Early gestation represents a period of vulnerability to environmental insult that has been associated with adult psychiatric disease. However, little is known about how prenatal perturbation translates into adult brain dysfunction. Here, we use a longitudinal study design to examine the effects of disruption of early gestational neurogenesis on brain volume in the non-human primate.Five Rhesus macaques were exposed to x-irradiation in early gestation (E30–E41), and four control monkeys were sham-irradiated at comparable ages. Whole brain magnetic resonance imaging was performed at 6 months, 12 months, and 3 and 5 years of age. Volumes of whole cerebrum, cortical gray matter, caudate, putamen, and thalamus were estimated using semi-automated segmentation methods and high dimensional brain mapping. Volume reductions spanning all ages were observed in irradiated monkeys in the putamen  and in cortical gray matter . Upon covarying for whole cerebral volume, group differences were reduced to trend levels . No group-by-age effects were significant.Due to the small number of observations, the conclusions drawn from this study must be viewed as tentative. Early gestational irradiation may result in non-uniform reduction of gray matter, mainly affecting the putamen and cerebral cortex. This may be relevant to understanding how early prenatal environmental insult could lead to brain morphological differences in neurodevelopmental diseases. Early gestation has been identified as a particularly vulnerable period for human brain development. For example, human populations exposed to atomic radiation from the World War II bombings or to radiation emissions from the Cherynobyl nuclear plant who were age 8–25 weeks of gestation exhibit brain dysfunction, with the most severe damage occurring at 8–15 weeks for the atomic irradiation In the present study we disrupted neurogenesis during early gestation in the macaque brain by exposure to x-irradiation. Although irradiation kills dividing neurons indiscriminately, temporal staggering of neurogenesis in the macaque brain allows for specificity in targeting different brain regions via differential timing of the radiation exposure. For example, the thalamus is generated early in gestation (E36–E45) The present study addresses the following questions: 1) Are brain volume reductions observed following early gestational exposure to radiation? 2) Are these volume reductions confined to subcortical structures in which newly generated neurons are killed by the radiation?, and 3) Are volume reductions present in infancy, or do they emerge at later maturational ages?in utero  or were sham irradiated  in utero  and thenIn utero exposure to x-rays was performed as described in detail by Algan and Rakic Following irradiation or sham irradiation, the status of pregnant monkeys was carefully monitored and viability of the fetus was confirmed by ultrasound throughout the remainder of the gestational period. All monkeys in this study, with the exception of CON-Lf and FIM-Am, were born naturally at term (∼E165) and allowed to stay with their mothers until 6 months of age. CON-Lf had to be delivered via cesarean section because the pregnancy went several days beyond term. FIM-Am, although delivered naturally, was rejected by his mother. Both of these animals therefore spent the first 6 months in the primate nursery in the Yale University School of Medicine. At 6 months of age, the monkeys were pair-housed (n = 7) or singly-housed (CON-Vf and FIM-Sf) in the adult non-human primate colony at Yale. Mean age and body weight did not differ significantly between groups at any of the four postnatal time points sampled .High-resolution 3D T1-weighted MR scans were collected at 6 months, 12 months, 3 years and 5–6 years. Due to the length of this study (>5 years), imaging was performed on three different scanner platforms. Within a given age group, animals were generally scanned on the same platform, except for one FIM (Am) and one CON (Lf) monkey who were approximately one year younger than the others. Scans for these two monkeys were performed one year later than same age scans of the other monkeys, and therefore scans at 12 months and 3 years of age were acquired on a different platform from the other monkeys at the same age. Specifically, the 6 month MR scans of all animals and the 12 month scans of all but those two animals (FIM-Am and CON-Lf) were acquired on a 1.5T LX GE Advantage scanner using a SPGR sequence. The 12 month scans of FIM-Am and CON-Lf, and the 3 year scans of all other animals were collected on a 1.5T Siemen’s Sonata scanner, using a 3D FLASH acquisition. The 3 year scans of FIM-Am and