using NIH ImageJ software.Green and red fluorescent cell tracker dyes were used for labeling and long-term separate imaging of cell types in mixed co-cultures. Membrane labeling green DiO (Invitrogen) was used to label cells excited at 468–475 nm with emissions filtered at 511–542 nm. Cytoplasm staining red CMPTX (Invitrogen) were used with 554–572 nm excitation filter and 597–675 nm emission filter. For double epifluorescence, separate excitations and emissions for either green or red dyes were provided by double band pass filters and double beam splitter arranged in a single filter cube , consecutively illuminated at the corresponding wavelengths with separate LEDs by Zeiss Colibri illumination system. The resulting single 8-bit images were subsequently assigned original dye colors and merged using NIH ImageJ software. The illumination system configured this way provided for overlap-free imaging of green DiO and red CMTPX.For single fluorescence the membrane labeling red DiI (Invitrogen) was used with single band pass excitation (530–560 nm) and emission (572–647 nm) filters .The drug NSC-23766 (Merck) was used in 5–100 µM concentrations in the standard medium to inhibit the small GTP-ase Rac1.dt as:i(t)X denoting the center of cell i at time t, 0t is an arbitrary reference frame of the image sequence analyzed. The empirical d(t) curves indicate a persistent random walk behavior in monolayer cell cultures studied. Based on the Ornstein-Uhlenbeck model pT is the persistence time and D is the diffusion coefficient of the long-term random behavior v, of a given cell i at time t was calculated ast is the difference of two consecutive steps of cell tracking and thus the time resolution data acquisition. To characterize the motility of an ensemble of cells, time-dependent average velocity v(t) was calculated asN(t) cell being in the cell population. Average velocity, v, was calculated by averaging v(t) over all time steps of cell tracking asv, is approximately equal to persistent speed, S, if the time resolution of tracking, Δt, is much smaller than persistence time, pT, however if Δt is comparable with or larger than pT, the directly measured average velocity is characteristically lower than persistent speed. The error of the manual tracking procedure is estimated to be ∼5 µm, which can also result in differences between the calculated average velocity and persistent speed for cases where Δt is smaller than pT while cell speed is low.Average cell velocity was also calculated directly from cell displacements in consecutive steps of cell tracking, summarized in 0R denotes the average diameter of clusters and K is a factor that is usually unknown as it depends on the distribution of the cluster shapes. By fitting a polynomial to the correlations we obtained the typical size of a cluster.To characterize the average size of clusters, the correlation length of two-point correlations was used. Only the pixels above a threshold were considered. First, we determined the two-point correlations for both colors in a given range (0–200 µm) and calculated the diameter of the clusters. For small distances, the two-point correlation can be approximated as a quadratic form of the distance:2) that do not participate in the segregation process. For these reasons, we applied a cutoff and we calculated the average area only of those clusters that were larger than 10000 µm2.We determined the average area of clusters. We used a simple depth-first algorithm to identify clusters and count their size. Since live cell images are generally noisy, simply counting the average size of clusters would result in very small values that could bias the analysis of the effect studied. Furthermore, during the experiments some unviable cells die forming small clusters , EPC fish keratocyte line, HaCaT human keratocyte line, C2C12 mouse myoblast cell line.(MPG)Click here for additional data file.Video S2Density dependent increase in the directional persistence of primary fish keratocytes. Phase contrast time-lapse video showing low density (left) and high density (right) primary fish keratocyte (PFK) cultures. Note the large increase in the directional persistence of the collectively migrating cell cluster in high density culture compared to single cells.(MPG)Click here for additional data file.Video S3Density dependent increase in the directional persistence of HaCaT keratocytes. Phase contrast time-lapse video showing low density (left) and high density (right) HaCaT keratocyte cultures. Note the increase in the directional persistence of cells in the confluent streaming monolayer compared to random migration of single cells at low density.(MPG)Click here for additional data file.Video S4Spontaneously segregating cell type pairs with low heterotypic adhesion and high homotypic adhesion. Merged phase contrast + fluorescent time-lapse video showing mixed co-cultures: primary goldfish keratocyte (PFK) + EPC keratocyte (red) (left) and HaCaT + EPC keratocyte (red) (right). Note the growth of homotypic cell clusters in both mixed co-cultures and the formation of the interface between the cell types.(MPG)Click here for additional data file.Video S5Spontaneously segregating primary fish keratocytes and EPC keratocytes in mixed co-culture. Merged double fluorescent+phase contrast time-lapse video showing a segregating co-culture of primary goldfish keratocytes  + EPC keratocytes (green). Note the fast growth of homotypic cell clusters.(MPG)Click here for additional data file.Video S6Spontaneously segregating EPC keratocytes and HaCaT keratocytes in mixed co-culture. Merged double fluorescent + phase contrast time-lapse video showing a segregating co-culture of EPC keratocytes (red) + HaCaT keratocytes (green). Note the growth of homotypic cell clusters.(MPG)Click here for additional data file.Video S7Spontaneously segregating HaCaT keratocytes and C2C12 myoblasts in mixed co-culture. Merged double fluorescent+phase contrast time-lapse video showing a segregating co-culture of HaCaT keratocytes (green) + C2C12 myoblasts (red). Note the slow growth of homotypic cell clusters.(MPG)Click here for additional data file.Video S8Inhibition of Rac1 enhances spontaneous segregation of primary fish keratocytes and EPC keratocytes in mixed co-culture. Merged double fluorescent + phase contrast time-lapse video showing a segregating co-culture of primary goldfish keratocytes  + EPC keratocytes (green) not treated (left) or treated with 30 µM NSC-23766 Rac1 inhibitor (right). Note the faster growth of cluster size of primary goldfish keratocytes treated with Rac1 inhibitor.(MPG)Click here for additional data file.