Odocoileus virginianus, a North American species, were successfully introduced in Finland in 1934 and the population has since been growing rapidly, but remained in complete isolation from other populations.Within-population genetic diversity is expected to be dramatically reduced if a population is founded by a low number of individuals. Three females and one male white-tailed deer Based on 14 microsatellite loci, the expected heterozygosity H was 0.692 with a mean allelic richness (AR) of 5.36, which was significantly lower than what was found in Oklahoma, U.S.A. , demonstrating that a bottleneck occurred. Observed H was in line with predictions from an individual-based model where the genealogy of the males and females in the population were tracked and the population's demography was included.Our findings provide a rare within-population empirical test of the founder effect and suggest that founding a population by a small number of individuals need not have a dramatic impact on heterozygosity in an iteroparous species. For example, there is a positive relationship between the size of a population and its genetic diversity across populations Odocoileus virginianus, a North-American species, was founded in Finland in 1934 by three females and one male. A second introduction was attempted in 1948 . This area belongs to the core distributional area of the white-tailed deer in Finland. Hunters cut a small piece of meat and place it into a small plastic bag along with information sheet about the animal and time and location. No hunters were encouraged or financially rewarded to collect samples to be used in this study. White-tailed deer hunting in Finland is state regulated (Hunting Act of 1 August 1993 (615/1993) with subsequent amendments; unofficial translation to English, URL http://www.finlex.fi/en/laki/kaannokset/1993/en19930615.pdf). Hunting groups are allowed to cull a particular number of adult male, female and fawn white-tailed deer, the number of which is decreed by the Ministry of Agriculture and Forestry. All animals culled fell under the license provided to the hunting groups. Animals were culled on private land, under permission of the land owner. In the first winter 79 samples were collected and in the second winter 100.Sampling of the white-tailed deer was done in south western Finland during two hunting seasons in winters 2009–2010 and 2010–2011. Samples were obtained by local hunters who shot the animals and collection involved several hunting groups operating over an area of approximately 400 km2O was used to elute DNA in the last step. The samples were amplified in Polymerase Chain Reaction (PCR) for twenty one microsatellite loci in four parallel panels. PCR was conducted with Phusion Flash master mix (Finnzymes), where one PCR reaction contained 5 µl of master mix-solution, 2 µl of extracted DNA, 1 µl of dH2O and 2 µl of primer mix. Panel 1 included primers INRA011 DNA was extracted from a small piece of meat following the method described in The loci that were successfully amplified were checked for Hardy-Weinberg equilibrium and for linkage disequilibrium with software Genepop 3.4 We aimed to compare our results to a study from Oklahoma USA, which was based on 72 fully genotyped individuals Expected heterozygosity and allelic richness were estimated using software FSTAT 2.9.3 A non-parametric Mann-Whitney U-test test was carried out using the program SPSS 17.0  to test whether the white-tailed deer in Finland and in Oklahoma USA differed in expected heterozygosity or allelic richness.We constructed a model which takes into account the demography and genealogy of the introduction of the white-tailed deer population. The model was based on tracking individual-specific genotypes in order to estimate expected heterozygosity across a number of scenarios. Because the composition of the founder population is not known, the initial population consisted of 3 females and 1 male with each 14 loci with an allelic richness equal to that observed in the population (see below for a scenario where this assumption was relaxed). Because information on the heterozygosity of founders is lacking, we considered both a scenario of maximal heterozygosity (H = 1) and one where heterozygosity was minimal given the number of alleles per locus. Individual-specific information on reproduction and survival was available for 1934–1938 and was included in the model in detail. Starting 1939, all females mated with a randomly-chosen male and reproductive output was assumed to follow age-specific probability values for producing 0, 1 or 2 fawns Apart from the above described scenario (scenario A) with maximal and minimal heterozygosity of founders, we further explored two alternative scenarios. A bottleneck reduces the allelic richness per locus, especially for loci with high allelic richness. Hence, the current allelic richness is a minimum of what the allelic richness in the founders would have been. In scenario B, we therefore assumed allelic richness in the initial population was maximal  which entails that the heterozygosity of the founders was 1. In scenario C, a putative second introduction was implemented in the model by adding 1 male and 3 females, each with a completely heterozygous, unique and novel genotype, at time step 15 ( = 1949). See One male and four female white-tailed deer calves were released in Finland in 1934 and were kept in an enclosure until 1938. One female died before reproducing in 1937 and the maximal number of individuals in the first introduction therefore was four . The four population founders were tame and the population stayed in the vicinity of the release site during the initial years. Detailed information of individual reproduction and survival was available during the period the animals were in the enclosure . EstimatR: Z = −2.565, one tailed p = 0.0045; HE: Z = −1.654, one tailed p = 0.052). Based on results from the program Bottleneck the founding event was seen in the Finnish populations . However, the graphic test gave a normal L-shaped distribution. Garza-Williamson gave a test statistic of 0.70 which is not under the threshold value (0.68) reported in the literature for a bottlenecked population.Not all loci amplified successfully. Loci ILSTS011, OCAM, BovPRL, BM415, BM4208, R and P had a success rate of less than 10% and were not used. The remaining 14 loci were found to be in Hardy-Weinberg equilibrium and unlinked. Locus-specific values are presented in Under the chosen parameter values, the dynamics of the simulated population resembled the available information on the abundance of white-tailed deer after its introduction in Finland . The whiIn general, the population sizes predicted by the model under the assumed reproductive and vital rates closely resembled the estimates of white-tailed deer population available in the literature . However, the difference in heterozygosity was minimal , although this reduction was marginally significant. In terms of classic population genetic theory , this hiThe main strength of our study is that it concerns a large mammal introduced from across the Atlantic. We can therefore be confident that the population has experienced no gene flow from another population and can exclude unmonitored re-stocking event of the introduced population. The only re-stocking on record consisted of four 1-year olds released in 1949. These individuals are thought to not have survived for a long period after their release A second caveat concerns our comparison of genetic diversity between Finland and Oklahoma U.S.A. The white-tailed deer introduced in Finland were from Minnesota. Since there was no published information on genetic diversity of white-tailed deer in Minnesota, we considered Oklahoma a good substitute. In Oklahoma , which has been conducted to investigate at which level the population size needs to be in the future to retain certain proportion of genetic diversity of today Ovis aries founded by only 2 individuals harbours a higher heterozygosity than predicted. Their hypothesis was that selection may have increased heterozygosity in this population above the predicted level. This study, however, only considered the consequences of the founders having variable  heterozygosity under the assumption that the founders had minimal (i.e. currently observed) allelic richness and did not study, as we did here, the consequences of founding a population with maximal allelic richness. Because loss of alleles leads to a reduction in heterozygosity Despite these recognised differences between classic and individual-based population genetic models, relatively few studies have taken an individual based approach to simulate extensively how diversity in populations can change. Examples include a simulation study of genetic diversity of copper redhorse introduction scheme of a large mammal, such as the white-tailed deer, should prioritise investing its likely limited resources in maximising the initial population growth of the introduced individuals. Prioritizing population growth could mean that financial resources are invested in restoring habitat, in providing the introduced individuals protection from predators or in providing them supplementary food rather than financially investing in introducing more individuals to increase the genetic diversity of founders. The case study of the introduction of the white-tailed deer in Finland clearly shows that a population founded by very few individuals can be highly successful provided it rapidly increases in size.In conclusion, founding a population on the basis of few iteroparous individuals may have little consequences for its genetic diversity, at least in terms of heterozygosity. This may seem surprising on the basis of general population genetic theory Table S1Extended tabulated summary of available information on the development of the population of white-tailed deer in Finland.(DOCX)Click here for additional data file.Table S2Reproduction and survival values, as implemented in the individual-based genetic model.(DOCX)Click here for additional data file.Table S3White-tailed population development and application of classic population genetic theory ( in the main text).(DOCX)Click here for additional data file.Table S4Locus-specific allele frequencies of the Finnish population, presented for both the 72 individuals of this study and for the maximal number of individuals available per locus.(DOCX)Click here for additional data file.Text S1Introduction of the white-tailed deer in Finland. Details of its introduction and further information on rationale behind model parameters.(DOCX)Click here for additional data file.Programme S1Zipped archive with MATLAB M-files forming the individual-based population genetics model used in this study to simulate the introduction of the white-tailed deer in Finland.(ZIP)Click here for additional data file.