CON-Lf and the 5–6 year scans of all animals, including FIM-Am and CON-Lf, were acquired on a 3T Siemen’s Trio scanner using a 3D MPRAGE acquisition. Resolution was 0.63×0.63×0.7 mm for the scans at 1.5T and 0.63×0.63×0.6 mm for the scans at 3T . When multiple T1-weighted scans were collected for a monkey at a given age, those volumes were registered and averaged prior to further analysis.Whole cerebral volumes (WCV) containing cerebral cortical gray matter (CGM) and white matter, subcortical gray matter, and diencephalon were manually segmented from the MR image of the entire cranium using Analyze 8.0 software A semi-automated procedure was used to measure volume of the thalamus, caudate, and putamen by a single author (KA). First, an MRI scan collected from a control Rhesus macaque, not included in this study, was used to construct a neuroanatomical template for the each of the three structures of interest. The neuroanatomical template was created by manually outlining the caudate nucleus, putamen, and thalamus in the template MR scan. The neuroanatomical boundaries of these structures were identified with the aid of an atlas Intra-rater reliability for the single rater for these data was assessed in two ways. First, following Kohn & Cheverud Structural volumes were analyzed in a repeated measures mixed model . Age was treated as a repeated measure class variable (rather than a continuous variable) to avoid assumptions about the trajectory of the changes over time, as would be required for random effect growth-curve modeling. Covariance of the model residuals across ages was modeled with a Huynh-Feldt covariance structure that was assumed to be the same for the FIM and CON groups. The Huynh-Feldt covariance structure (“type = hf" in PROC MIXED) allowed the estimation of separate variances at each age, with the covariance between ages constructed from the arithmetic means of these variances. Degrees of freedom were calculated using the method of Kenwood and Roger To investigate the possible impact of changes in scanner platform on our results, we examined models with group, age, and scanner as fixed effects. Inclusion of scanner platform had no appreciable impact on the p-values for the effect of group for any of the structures (relative to the model with just group and age as effects), and while the significance of the effect of age was generally reduced, it remained significant for the whole brain, cortical gray matter, and caudate (p<0.03). Notably, the effect of scanner itself was non-significant for all structures (p>0.15).Raw volume data are plotted in WCV did not differ significantly between groups . However, when WCV was included as a covariate, the difference was reduced to trend levels (p = 0.08). CGM was reduced in FIMs approximately 6% at 6 months, 13% at 1 year, 15% at 3 years, and 14% at 5 years.Thalamic volume exhibited trend reductions in FIMs at all ages . The difference between groups did not reach statistical significance without covarying for WCV (p = 0.09); inclusion of WCV as a covariate nullified this trend (p = 0.73).Caudate volume did not differ significantly between groups .Putamen volume was reduced in FIMs on average at all four ages . This reduction was statistically significant without WCV included as a covariate (p = 0.01) and reduced to trend levels with WCV as a covariate (p = 0.07).Macaques exposed to radiation in early gestation appear to exhibit non-uniform reductions in gray matter volume, observed most prominently in the putamen and cerebral cortex. Volume differences were present at the youngest age (6 months) examined even in the cerebral cortex. The presence of cortical reductions following early gestational disruption of neurogenesis suggests vulnerability of brain circuits to early gestational environmental insult.This study is based on a relatively small number of subjects: 5 FIM and 4 CON. The lengthy experimental paradigm, spanning 6 years and involving prenatal irradiation and repeated postnatal MRI scans, limited the numbers of subjects in this study. Although mean volumes were consistently lower in FIMs across all brain structures, including whole cerebral volume, significant differences between groups were not always found. The lack of significance may in part have been due to what appears to be a higher degree of variability in brain volume in the irradiated group compared to controls. Thus, with a larger number of subjects, additional significant findings might have been observed.It should be emphasized that the FIMs examined in the present study were healthy animals. Their mean body weight did not differ from that of CONs. One FIM had to be raised in the Yale primate nursery after being rejected by his mother, as was one CON who had been delivered by Caesarian section and not able to stay with her mother. Both were hand fed in the nursery and thrived. In fact, FIMs were virtually indistinguishable from CONs throughout their postnatal maturation. They exhibited normal home cage behavior, including feeding and drinking. The only abnormalities detected in FIMs were circling behavior and restricted cognitive deficits, the latter emerging only in adulthood Imaging was performed on three different scanner platforms over the 6 year course of this study, due to a combination of institutional decisions (necessitating the switch to the Siemen’s Sonata) and a desire to collect the highest quality anatomical images (the switch to the Siemen’s Trio). The changes in scanner platform and imaging sequence may have introduced some bias and increased the overall variance of the volume measurements We have analyzed brain volumes with and without covarying for whole cerebral volume. Significant group differences were not found.when volumes were covaried for whole cerebral volume. Although covarying for brain volume has become standard procedure for taking account of individual variability in brain size in neuroimaging studies of human disease, correcting for whole brain volume when studying developmental disorders, or in this instance the impact of prenatal irradiation, that lead to smaller overall cerebral volumes prevents detection of the actual pathology since all individuals in the experimental group would be expected to have smaller brain volumes than the controls Reduction of gray matter volume in the putamen may be due to the direct lethal effect of radiation on newly born neurons in the putamen We hypothesized that the cortex might be spared at early postnatal ages since the early gestational irradiation would not have directly killed newly generated cortical cells Volume plots for the control monkeys in this study may serve to illustrate the normative brain growth pattern for the macaque monkey. Plots for the prenatally irradiated monkeys generally paralleled curves for the sham-irradiated monkeys though volumes were smaller for the irradiated monkeys. To our knowledge, there has been only one other longitudinal analysis of brain volume in the non-human primate, and that study did not parcellate brain regions Our findings suggest that a relatively mild insult in early gestation may result in multifocal reductions of gray matter volume. These findings highlight the vulnerability of the developing brain in the early embryonic period. As such, these findings might be relevant to human psychiatric conditions, including schizophrenia, ADHD, and autism, in which mounting evidence suggests a link to adverse prenatal events.


Over 3000 microbial  genomes have been made publically available to date, providing an unprecedented opportunity to examine evolutionary genomic trends and offering valuable reference data for a variety of other studies such as metagenomics. The utility of these genome sequences is greatly enhanced when we have an understanding of how they are phylogenetically related to each other. Therefore, we here describe our efforts to reconstruct the phylogeny of all available bacterial and archaeal genomes. We identified 24, single-copy, ubiquitous genes suitable for this phylogenetic analysis. We used two approaches to combine the data for the 24 genes. First, we concatenated alignments of all genes into a single alignment from which a Maximum Likelihood (ML) tree was inferred using RAxML. Second, we used a relatively new approach to combining gene data, Bayesian Concordance Analysis (BCA), as implemented in the BUCKy software, in which the results of 24 single-gene phylogenetic analyses are used to generate a “primary concordance” tree. A comparison of the concatenated ML tree and the primary concordance (BUCKy) tree reveals that the two approaches give similar results, relative to a phylogenetic tree inferred from the 16S rRNA gene. After comparing the results and the methods used, we conclude that the current best approach for generating a single phylogenetic tree, suitable for use as a reference phylogeny for comparative analyses, is to perform a maximum likelihood analysis of a concatenated alignment of conserved, single-copy genes. Until relatively recently, the evolution of species was largely assumed to be a strictly bifurcating, tree-like process More than 3000 bacterial and archaeal genomes have been sequenced and deposited in public databases to date, including the results of a large-scale effort to choose organisms for genome sequencing based on their