Because functionally important sites are under stronger purifying selection, site-specific substitution rates tend to be lower than usual at these sites. A large number of phylogenetic models have been developed to estimate site-specific substitution rates in proteins and the extraordinarily low substitution rates have been used as evidence of function. Most of the existing tools, e.g. Rate4Site, assume that site-specific substitution rates are independent across sites. However, site-specific substitution rates may be strongly correlated in the protein tertiary structure, since functionally important sites tend to be clustered together to form functional patches. We have developed a new model, GP4Rate, which incorporates the Gaussian process model with the standard phylogenetic model to identify slowly evolved regions in protein tertiary structures. GP4Rate uses the Gaussian process to define a nonparametric prior distribution of site-specific substitution rates, which naturally captures the spatial correlation of substitution rates. Simulations suggest that GP4Rate can potentially estimate site-specific substitution rates with a much higher accuracy than Rate4Site and tends to report slowly evolved regions rather than individual sites. In addition, GP4Rate can estimate the strength of the spatial correlation of substitution rates from the data. By applying GP4Rate to a set of mammalian B7-1 genes, we found a highly conserved region which coincides with experimental evidence. GP4Rate may be a useful tool for the  To understand how a protein functions, a critical step is to know which regions in its protein tertiary structure may be functionally important. Functionally important protein regions are typically more conserved than other regions because mutations in these regions are more likely to be deleterious. A number of phylogenetic models have been developed to identify conserved sites or regions in proteins by comparing protein sequences from multiple species. However, most of these methods treat amino acid sites independently and do not consider the spatial clustering of conserved sites in the protein tertiary structure. Therefore, their power of identifying functional protein regions is limited. We develop a new statistical model, GP4Rate, which combines the information from the protein sequences and the protein tertiary structure to infer conserved regions. We demonstrate that GP4Rate outperforms Rate4Site, the most widely used phylogenetic software for inferring functional amino acid sites, via simulations with a case study of B7-1 genes. GP4Rate is a potentially useful tool for guiding mutagenesis experiments or providing insights on the relationship between protein structures and functions. An important question in biology is the identification of functional residues in proteins. This information can help us understand the relationship between protein structures and functions as well as guide us to design new proteins by genetic engineering. However, experimental techniques for identifying functional sites, e.g. mutagenesis, are time consuming and expensive, which prohibits the brute force scanning of functional sites by experiments. Therefore, bioinformatics tools are useful, because they can narrow down the candidate sites for experimental investigation. Evolution operates similar to a high-throughput mutagenesis experiment: spontaneous mutations introduce protein variants in each generation and then the functional effects of the spontaneous mutations are “measured” by natural selection A number of bioinformatics tools based on phylogenetics have been developed to infer functional sites by the simple idea that functionally important amino acid sites tend to be more conserved than unimportant ones However, these existing methods are far from flawless. Most of the popular methods, e.g. Rate4Site dN/dS ratios at the codon level, to overcome the drawbacks of the i.i.d. assumption dN/dS ratio at a focal site is approximated by the average substitution rate in a set of neighbor sites in the protein tertiary structure Several methods have been developed to incorporate the spatial correlation of evolutionary patterns, e.g. substitution rates at the protein level or dN/dS ratios over a protein tertiary structure. This model solved many problems of the sliding window framework. However, the prior distribution in Watabe and Kishino's model is unnormalized Very recently, a Bayesian model which combines the Potts model in statistical physics and the phylogenetic model has been proposed by Watabe and Kishino to infer protein patches under positive selection in protein tertiary structures Here we propose to incorporate a Gaussian process with the phylogenetic model to overcome the drawbacks of the existing methods. The Gaussian process has been widely applied in geostatistics and machine learning to capture the spatial correlation of interesting features We name this kind of hybrid model of Gaussian processes and phylogenetics as a phylogenetic Gaussian process model (Phylo-GPM). In the Phylo-GPM framework, the spatial correlation of substitution rates can be naturally described and the strength of spatial correlation can be learned from the data. Therefore, it overcomes the common drawback of the sliding window methods that the window size must be manually specified. Unlike Watabe and Kishino's model Simulations were implemented to evaluate the performance of GP4Rate and to compare it with the widely used software, Rate4Site To generate simulated alignments, we need a phylogenetic tree to describe the evolutionary relationship between simulated sequences, a protein structure to calculate the pairwise Euclidean distances between sites, a substitution model, and a vector of substitution rates. Note that the following discussions will be mainly based on the substitution rates rather than their log values. A simple phylogenetic tree was used in all simulations . The treIn the 2D toy protein model, the protein tertiary structure was described by a 20 by 20 regular 2D grid, in which each dot corresponds to an amino acid in the toy protein structure . In addi heatmap . TherefoTwo different spatial configurations of site-specific substitution rates were used in the 2D toy protein simulations. In the first configuration, the 20 by 20 grid was divided into 4 non-overlapping blocks, each of which was a 10 by 10 grid . Sites wA program based on Bio++ We firstly randomly sampled two simulated alignments, one for each configuration, as examples to get insights on the performances of GP4Rate and Rate4Site. As shown in versus the average false positive rates using the ROCR library in R To quantitatively evaluate the performance of GP4Rate and Rate4Site, we used receiver operating characteristic (ROC) curves to measure the power of the two methods. ROC curves are widely used to evaluate the accuracy of binary classifiers. The area under a ROC curve is usually used as a measure of the power of the corresponding method. To apply ROC curves to the simulated datasets, we must divide the amino acid sites into two categories, functional sites and nonfunctional sites, before generating simulated alignments. The functional sites are used as true positives while the nonfunctional sites are used as true negatives. In the 2D toy protein simulations, functional sites evolved at the lower rate (0.2) while nonfunctional sites evolved at the higher rate (1.8). Then, the ROC curves were created by plotting the average true positive rates i, respectively. The log values of site-specific substitution rates are used in the right-hand side of ROC curves measure whether a model can distinguish slowly evolved functional sites from the other sites. If a model can assign relatively low substitution rates to slowly evolved sites and relatively high rates to the other sites, it will perform well in the evaluations based on ROC curves. However, ROC curves cannot capture potential systematic biases of the model. For example, if the model adds a constant bias to the site-specific substitution rates, its ROC curves will be exactly the same regardless of the magnitude of the constant bias. Therefore, we used a simple loss function complementary with the ROC curves to capture any potential systematic biases of the estimated site-specific substitution rates. The loss function is defined by the following formulaWe plotted the losses of both GP4Rate and Rate4Site in the 2D toy protein simulations. As shown in GP4Rate has two hyperparameters, i.e. the characteristic length scale In summary, when spatial correlation of substitution rates exists in proteins, GP4Rate always outperforms Rate4Site. However, the spatial correlation of site-specific substitution rates may be insignificant in some proteins. Therefore, we also evaluated both GP4Rate and Rate4Site in simulated alignments in which the spatial correlation of site-specific substitution rates is absent. These simulated alignments were generated by randomly shuffling the columns in each alignment in the first spatial configuration of substitution rates . The perWe generated a second set of simulated alignments based on more realistic assumptions. The basic idea is that if we have a large number of highly diverged sequences, a simple method which does not consider the spatial correlation of substitution rates may accurately estimate the site-specific substitution rates because of the rich information in a very large dataset. We may generate simulated alignments based on the real protein tertiary structure and the presumably accurately estimated site-specific substitution rates. These simulated alignments may have similar features as real proteins.In this set of simulations, we used the same phylogenetic tree  and the −4). Therefore, GP4Rate still outperforms Rate4Site in the realistic simulations.To evaluate the performance of GP4Rate and Rate4Site by ROC curves, we divided the sites into two categories before generating simulated alignments: slowly evolved functional sites and others. Based on the site-specific substitution rates reported by ConSurf-DB, the 10 percent most slowly evolved sites were considered to be functional while the others were not. As shown in The B7-1 (CD80) family is a member of the immunoglobulin superfamily (IgSF) and is critical for the regulation of immune responses We used the human B7-1 protein structure  In addition to modeling the spatial correlation of site-specific substitution rates, protein tertiary structures have been used to improve phylogenetic models and the estimation of site-specific substitution rates in a few other studies The Phylo-GPM framework proposed in this paper may be used as a general tool to model the spatial correlation of patterns in the protein tertiary structure. The phylogenetic hidden Markov model (Phylo-HMM) is a popular method which combines the hidden Markov model and statistical phylogenetics http://info.mcmaster.ca/yifei/software.html. GP4Rate combines the protein alignment and the protein tertiary structure to infer groups of close-located functional sites evolved at low rate. We assume that the protein alignment, the phylogenetic tree, and the tertiary structure of one protein in the alignment are provided by users. In GP4Rate, both the topology and the branch lengths of the phylogenetic tree are fixed to improve the speed of the program. In addition, we assume that the protein sequences in the alignment belong to the same gene family and have very similar functions, which implies that the functionally important sites do not vary among sequences and the site-specific substitution rates do not change over time. However, we do assume that the substitution rates can vary across different sites. The site-specific rates are used as proxies of functionality: very low substitution rates suggest the corresponding sites are functionally important.GP4Rate is an open-source software application written in C++ and its source code is freely available from In most molecular phylogenetic programs, e.g. Rate4Site i-th column, and i, which is a function of the site-specific log substitution rate at site i. In GP4Rate, the parameters are estimated using the Bayesian principle. In Bayesian statistics, the parameters are random variables and the conditional distribution of parameters given data, i.e. the posterior distribution, gives us an estimation of parameters. For simplicity of presentation, first we focus on the vector of site-specific log substitution rates, which is the collection of log values of substitution rates at all amino acid sites, and defer the discussions on the other parameters. The posterior distribution of the vector of log site-specific substitution rates can be defined by the following equation,A realistic i and site j in the protein tertiary structure. i and site j are the same site and equal to 0 otherwise. The covariance function contains two free parameters, As mentioned above, ribution . In the In addition to the Matérn 1.5 covariance function, GP4Rate has two alternative covariance functions for users to choose. One is the Matérn 2.5 covariance function,The three covariance functions are all special cases of the general Matérn covariance function The hyperparameters in the covariance functions, i.e. i before the interpolation. By default, GP4Rate calculates and caches the site-specific log likelihoods at 4000 evenly spaced substitution rates, ranging from To fully define the unnormalized posterior distribution , the likGP4Rate uses MCMC simulations to sample parameters from their posterior distribution. The algorithm follows previous studies by Neal To update Instead of updating In practice, the update of Table S1The list of most slowly evolved sites predicted by GP4Rate and Rate4Site in the case study of B7-1 genes.(XLS)Click here for additional data file.Text S1The comparison of GP4Rate and Rate4Site in the simulations without spatial correlation of substitution rates and the Bayesian model comparison of GP4Rate and Rate4Site in the case study of B7-1 genes.(PDF)Click here for additional data file.