phylogenetic diversity supermatrix method), or used individually to infer a tree for each gene and then combining the gene trees to build a single organismal phylogeny (supertree and simultaneous gene tree/species tree methods). Both approaches have trade-offs and shortcomings that have been discussed extensively elsewhere This work is motivated by the need for a single, fully resolved, best estimate of the phylogeny of bacteria and archaea that will be used to perform various comparative analyses in a phylogenetic context. Here, we employ several phylogenetic methods to infer the phylogeny of the bacteria and archaea for which genome sequences are available, using a set of 24 single-copy, ubiquitous genes . AlignmePrevious work on large-scale microbial phylogenetics has primarily relied upon the supermatrix approach Many factors can contribute to the incongruence of phylogenetic trees inferred from different genes. In bacteria and archaea, horizontal gene transfer is the most common explanation offered in the literature, but incomplete lineage sorting, model violation, convergence, long branch attraction, and lack of phylogenetic signal can also contribute to phylogenetic incongruence. Several methods, including *BEAST https://github.com/gjospin/PhyloSift) was run on the database of all bacterial and archaeal draft and complete genomes available from NCBI and IMG as of April 2011. PhyloSift . A multiple sequence alignment was generated for each of the 38 genes with hmmalign PhyloSift  without contributing useful information to the inference of relationships among the closely-related species. Therefore, a subset of taxa was chosen that is representative of the total organismal diversity. In order to do this, we first built a phylogeny using FastTree In addition to eliminating redundant taxa, a related concern was one of minimizing the amount of missing data. Missing data are particularly problematic for the implementation of BUCKy because it requires that each taxon is present in every single-gene analysis. In order to apply BUCKy to data where a taxon is missing a gene, phylogenetic analysis for that gene must include that taxon with all sites coded as missing data. If many taxa are missing data, the result is a lot of uncertainty, causing a diffuse posterior distribution of topologies and inaccurate results An additional 41 taxa were manually added to the initial list of 800 genomes. These additional genomes were either publicly unavailable (not yet deposited into Genbank) or of particular interest (because they represent under-sampled lineages) and missing one or more of the 24 marker genes. See In order to ensure that the results from the supermatrix and supertree approaches would be directly comparable, we used a concatenated alignment of the same 24 marker genes with the same 841 taxa as input for a Bayesian phylogenetic inference with MrBayes v. 3.2.0 Alignments of the 24 marker genes selected to minimize the amount of missing data (as described above) were analyzed with MrBayes using the parameters above, for 1 million generations, requiring approximately 9 months of CPU time for each marker.A concatenated alignment of 24 genes for 841 taxa was used as input for maximum likelihood inference with RAxML v 7.2.8 Alignments of each of the 24 markers that were selected to minimize the amount of missing data were analyzed with RAxML as described above for the concatenated alignment. However, because some taxa were composed entirely of missing data for some of the single-gene analyses, the RAxML source code was modified to permit the analysis to run with these taxa included. We used the –f a option of RAxML to generate 100 rapid bootstrap replicates 16S rRNA sequences for each of 841 organisms were either downloaded from NCBI or retrieved manually from the IMG database For reasons discussed below in the results, we did not use BUCKy with the results of the single-gene Bayesian phylogenetic analyses. Instead, we used BUCKy to generate a primary concordance tree (referred to hereafter as the “BUCKy” tree) from the single-gene RAxML bootstrap replicates. To convert RAxML output into a format acceptable as input for BUCKy, a custom perl script, dependent on R, is provided in bool buildPopulationTree = false;was added immediately after the line:string quartetTree, quartetTreeWithWts;We evaluated the effect of choosing different prior values for the alpha parameter on the results generated by BUCKy. We ran BUCKy using the default prior of 1, which for our data, centers the prior distribution on the number of distinct gene trees around 3.5. We also used alphas of 10, 50, and 100, for which the prior distribution is centered