Microviridae (a family of ssDNA bacteriophages) might play an important role in a broad spectrum of environments, as they were found in great number among the viral fraction from seawater and human gut samples. 24 completely sequenced Microviridae have been described so far, divided into three distinct groups named Microvirus, Gokushovirinae and Alpavirinae, this last group being only composed of prophages. In this study, we present the analysis of 81 new complete Microviridae genomes, assembled from viral metagenomes originating from various ecosystems. The phylogenetic analysis of the core genes highlights the existence of four groups, confirming the three sub-families described so far and exhibiting a new group, named Pichovirinae. The genomic organizations of these viruses are strikingly coherent with their phylogeny, the Pichovirinae being the only group of this family with a different organization of the three core genes. Analysis of the structure of the major capsid protein reveals the presence of mushroom-like insertions conserved within all the groups except for the microviruses. In addition, a peptidase gene was found in 10 Microviridae and its analysis indicates a horizontal gene transfer that occurred several times between these viruses and their bacterial hosts. This is the first report of such gene transfer in Microviridae. Finally, searches against viral metagenomes revealed the presence of highly similar sequences in a variety of biomes indicating that Microviridae probably have both an important role in these ecosystems and an ancient origin.Recent studies suggest that members of the  Microviridae family is one of the most commonly retrieved.Viruses, particularly bacteriophages (viruses of bacteria), are the most numerous biological entities on Earth, retrieved from all sorts of biomes . Among these, phages with double-stranded DNA (dsDNA) genomes have been the most thoroughly studied Microviridae are small icosahedral viruses with circular single-stranded DNA genomes. This family has been thoroughly studied from numerous perspectives – from virion structure and assembly Microvirus) and gokushoviruses (subfamily Gokushovirinae) Alpavirinae, was found through bacterial genome analysis Microvirus exclusively infect Enterobacteria and have been extensively studied through the archetype of this familly, the bacteriophage ΦX174 Gokushoviruses are currently known to infect only obligate intra-cellular parasites, members of bacterial genera Chlamydia, Bdellovibrio and SpiroplasmaGokushovirinae genomes are currently available: 6 Chlamydia phages and 1 genome assembled from seawater viromes Bdellovibrio phage phiMH2K Spiroplasma phage 4 Alpavirinae is restricted to prophages residing in the genomes of bacteria belonging to two genera of the phylum Bacteroidetes: Prevotella and Bacteroides. The latter study was the first to implicate the Microviridae in lysogenization of their hosts and also to associate this virus group with BacteroidetesMicroviridae-like sequences were found in large numbers in different ecosystems, ranging from microbialites Microviridae-like sequences. As the known members of the Microviridae family exhibit small genomes (3–7 kb), two complete Microviridae genomes could be assembled from the Sargasso Sea virome Microviridae viruses in the environment, we reanalyzed a set of previously published viromes by assembling the reads from each of these viromes and then searching the resultant contigs for the presence of complete genome sequences related to Microviridae. We were able to assemble 81 complete circular genomes related to members of the Microviridae from 95 public viromes. Phylogenetic and genomic organization analyses of these new viruses revealed a new Microviridae subgroup (the Pichovirinae), enriched the genome collection of Gokushovirinae, and, for the first time, confirmed the existence of extrachromosomal complete genomes from Alpavirinae virion particles. Microviridae core genes could be more thoroughly studied, especially the structure of the major capsid protein. Horizontal gene acquisition events are also documented for the first time in this viral family. Finally, as the viromes analyzed in the current study cover a wide range of ecosystems, the distribution of the new genomes inside the Microviridae tree provides a better understanding of both the diversity and the evolution of Microviridae family.To gain insights into the diversity of the Microviridae are found in a large number of viromes, assembly of complete Microviridae genomes was described in only one dataset, the Sargasso Sea virome Microviridae was first created by Angly et al., Microviridae were assembled in a recent analysis of the same dataset using up-to-date assembly software Microviridae-like circular genomes. As a result, 81 contigs representing putative complete Microviridae genomes were obtained from 25 out of the 95 viromes tested .Even though sequences from s tested . Out of equences  but the equences , with thMicroviridae sequences were used as queries in searches against the complete bacterial genomes from the NCBI and the Human Microbiome Project Prevotella multiformis strain, highly similar to other Microviridae prophages detected in Prevotella.In order to detect potential new prophages , the newly assembled Microviridae family and its genome evolution, a phylogenetic tree was computed from the major capsid protein (VP1) sequences and correlated with the corresponding genome maps , while the fourth one is exclusively composed of genomes generated in this study. The assembled viromes considerably expanded the available complete viral genome pool for Gokushovirinae and Alpavirinae, while not a single new virus was affiliated to the genus Microvirus. These four groups are further described below:Four well-supported clades  are formed on the VP1 phylogenetic tree: three of these correspond to the previously described taxonomic groups  detected in this study is more similar to prophages from other Prevotella genomes. Interestingly, a new Microviridae, generated from free viral particles from a human lung sample, is interspersed on the tree among these Prevotella prophages. Bacteroides prophages form a monophyletic group with 11 new genomes sampled from human gut flora.The Gokushovirinae group , 12 from freshwater lakes (11 from Lake Bourget and 1 from Lake Pavin), 1 from marine environment , 1 from human lung (SectLung2LLL_002) and 1 from microbialites . This group can be internally divided : Gokushovirinae assembled from aquatic samples are most closely related to Bdellovibrio phage ΦMH2K, whereas sequences assembled from human gut samples are divided into two subgroups, one around Spiroplasma phage 4 and Microvirus ΦCA82, and another group close to Chlamydia phages.The new group .The Microviridae genomes were composed of 3 to 9 predicted genes. These gene numbers are consistent with the known reference genomes, and were similar for each subgroup. The Microviridae core genes  are detected in all Microviridae genomes but two  being significantly similar to those of Microviridae from other genera/subfamilies. Two new genomes (Human_gut_24_085 and Human_gut_32_015), sampled from two different individuals, lack an ORF significantly similar to VP2, but instead possess a similarly-sized ORF at a position equivalent to that occupied by VP2 in all other members of the Alpavirinae , with the exception of Spiroplasma phage 4, Microvirus ΦCA82, Sargasso sea phage ssΦ2 and 12 new genomes from human gut, for which VP3 and/or VP5-like genes could not be identified using standard sequence analysis methods. However, upon a closer examination using a sensitive profile-profile comparison algorithm implemented in FFAS03 Chlamydia phage Chp2 scaffold protein VP3 superfamily, pfam id : PF09675; FFAS03 mean score: −29.2). An internal separation of the Gokushovirinae assembled in this study into two subgroups can be deduced from the gene order conservation within these subgroups, consistently with the phylogenetic information. A first subgroup is found near Bdellovibrio phage ΦMH2K, and encompass only genomes assembled from aquatic environments . This subgroup displays a specific gene order : VP4-VP5-VP1-VP2-VP3  is found near the known Chlamydia phages, and presents a gene order similar to the aquatic Gokushovirinae assembled in this study . Nevertheless, Pichovirinae genomes are the only ones within the Microviridae family where the gene order of the core genes is altered: whereas all Microviridae present a VP4 - VP1 - VP2 organization, the gene order in all Pichovirinae is VP4 - VP2 - VP1 , only those of microviruses are decorated with pentameric major spike protein complexes positioned at each of the 12 five-fold vertices Microviridae revealed that the average identity at the protein level varies from 40 to 80% within a group, and from 20 to 40% between the groups, with an exception of the Enterobacteria-infecting phages, which generally present no significant sequence identity with MCPs from other clades of the Microviridae . ΦX174-like microviruses possess smaller MCPs (average length 427 aa), while those of gokusho-, alpa- and pichoviruses are significantly larger  and Alpavirinae (from 45 to 209 aa). Interestingly, this variation appears to be ecosystem- rather than virus subgroup-dependent. The insertion length variation was much less pronounced for VP1 proteins of viruses residing in aquatic environments (including both gokushoviruses and pichoviruses) than it was for viruses from human samples   . The samAlpavirinae . In order to verify whether such extensive insertions would not interfere with normal virion formation, we fitted our three-dimensional model of the alpaviral VP1 into the pseudoatomic model of SpV4 (PDB ID:1KVP) and mapped the location of all the insertions exceeding 15 aa. We identified 6 MCP hot-spots where large insertions were tolerated in alpaviruses . Nevertheless, the three conserved motifs of superfamily I rolling cycle replication proteins are all conserved . The ΦX174 protein H is a multifunctional structural protein (12 copies per virion) required for piloting the viral DNA into the host cell interior during the entry process, and de novo synthesis of protein H is required for efficient production of other viral proteins Gokushovirinae, Enterobacteria phages, Alpavirinae and Pichovirinae,Chlamydia phages  of this protein might be tightly linked to the identity of the host.The last gene retrieved in all Microviridae. Analysis of the complete microviral genomes assembled in this study has unexpectedly revealed 11 genes from human-associated Microviridae . Gokushovirinae peptidases are affiliated to Firmicutes : Faecalibacterium prausnitzii (4 of 5), and Gamma-proteobacteria . These closest homologues of the microviral proteins are found next to phage-like genes in several bacterial genomes  and a gene of unknown function, with a homologue present as part of the lysis gene cluster in Streptococcus phage 858  in the Human_gut_33_003 genome is encoded on the complementary strand. Such orientation is highly unusual; to our knowledge, no other cases of complementary strand genes have been reported in Microviridae.The closest homologues detected by BLAST for these 11 genes are from bacterial genomes, except for Human_feces_A_016, for which the closest homologue is found in a tailed dsDNA phage genome . Half of genomes . For exaiophages . Notablyhage 858 . All thiMicroviridae phages, a phylogenetic tree was computed from a multiple alignment including the Microviridae peptidases and their closest homologues in both bacterial and viral genomes  and the five peptidase genes from Gokushovirinae (in blue). Within the Alpavirinae, the peptidase tree topology can be associated with the location of the peptidase gene integration within these viral genomes. Peptidases genes are inserted in two different positions in Alpavirinae genomes : between VP2 and VP4 , and between VP4 and VP1 (Human_feces_A_016 and Human_gut_33_005)  and Proteobacteria . The most likely explanation for such clustering is that peptidase genes were horizontally acquired by several members of the Microviridae on multiple occasions. The presence of dsDNA phage peptidases near the Microviridae sequences on the tree suggests that ds and ssDNA phages might be engaged in gene exchange, either directly during a co-infection or via infection of a prophage-bearing host cell.In order to shed light on the evolutionary event(s) leading to the acquisition of M15 peptidase genes by  genomes , Fig. S9_33_005) . ConsistVP1 tree , Fig. 1.Microviridae genomes was investigated in light of this acquisition of a peptidase gene by several human gut Microviridae. For that, we performed a phylogenetic analysis of the two most conserved Microviridae proteins, VP1 and VP4, for each of the four subgroups . No signs of recent gene transfer event could be detected, confirming the hypothesis that gene transfer between Microviridae are rare, even within the temperate members of the group Finally, the possibility of horizontal gene transfer between Microviridae genomes could be assembled  and in human microbiome : genomes noted as “Human feces”, sampled in South Korea Microviridae, which would have colonized a wide range of habitats, from human microbiome to seawater, freshwater, and sedimentary structures like Microbialites.The wide distribution of samples from which complete ssembled  makes itMicroviridae genomes from random environmental sequences requires a large number of reads, especially for viromes composed of reads not exceeding 100 bp. Thus, only 3 complete Microviridae genomes have been generated from the three viromes with reads of ∼100 bp  sequences was built encompassing all published and newly assembled Microviridae. These sequences were used to search for VP1 homologues in the unassembled virome reads.Nevertheless, assembling complete Microviridae complete genomes. These 498 metagenomic sequences span 36 of the 95 viromes, from 5 different types of ecosystem . Microviridae remains undetected in hypersaline and hyperthermophilic environments , and is found in all Microviridae-containing biomes (Pichovirinae are less frequently detected (12 viromes), but are also retrieved from different types of biomes. On the contrary, Alpavirinae are exclusively detected in human sample viromes (16 samples). Finally, only one sequence affiliated to the genus Microvirus was detected in a seawater virome. Yet, the low BLAST bit score (50.1) and the fact that this is the only microvirus-like sequence retrieved indicates that microviruses are likely to be extremely rare in such environments.A total of 498 sequences were found to be significantly similar (BLASTx bit score greater than 50) to the VP1 protein of at least one of the ronments . In ordeg biomes . PichoviMicroviridae and considerably expanded the existing knowledge on genome evolution, diversity and environmental distribution of this viral family. Using already published viromes that have never been analyzed for the presence of complete Microviridae genomes, this study more than tripled the number of complete genomes available for this family.Microphages are progressively retrieved from a broad range of environmental samples from various locations. In this study, the focus on this family through a search in viral metagenomes made it possible to describe a new subgroup of Microviridae seems more abundant and frequently retrieved in particular ecosystem types (as in the human gut), all biomes were not evenly sampled. Indeed, the published viromes used in this study were generated independently and with different sequencing technologies. For instance, viromes from the human gut contain approximately 2 times more base pairs than viromes from sea water, and 6 times more than those from hypersaline ponds. Third, the methodology used to generate a virome greatly influences the type of viruses which will be retrieved. Viromes prepared through LASL (Linker Amplified Shotgun Library) are not supposed to contain any ssDNA genomes as this technique only recover dsDNA fragments. Not surprisingly, no Microviridae were detected in the two viromes prepared through LASL in this study, both sampled from hyperthermophilic environments. Last, the quantitative importance of ssDNA viruses in general, and of Microviridae more specifically, remains an open question. The abundance of Microviridae was first considered to be higher than predicted when the affiliation of virome reads was normalized by the mean genome length, i.e. when viruses were compared in terms of “number of viral particles in the original samples” Microviridae is still unknown, the fact remains that a considerable part of reads in numerous viromes could be affiliated to Microviridae, leading to the assembly of 81 complete Microviridae genomes.Several points have to be discussed regarding both bioinformatical and biological issues in order to better understand the results obtained in this study. First, sequences reconstructed here from metagenomic data represent consensus sequences of individual microviral populations and thus, DNA sequence variations within each population are masked. Nevertheless, the assembly criteria used here (98% similarity on 35 bp) are considered stringent enough to gather only sequences from the same viral species Microviridae is a consistent and homogeneous viral family. All but two genomes contained ORFs significantly similar to the three core genes of Microviridae . Moreover, the division of the Microviridae family into sub-families, which has been previously presented Enterobacteria-infecting phages (genus Microvirus), and the other known Microviridae. Members of the genus Microvirus are considered as Microviridae archetypes, and are the most studied Microviridae so far, yet none of the genomes assembled in this study is associated with this genus. This apparent paradox might be due to culture bias, as Microviruses are cultivated on E. coli strains, the most widely used and studied prokaryotic model organism. The present study indicates that this genus is rare among the biosphere, and likely constitute a very specific type of Microviridae especially in terms of gene content and capsid structure. The other Microviridae appear to be internally divided into three main subgroups, namely Gokushovirinae, Alpavirinae, and a new subgroup, which we propose to name Pichovirinae. This new clade appear to be more related to Gokushovirinae than to Enterobacteria phages, and could represent an intermediate group, like Alpavirinae, which could fill the gap between the two currently recognized sub-families. Moreover, this new group has a unique genome organization, as VP2 is located between VP1 and VP4 for all the genomes in this group. Based on this synteny break, and the fact that Pichovirinae-like genomes have been assembled from very different samples , this clade has probably diverged from the common Microviridae ancestor a long time ago. Consistently, significant sequence similarity between pichoviruses and the other Microviridae is confined to the