around 12.5, 18, and 22 trees, respectively.We used the Robinson-Foulds (RF) metric In large part because not every genome used in this analysis was completely finished, if we had restricted our analysis to genes that were universally present in all the genomes analyzed, we would have been left with only four ribosomal proteins. Given the amount of total phylogenetic diversity among these organisms, a phylogeny reconstructed using only four ribosomal protein (i.e. short) genes would certainly be too poorly resolved for our purposes. We did attempt to minimize the impact of missing genes by limiting the number of missing sequences per gene to no more than six.841 organisms, including four plastid and three mitochondrial genomes and a subset of 24 of the PhyloSift phylogenetic markers were used for all initial analyses. In an attempt to minimize artifacts due to long-branch attraction (discussed below) at the split between the bacteria and archaea, we also built trees from bacteria-only alignments of 761 taxa. See The total phylogenetic diversity (branch length) contained in a tree of all 16S rRNA sequences maintained in the Greengenes database Mycoplasma suis, Candidatus Carsonella ruddii, and Candidatus Hodgkinia cicadicola. These organisms are either missing up to 10 of the marker genes (because they are intracellular symbionts with streamlined genomes or single-cell isolates with incomplete genomes) or have accelerated rates of sequence evolution, or both.Long branch attraction (LBA) is a well-known phylogenetic artifact that causes sequences that are on long branches (which can occur because the lineages have accelerated evolutionary rates or because they are on isolated evolutionary branches) to incorrectly appear to be closely related Mycoplasma species moved, with high bootstrap support (100%) to a clade with the other Mycoplasma species. The two insect endosymbionts, Hodgkinia and Carsonella, moved to within the Proteobacteria, but there they form a poorly-supported clade with another long-branch taxon (candidate division TM7 genomosp. GTL1), suggesting that LBA may still be an issue with the placement of these taxa. The two TM7 genomes were moved to different locations in the tree, as discussed in the TM7 section. In the BUCKy tree with only bacterial taxa, the Mycoplasma species moved to the Mycoplasma clade; the two TM7 genomes do have a sister relationship, and they form a lineage basal to the Actinobacteria; Hodgkinia becomes the basal lineage of the alpha-proteobacterial clade; and Carsonella is sister to Candidatus Zinderia insecticola, a beta-proteobacterial insect endosymbiont. Carsonella+Zinderia form a clade with the mitochondrial genomes, and that clade is sister to the alpha+beta-proteobacterial clade.One way to reduce the impact of LBA, in terms of drawing long branches to the root, is to remove the distantly-related outgroup taxa. We repeated the RAxML analysis with all of the archaeal species removed from the concatenated alignment. When we did this, each of these six long branches moved to different locations in the concatenated ML tree. The two Aside from the TM7 genomes, which have never been phylogenetically placed with confidence, each of these moves were to positions that were at least closer to expectation given the 16S rRNA tree and other, independent, physiological lines of evidence For both the concatenated alignment and for all single-gene alignments, after approximately 18 months of CPU time, two million MCMC generations had completed in MrBayes. However, the average standard deviation of split frequencies (ASDSF) for all runs was > = 0.16, which is well above the level (0.01) suggested by the authors of the software to be indicative of convergence. And, in majority-rule consensus trees, generated after discarding the first 25% of the trees sampled as burn-in, not a single node had greater than the 0.5 posterior probability required for inclusion in a majority rule consensus tree.Because it is impossible to know if or when these analyses would ever converge upon an answer, given our computational resources, we opted to abandon them. Recent work in Bayesian phylogenetics has demonstrated new algorithms that can run several orders of magnitude faster than MCMC on large datasets like ours We concluded that the best approaches to reconstructing the phylogeny of the bacteria and archaea, given our time limits and computational resources were by 1) running a Maximum Likelihood search using RAxML on a partitioned, concatenated alignment of the 24 single-copy phylogenetic marker genes and 2) using bootstrap replicates for each of the 24 phylogenetic marker genes as input for a