major capsid protein VP1 and the replication protein VP4, while the pilot proteins (VP2) displays only limited sequence similarity to corresponding proteins from the Alpavirinae prophages.The new genomes assembled in this study confirmed that Microviridae family confirms that the microphage diversity was under-estimated. The detection of sequences homologous to Pichovirinae and Gokushovirinae in very different environments (Pichovirinae on one side (VP1-VP4-VP2) and the remaining Microviridae subgroups on the other (VP1-VP2-VP4). The finding that microphages belonging to different subgroups occupy a variety of different ecological niches suggests that the association of microphages with bacteria is ancient, possibly predating the divergence of this cellular domain into the contemporary lineages.This taxonomic structure of the ronments  suggestsMicroviridae might share a common origin with eukaryotic viruses from the families Circoviridae, Geminiviridae and ParvoviridaeMicroviridae and parvoviruses possess long insertion loops connecting the β-strands  that on average possess larger and more numerous insertions within their MCPs , and more likely through at least two independent transfer events, shows that Microviridae are able to integrate genes of interest from external sources into their genomes, even if such transfers are rare. The different uncharacterized ORFs detected in the new Microviridae genomes are then of great interest, since they could represent other horizontally acquired genes. On the contrary, no direct gene transfers between two Microviridae genomes could be detected in our dataset. Notably, phylogenetic analysis of the 47 closely-related Escherichia coli-infecting microviruses illuminated a few cases of HGT between these viruses that probably occurred by homologous recombination Microviridae is limited by the genetic distance between the donor and the recipient virus species. Consequently, larger datasets of closely related virus genomes might be needed to better understand the prevalence of homologous recombination-driven HGT events in Microviridae.Until now, genes encoding novel functions in microviral genomes were thought to emerge from pre-existing genomic regions through accumulation of point mutations Listeria phage A500 Microviridae M15 peptidase might also be involved in dissolution of the host cell wall at the end of the phage life cycle.ΦX174 is the only microvirus for which the mechanism of host cell lysis has been elucidated. Unlike dsDNA phages that typically encode a holin-endolysin system, where holin perforates the cytoplasmic membrane and endolysin digests the peptidoglycan, microviruses depend on a single-gene lysis system Microviridae, only one subgroup (Alpavirinae) was found to contain temperate members (i.e. detected as prophages). This could be linked to a relatively low number of complete bacterial genomes from aquatic environments. However, the absence of Microviridae prophages in Enterobacteria, which have been far more thoroughly studied, as well as the absence of detection of any new prophage even with the new Microviridae genomes described here, suggests that the use of the lysogenic cycle is likely to be rare among Microviridae.From the current knowledge on Alpavirinae and the presence of a horizontally transferred peptidase in several of their genomes made it possible to deduce potential host organisms for these phages. As Alpavirinae prophages have been found only within Bacteroidetes genomes, and the Alpavirinae peptidase genes are most similar to genes from Bacteroidetes genomes as well, members of the Alpavirinae group are likely to infect members of this bacterial phylum. Interestingly, free-living Alpavirinae closely-related to Prevotella prophages were only found in a lung sample, whereas Alpavirinae related to Bacteroides prophages were found in different human stool samples. This finding is consistent with the fact that most of the sequenced Prevotella strains have been isolated from oral samples, while Bacteroides are thought to be primarily associated with gut flora.The lysogenic cycle of some of the Gokushovirinae makes it impossible to be conclusive regarding the potential host(s) of these viruses. Yet, 4 peptidases from human gut gokushoviruses form a common clade with genes from a marine bacterium (Ahrensia sp. R2A130) and 7 human gut bacteria belonging to genera Faecalibacterium and Anaerofustis , thus it is tempting to speculate that at least some members of the Gokushovirinae might infect Gram-positive bacteria.The absence of described prophage for rofustis , Fig. S9A set of 95 viromes available in public databases were downloaded and used in this study . Lake PaMicroviridae genes . Viromes were assembled using Newbler 2.6 (454 Life Sciences), using the stringent threshold of 98% identity on 35 bp. In addition to the contigs assembly, Newbler software detect putative links between different contigs, usually used to create scaffolds. The contigs linked to themselves (i.e. the end of the contig is similar to the start of it) were thus considered as circular DNA sequences, and searched for Microviridae-like genes via tBLASTx (threshold of 50 on bit score). After a first iteration of this search step, all Microviridae genomes retrieved were used as query in a second iteration, to detect more distant homologies . A fasta file containing the raw sequences of the 81 Microviridae genomes assembled in this study, alongside the annotation of each genome in separated genbank-formatted files, in a zip archive .All viromes were assembled , and scrMicroviridae-like genes via tBLASTx (threshold of 50 on bit score) in order to identify new prophages related to Microviridae.In addition, complete bacterial genomes from Refseq database and genomes currently assembled from the NCBI were looked for Microviridae genome. The predicted ORFs were compared to the sequence database NR using BLASTp and best BLAST hit were conserved. In order to identify and annotate genes not predicted by Glimmer, intergenic regions of the genomes were also compared to NR using BLASTx.An ORF prediction was processed using Glimmer 3.02 Gokushovirinae, Protein F in Microvirus) were retrieved from known sequenced genomes and complete genomes generated from viromes. The VP1 sequence retrieved as prophage in Prevotella bergensis genome was not included in the analysis, since the prophage is split among two scaffolds Sequences similar to the Major Capsid Protein  modeling with MODELLER v9.10 ab initio and homology-based approaches for structural modelling VP1 homologues from each of the analyzed Microviridae peptidases (threshold of 90 on bit score). Peptidases from Bacteroides and Prevotella genomes were added to the dataset, as Microviridae prophages had been detected in each of these genera. The multiple alignment was computed using Muscle Reference peptidase sequences were taken from the NR database, based on a BLASTp of the Microviridae genomes both published and assembled in this study (threshold of 50 on BLAST bit score).The unassembled reads from the set of viromes used in this study were screened for sequences homologous to major capsid protein, and these sequences were affiliated via a best BLASTx hit against a database formed of all VP1 from the complete Figure S1Boxplot of genome sizes within each clade. Affiliations were based on the major capsid protein phylogenetic tree .(TIFF)Click here for additional data file.Figure S2Heatmap based on the percentage of identity computed from the major capsid protein multiple alignment. Scale is indicated on the top left, with the distribution of the percentages of identity. The genome affiliation is indicated above the map, and groups are framed on the heatmap.(TIFF)Click here for additional data file.Figure S3Multiple amino acid alignment of the major capsid protein. Large insertions (more than 10 aa) are framed and identified from A to G. The insertion retrieved in all Microviridae but Enterobacteria phages known to induce mushroom-like structure is identified as the insertion E. One or several sequences were taken for each group, φX174 for Enterobacteria phages, CF7ML00001 and Prevotella Buccalis for Alpavirinae, Pavin_00723 for Pichovirinae, Chlamydia phage Chp2 and Bourget_00154 for Gokushovirinae and Spiroplasma phage 4.(TIFF)Click here for additional data file.Figure S4A boxplot illustrating length variation of the ‘mushroom-like’ protrusion-forming insertions in the major capsid proteins of Gokushovirinae, Alpavirinae, and Pichovirinae. The insertion lengths are plotted as a function of the Microviridae subgroup (A) and ecosystem type (B).(TIFF)Click here for additional data file.Figure S5Alpaviral VP1 in the context of the entire virion. Pseudoatomic model of the gokushovirus SpV4 virion (PDB ID:1KVP) with one of the capsomers substituted with the structural model of the alpaviral VP1 . The hot-spots in the alpaviral VP1s where specific insertions (>15 aa) with respect to the BMV5 VP1 sequence were detected are indicated with orange spheres. The length of the largest insertion at each of the hot-spots is indicated along with the name of a corresponding viral genome. HF, human feces.(TIFF)Click here for additional data file.Figure S6Alignment of the conserved motifs of the superfamily I rolling-circle replication protein.(TIFF)Click here for additional data file.Figure S7Heatmap based on the percentage of identity from the replication protein multiple alignment. Scale is indicated on the top left, with the distribution of the percentages of identity. The genome affiliation is indicated above the heatmap, and groups are framed on the heatmap.(TIFF)Click here for additional data file.Figure S8Heatmap based on the percentage of identity detected on the capsid assembly protein multiple alignment. Scale is indicated on the top left, with the distribution of the percentages of identity. The genome affiliation is indicated above the heatmap, and groups are framed on the heatmap.(TIFF)Click here for additional data file.Figure S9Maximum-likelihood phylogenetic tree based on peptidase M15_3 protein sequences. Each reference sequences is identified by its name, followed by its gene id. Alpavirinae sequences are highlighted in pink, Gokushovirinae in blue, and viral reference sequences are marked with a black circle.(TIFF)Click here for additional data file.Table S1List of viromes assembled. For each virome, the number of circular contigs identified as complete Microviridae genome is indicated. The web-server hosting the datasets are : NCBI (www.ncbi.nlm.nih.gov), MG-Rast (http://metagenomics.anl.gov), and Metavir (http://metavir-meb.univ-bpclermont.fr). When available, the methodology used to purify viral particle is indicated . *2 contigs were detected for virome 35 Marine_Sar_Vir, but they corresponded to the 2 contigs already assembled from this virome, described in Tucker et al., 2011, and were thus discarded.(DOC)Click here for additional data file.Table S2List of circular contigs similar to complete genomes of Microviridae. For each major protein, the gi of the best BLAST hit is indicated with the bit score of the corresponding BLAST. All the sequences and corresponding annotations are available through Dryad Digital Repository, doi:10.5061/dryad.8ht80; http://dx.doi.org/10.5061/dryad.8ht80.(DOC)Click here for additional data file.Table S3List of the Microviridae peptidase genes detected, with their best BLAST hit against NR database.(DOC)Click here for additional data file.


Plasmodium falciparum. However, Percoll, a hyperosmotic agent, may have harmful effects on the parasites. Magnetic bead column (MBC) separation has been used as an alternative. This is a report of a head-to-head comparison of the in vitro invasive capabilities of parasites isolated by either of the two methods.Percoll gradient centrifugation is often used for synchronization, enrichment, or isolation of a particular stage of P. falciparum laboratory strain isolate 7G8 was grown in vitro using standard procedures and synchronized using 5% sorbitol. On separate days when the schizont parasitaemia was >1%, the culture was split and half was processed by Percoll gradient centrifugation and the other half by magnetic bead column separation. Both processed parasites were placed back in culture and allowed to invade new uninfected erythrocytes.The t test with two tails was used for these comparisons.In 10 paired assays, the mean efficiency of invasion of 7G8 parasites treated by Percoll gradient centrifugation was 35.8% that of those treated by magnetic bead column separation  A paired In this comparison, magnetic bead column separation of 7G8 schizonts resulted in higher viability and efficiency of invasion than utilizing Percoll gradient centrifugation. To achieve synchronization several methods have been developed. One of the earliest reported is based on the use of a gelatin [in vivo by Thi Trang [Plasmodium parasites utilize the haemoglobin of the red blood cells as a nutrient and transform the iron containing haem group into haemozoin, a pigment that remains in the food vacuole. Therefore, schizonts possess an abundance of the iron-containing haemozoin. Because the iron in haemozoin is in the Fe3+ state, it has a stronger paramagnetic effect than the iron in haemoglobin, which is in the Fe2+ state. This translates into a stronger magnetic attraction under a magnetic field for haemozoin than for haemoglobin. Because not all strains of P. falciparum seem to withstand well the Percoll treatment [in vitro retained capability of invasion of new uninfected erythrocytes of the 7G8 strain parasites after purification either by Percoll gradient centrifugation or magnetic bead column separation was compared.Purification of schizonts from  gelatin , which r gelatin , since o gelatin . The mos gelatin , which d gelatin -7. Anoth gelatin  with or  gelatin , and thehi Trang . The usereatment , the in Plasmodium falciparum chloroquine-resistant and pyrimethamine-resistant strain 7G8 [et al [rain 7G8  was used8 [et al  using O+Cultures with parasitaemia of 4-20% were divided into two equal portions. One half was used for Percoll gradient centrifugation and the other for magnetic bead column separation. The Percoll gradient consisted of three layers of Percoll in RPMI (from top to bottom: 40% (4 ml), 70% (6 ml) and 90% (8 ml) Percoll) placed in 30 ml OAK RIDGE polycarbonate screw cap centrifuge tubes. The culture was centrifuged at 3000 rpm for 5 min at 4°C, the pellet resuspended in RPMI-1640 (1:1 vol/vol), and carefully placed on top of the gradient. The tubes were centrifuged at 10,000 rpm for 20 min at 20°C. The cells at the 40-70% interface, containing mostly schizonts, were collected, washed twice in RPMI-1640, resuspended in culture medium, counted in a haemocytometer and used as inoculum for the overnight cultures. For magnetic column separation, we used MACS Separation Columns "LS" . Pre-equilibration, washings, and elution were all carried out at RT with CM. Upon applying a magnetic field, three 8 ml loads of culture were added to the column and the flow-through, which usually contained uninfected erythrocytes, ring-infected erythrocytes, and young trophozoites, was collected and used to reload the column or was placed back in culture. After one wash with 8 ml of CM (since then we have optimized the purity obtained with the procedure by washing first with one ml followed by 5 ml of CM), the column was removed from the magnetic field and its contents eluted with 3 ml of CM. The eluted parasitized RBCs were centrifuged at 1,500 rpm for 5 min at RT, the supernatant discarded, and the pellet resuspended in 150-250 μl of CM plus 10% human serum (CMS), depending on the concentration desired. The number of parasites captured was calculated by haemocytometer determination, as with the Percoll treatment.et al [Schizont-infected erythrocytes from both magnetic beads column or Percoll gradient separation were incubated for 16-22 hrs at a haematocrit of 1-2% in CMS, in duplicate wells. Final schizont-infected parasitaemia ranged from 1 to 10%. For light microscopy readings, Giemsa-stained thin smears were prepared from each well and the number of ring-infected erythrocytes in 1,000 erythrocytes was counted. The microscopist was always blinded to the experimental group designation of each slide. For the flow cytometry readings, infected red blood cells were stained with Hoechst 33342 and read with a UV laser in a BD LSRII equipment following procedures as described by Spadafora et al . Each exThe percentage of efficiency of invasion compared to the MCB method is the result of the following formula:A paired t-test analysis was performed, where each sample from Percoll and from MBC done on the same day was compared to each other for their invasion percent into new red blood cells. Only experiments with purity of isolation above 65% were considered for the calculations.Table It is evident that schizonts purified by magnetic bead column separation were more viable than those purified by Percoll gradient centrifugation. The reasons for this are not clear. However, there are several possible explanations. It hs been reported that permeabilization of the host erythrocyte membrane appears s early as 6 h after parasite invasion and increases gradually with parasite maturation . Since tet al [P. falciparum treated by two methods of isolation, MCB and a centrifugation gradient obtained with Percoll to which sorbitol has been added, did not show any difference in their invasion capabilities. However, they reported that when they performed the same analysis with field isolates their results were' similar to those we show here, with the MCB treated ones invading at higher rates. It seemed to them that the adaptation to the laboratory conditions was the major factor determining if the parasites would be affected or not by the Percoll/sorbitol treatment. In view of the present experiments, these results are better explained by the difference in susceptibility to external chemicals and solvents inherent to different strains. Reed et al [mef could not be isolated using the Percoll method, which would also point out to different strains behaving differently to the Percoll treatment, for reasons that are still unknown, and that could include different membrane porosity and osmotic pressure sensitivity. In the case of 7G8, the isolation is possible but the invasion efficiency is reduced, something that could be avoided using the magnetic beads based technique.Bates et al  reporteded et al  reportedIn addition to increased viability, magnetic bead column separation offers the advantage that the parasitized red cells remain in an iso-osmotic environment. This means that the red cells can be introduced back into the culture media without concern for abrupt changes in the osmolarity, which could affect some strains more than others.Magnetic column enrichment of schizont and late stage trophozoite-infected red cells is a very viable alternative to Percoll gradient centrifugation especially for the purpose of preparing red cell invasion assays with the 7G8 strain. This technique can be recommended for several reasons: the procedure itself has the advantage of not being invasive on the parasites since it involves no contact with any foreign chemical reagent to the culture media, it requires no expensive centrifuges, and 7G8, as well as some other strains seem to be affected by the use of Percoll gradient centrifugation.The authors declare that they have no competing interests.in vitro culture, carried out most parts of the laboratory experiments, prepared and analyzed the data obtained, drafted the paper, and finalized the manuscript for submission with contribution from authors. KMK was involved in the experimental design, participated in the coordination of the project under which this study was conducted, assisted in the management of culture, helped supervise, and contributed to the preparation of the manuscript. LG contributed with maintaining parasite culture and assisted with interpretation of smears. All authors read and approved the final manuscript.CS suggested the idea, developed the protocol for the magnetic beads separation, maintained


Individual radiosensitivity has a crucial impact on radiotherapy related side effects. Our aim was to study a breast cancer collective for its variation of individual radiosensitivity depending on the patients’ age.in situ hybridization.Peripheral blood samples were obtained from 129 individuals. Individual radiosensitivity in 67 breast cancer patients and 62 healthy individuals was estimated by 3-color fluorescence Breast cancer patients were distinctly more radiosensitive compared to healthy controls. A subgroup of 9 rather radiosensitive and 9 rather radio-resistant patients was identified. A subgroup of patients aged between 40 and 50 was distinctly more radiosensitive than younger or older patients.In the breast cancer collective a distinct resistant and sensitive subgroup is identified, which could be subject for treatment adjustment. Preliminary results indicate that especially in the range of age 40 to 50 patients with an increased radiosensitivity are more frequent and may have an increased risk to suffer from therapy related side effects. The advantage of this approach is that chromosomal aberrations are a late endpoint in radiation damage processing. Chromosomal aberrations cover the entire ability of cells to recover from DNA damage and to process these damages. It includes: (i) DNA damage repair, (ii) mediating a proper signal transduction, (iii) achieving an appropriate cell cycle control and (iiii) induce cell death, if necessary. The analysis of this late endpoint could be favorable compared to assays detecting earlier endpoints like DNA-double strand breaks (γH2AX), apoptosis  or cell-cycle control (PI-flow cytometry).The very demanding task of a radiotherapy is to kill all cancer cells and at the same time to spare the surrounding normal tissue. However, there is a spectrum of confounding parameters which can affect normal tissue tolerance. One parameter is the individual radiosensitivity of the patient.et al.2Nevertheless, even if the individual radiosensitivity could be determined with great accuracy, it is not certain that a study allows proving the relation between radiosensitivity and side effect. There have been several studies with disappointing results. The reason is based on the observations of Jung in situ hybridization approach (3-color-FISH) in a prospective study. Therefore the frequency of chromosomal aberrations in blood lymphocytes after in vitro irradiation with 2 Gy was detected. Additionally the influence of age on individual radiosensitivity was studied.Our aim was to determine the sensitive, intermediate and resistant groups of a breast cancer collective by a 3-color-fluorescence in vitro with 2 Gy on a 6 MV linear accelerator . This study was approved by the ethics review committees of the Friedrich-Alexander-Universität Erlangen-Nürnberg (No. 2725), and informed consent was obtained from all patients and healthy volunteers. None of the patients used medications which are suspected to increase radiosensitivity.67 breast cancer patients and 62 healthy controls were included in the prospective study . PatientChromosomal aberrations were detected using 3-color FISH with whole chromosome painting of chromosomes 1, 2 and 4 as described earlier.14in vitro irradiated slides.A fluorescence microscope  was used to detect chromosomal aberrations, such as breaks, deletions, translocations, dicentrics, insertions and rings . 500 metData analysis and statistics were performed using SPSS 16 for Windows . For comparing the results referred to the different groups, the Kolmogorov–Smirnov test and Lilliefors test were applied for testing normality and data were fitted by a Gaussian distribution. Standard deviations of the Gaussian distributions have been used to designate an individual’s categorization. The cut-off values of 2 and 3 standard deviations are equivalent to the 95% and 99% confidence intervals. Different groups were compared using the two-sample t-test. Graphics were plotted with TechPlot for Windows 3.0.11 .in vitro irradiation with 2 Gy.Our intention was to study the individual radiosensitivity in a prospective collective of breast cancer patients. Three-color-FISH was used to estimate radiosensitivity . All abei.e. 150 metaphases from lymphocytes irradiated in vitro with 2 Gy and 500 metaphases from non-irradiated lymphocytes. Breaks per metaphase in the patients’ group were slightly higher than in the group of healthy individuals. An in vitro irradiation with a 2 Gy dose leads to a distinct higher amount of breaks. Yet there was only a trend to an increase of breaks in the patients’ group compared to the controls  with older individuals (69.2 years) and a spontaneous increase of chromosomal aberrations by a factor of 5.In in vitro irradiation of peripheral blood lymphocytes, which could be subject for treatment adjustment. Especially in the range of age 40 to 50 there is an increased fraction of patients having an increased radiosensitivity. These patients have an increased probability to suffer from therapy related side effects.In the breast cancer collective a distinctly resistant and sensitive subgroup is identified by using 3-color-FISH after 2 Gy


A new modality is necessary to prevent recurrence of superficial bladder cancer after complete transurethral resection because of the high recurrence rate even with current prophylaxis protocols.In order to analyze the predictive value of cyclooxygenase-2 (COX-2) expression and tumor infiltrating lymphocytes (TILs) in recurrence of this disease tumor specimens from 127 patients with solitary papillary non-muscle invasive bladder cancer (NMIBC), 78 with recurrent disease and 49 without recurrence during follow up of minimum 5 years, were retrieved for tissue microarrays construction and immunohistochemical analysis. COX-2 expression was scored according to Allred’s scoring protocol, while presence of TILs was categorized as absent (no) or present (yes) on whole tissue sections.COX-2 immunoreactivity was presented in 70 (71%), weak in 16% and strong in 55% of cases, while 29 (29%) tumors were negative. TILs were present in 64 (58%) NMIBC, while 44 cases (41%) did not reveal mononuclear infiltration in tumoral stroma. Statistical analysis demonstrated a higher proportion of patients with recurrence in the group with the COX-2 score 0, and lower in the group with score 2 . In addition, a higher proportion of recurrent patients in the group with no TILs, and lower proportion in the group with TILs were found . Univariate and multivariate analysis revealed overexpression of COX-2 and presence of TILs as negative predictors.Patients with lower COX-2 expression and absence of TILs in NMIBC need to be followed up more vigorously and probably selected for adjuvant therapy.http://www.diagnosticpathology.diagnomx.eu/vs/1411318819790406The virtual slide(s) for this article can be found here:  Urinary bladder cancer (UBC), on the average, includes 2% of all the malignant diseases with male-to-female ratio being about 4:1. The incidence of UBC increases with age ,4. BCG iIn fact, 70% of patients treated with transurethral resection (TUR) experience a relapse of the underlying disease and 15-25% of patients will progress over time to muscle-invasive cancer -10.Standard prognostic factors like histological grading are limited in predicting possible recurrence of the disease. So, understanding of molecular processes that could reflect individual biological potential and clinical behavior is important. For that purpose, various biomarkers have been investigated since they have potential in decoding unique biological features in identifying patients with high risk of progression after the local treatment, as well as in more reliable prognosis and treatment of UBC . In the To determine if COX-2 overexpression could be causally related to recurrence of superficial UBC the present study was designed with pathologically homogenous group of solitary papillary non-muscle invasive bladder cancer (NMIBC) presented in patients according to development of the recurrent disease. More specifically, the aim was to assess and compare the COX-2 expression between the patients with and without recurrence of disease and, moreover, to analyze association between COX-2 expression and tumor infiltrating lymphocytes (TILs). We hypothesize that such investigation can be used to predict recurrence and help identify patients who require adjuvant treatment.The tumor specimens analyzed in this study were obtained from a total of 127 patients with solitary papillary NMIBC treated with initial transurethral resection (TUR), as a standard procedure at the Department of Urology, Rijeka University Hospital Center in Rijeka, between 1996 and 2006. None of the patients received adjuvant chemotherapy or immunotherapy or any other medical intervention after the initial TUR. All patients with multiple tumors and patients with a solid or flat aspect of the tumor were excluded from this study. The study was approved by the University of Rijeka Ethics Committee.All the sections were reviewed to confirm the original diagnosis and were staged according to the 2002 American Joint Committee on Cancer guidelines  and gradHematoxylin and eosin stained tumor sections were used in order to evaluate the presence of inflammatory cells and to mark the areas with the most pronounced inflammatory infiltrate for construction of tissue microarrays. Positive inflammatory cells were semi quantitatively graded and presence of TILs was categorized as absent (no) or present (yes) as shown in Figure Paraffin blocks were available for all 127 cases, and TMAs were constructed by using a manual tissue arrayer . Three tissue cores, each 1 mm in diameter, were placed into a recipient paraffin block. Normal liver tissue was used for orientation. Cores were spaced at intervals of 0.5 mm in the x- and y-axes. One section from each TMA block was stained with hematoxylin and eosin to confirm the presence of tumor tissue. Serial sections were cut from TMA blocks for immunohistochemical staining. Three to four μm thick sections were placed on adhesive glass slides , left to dry in oven at 55°C overnight, deparaffinized and rehydrated.Tumor samples were processed for immunohistological analysis in a Dako Autostainer Plus  according to the manufacturer’s protocol using Envision peroxidase procedure .′-diaminobenzidine tetrahydrochloride chromogen , under microscope control. The sections were counterstained with Mayer’s Hematoxylin. Quality control performed by external and internal negative and positive controls was necessary to monitor the accuracy of tissue processing, staining procedures and reagents effectiveness. The primary antibody specificity sought to be assessed by their negative controls.The sections were incubated with the primary antibodies, anti-COX-2 mouse monoclonal antibody , dilution range 1:100. The immune reaction was amplified using the appropriate secondary antibody and the Streptavidin–Biotin–Peroxidase HRP complex . Sections were then developed using 3,3COX-2 immunohistochemical expression was quantified and scored by assessing a proportion of percentage as an intensity score according to Allred’s scoring protocol . The assFor the purpose of further statistical analysis, the mean values of immunohistochemical staining of all three tissue microarrays were used.Statistical analysis was performed using MedCalc for Windows, version 12.3.0.0 . In the first part of the work the classical descriptive methods were used as well as the Chi-square test for the comparison of proportions. Mann–Whitney test was used to compare the medians. The method of logistic regression was used in order to compute the odds ratio for predictors of recurrence in univariate and multivariate manner. The AUC value was used to evaluate the quality of predictive model. The significance level in all tests was 0.05.Clinicopathological characteristics of 127 patients with solitary, papillary NMIBC unrolled in the present study are summarized in Table Immunohistochemical analysis revealed COX-2 expression predominantly granular localized to the cytoplasm of tumor cells. Nuclear staining was not observed. In a few cases immunoreactivity was detected in endothelial cells within the tumor. Low intensity staining was also observed in smooth muscle tissue in some sections. There was noticeable heterogeneity in the intensity and percentage of COX-2 positive cells within the tumor tissues. Positive COX-2 immunoreactivity scored according to Allred was presented in 70 (71%) while 29 (29%) tumors were negative. Among positive tumors 16 (16%) show weak and 54 (55%) strong immunostaining. Representative cases of tumors with moderate and strong COX-2 staining are presented in Figure Tumor infiltrating lymphocytes as shown in Figure Main objectives of the present study were to examine possible association between COX-2 expression and TILs with regard to recurrence of disease, as well as their mutual relationship which revealed no significant association (p=0.892).Clinicopathological parameters included in the above mentioned analysis are presented in Table Moreover, statistical analysis demonstrated a difference in tumors COX-2 expression between the analyzed groups, e.g. between recurrent and non-recurrent patients. Possibly, due to small sample size in the group with the score 1 no significant difference was found between recurrent and non-recurrent patients (p=0.2196). In the group with the score 0 and the group with the score 2 there was a significant difference in the proportion of patients with and without recurrence . From these results we can expect that lower score is predictor of recurrence, since there is higher proportion of recurrent patients with score 0 and lower proportion of recurrent patients with score 2.A significant difference between the recurrent and non-recurrent patients in both groups, with absent and present TILs was also found . More precisely, higher proportion of recurrent patients in the group with no TILs and lower proportion in the group with TILs were found. Therefore, we can expect the absence of TILs to be the predictor of recurrence.Univariate and multivariate analysis of the influence of COX-2 expression, TILs, tumor size and disease stage on prediction of recurrence (PR) are shown in Table The odds ratio for each predictor was computed using the method of logistic regression. As we can see the tumor size is not the significant predictor in univariate, as well as is in multivariate manner. Disease stage is in both analysis significant and positive predictor, which means that the odds for a recurrence in cases with stage 1 are 2.39 times higher than in cases with stage 0. In both manners the TILs and COX-2 are negative predictors. The positive change in score for COX-2 (score higher for 1) produces 0.36 times lower risk, or that the odd for not getting a recurrence is 1/0.36=2.77 times higher. The TILs is also significant negative predictor of recurrence with OR 0.31 in univariate manner and 0.23 in multivariate analysis. The AUC value as a classification parameter which gives us the quality of predictive model was used. In our case we have the AUC 0.875 which means that our model has very high discriminating power.There is large evidence implicating COX-2 activity in the development of UBC. Yet, the results obtained in the present study demonstrated that low COX-2 instead of high COX-2 score could predict the recurrence of NMIBC. These results open many questions related to the methodology used in various studies, as well as the complexity in understanding the role of different biological markers in tumorigenesis.In the present study we used TMA instead of whole tissue section for immunohistochemical analysis. Gudjonsson et al. in their study analyzed COX-2 among different markers on TMA and they found that proteins assessed had no predictive value for recurrences of Ta bladder cancer (BC) . The conPrevious studies have been conducted mostly on heterogeneous groups of patients with superficial and invasive UBC. One of the first papers in which the expression of COX-1 and COX-2 were analyzed in human invasive TCC of the urinary bladder samples was by Mohammed and coworkers . COX-2 wOpposite to the role of COX-2 in tumor progression there are also studies where COX-2 expression was not associated with primary tumor stage, lymph node status, histological grading, overall and disease-free survival . WulfingThere are also several studies analyzing COX-2 expression only in NMIBC. In the CIS group, COX-2 expression was significantly associated with disease recurrence and progression, but not with BC related survival , while in the stage T1 the TCC COX-2 expression was not associated with clinical or pathological parameters or clinical outcome . Kim andOur results differ from the above mentioned because loss of COX-2 was a predictive marker of tumor recurrence. We believe this can be explained by very complex, even different role of this enzyme during tumorigenesis. There is no doubt that COX-2 expression is sequentially up-regulated from normal to chronic cystitis and to malignant changes . Also thAlthough further studies are necessary to fully elucidate the mechanism involved, the present study showed that higher COX-2 expression and present TILs resulted as a negative predictor of recurrence in NMIBC. On the other hand, patients with lower value of both analyzed parameters should be probably selected for adjuvant therapy.The authors declare that they have no competing interests.TT participated in study design, conceived the study and participated in coordination. KK added clinical data. SŠ actively performed part of immunohistochemical analysis. EB conceived the study and participated in coordination and performed statistical analysis. ŽF participated in study design and coordination. NJ advised and led the whole group as coordinator. All authors read and approved the final manuscript.