Bayesian Concordance Analysis using BUCKy to produce a primary concordance tree We performed 1000 bootstrap replicates for each gene, and assessed convergence using the –I autoMRE option in RAxML. Bootstrapping converged for all genes after no more than 500 replicates .The tree in BUCKy was designed to take as input the posterior distribution of tree topologies generated by MrBayes for each gene. Because the single-gene Bayesian analyses could not be run to convergence due to computational limitations, we used an alternative approach suggested by The trees produced using alpha = 10, 50, and 100 were identical, both in topology and the values of the concordance factors at every node. Because we know that there is considerable uncertainty in the single-gene trees, we chose to use the primary concordance tree that was generated using a larger prior than the default of 1. The RF distance between the alpha = 1 and alpha = 10 primary concordance trees was (72/4.76%) and the average concordance factor value was (0.360) in the alpha = 1 tree, versus (0.368) in the alpha = 10 tree. The main (minor) difference between the two trees is the degree of resolution: the alpha = 1 tree has 1508 splits and the alpha = 10 tree has 1512 splits. For reference, a fully resolved tree has 1522 splits.The BUCKy tree  showed aFor many organisms, especially those that have been selected for genome sequencing due to their phylogenetic novelty or are otherwise from relatively under-sampled clades, the best estimate of their phylogenetic history has been derived from their position in the 16S rRNA gene phylogeny. Our results show that there is a great deal of congruence/agreement between the phylogenetic trees obtained by analysis of the 16S rRNA gene and that of the 24 genes used here, especially in terms of recovering known phyla see  and 2. HChlamydiae/Verrumicrobia group with 86% bootstrap support.The placement of the Planctomycetes has been somewhat controversial Thermodesulfobium narugensedsrAB and apsA. These two genes are found in only a few bacterial and archaeal clades, so they were not useful for confirming the phylogenetic placement of T. narugense, but they do have conflicting topologies that the authors state may be due to HGT of the sulfate reduction genes (which has been proposed to be frequent). It appeared to have a “distinctive” dsrA gene, while its ApsA gene appeared to have been most closely related to that of D. hydrogenovorans (a delta-proteobacterium). In our 16S rRNA tree of all sequenced genomes, it is a part of a basal polytomy of the bacteria. In the concatenated ML tree, it is sister to Dictyglomus (boostrap support = 60%) and those two are sister to the Thermotogae+Coprothermobacter with moderate (53%) bootstrap support. This corroborated a recently published anlysis of 44 orthologous protein sequences, which demonstrated a sister relationship between Dictyglomus and Thermotogea, but did not include ThermodesulfobiumThermodesulfobium+Dictyglomus) and (Thermotogae+Coprothermobacter) have a strongly-supported (bootstrap = 83%) sister relationship to a clade containing the Deinococcus-Thermus group and the Aquificaceae.The published description of Hippea maritima is a thermophilic, sulfur-reducing bacterium that was isolated from shallow submarine hot vents Desulfurellaceae based on its 16S rRNA sequence similarity (89.6%) to Desulfurella multipotens and placement as sister to the Desulfurella clade of the delta-proteobacteria Carsonella rudii, which is on a long, unstable branch (see discussion of LBA above), the Acidithiobacillus species are the only gamma-protobacterial taxa that are not contained within a well-supported (bootstrap support = 70%), monophyletic clade. The two Acidithiobacillus species instead represent a distinct lineage, basal to the clade (bootstrap = 48%) containing the beta+alpha-proteobacteria. In the BUCKy tree, they are the basal lineage of the beta-proteobacterial clade. The placement of this group has historically been problematic In all trees presented here, the gamma-proteobacteria is a paraphyletic group. In the concatenated ML tree, with the exception of Candidatus Thermodesulfobacterium has been proposed to represent a deeply-diverging division-level lineage dsrAB genes of Thermodesulfobacterium species are most closely related to those of the delta-proteobacteria. The concatenated ML tree provides strong support (bootstrap = 100%) for the sister relationship of Thermodesulfobacterium commune+Thermodesulfatator indicus to the Desulfovibrionales, a family within the delta-proteobacteria. This result, along with the fact that these organisms have a similar metabolism to other DesulfovibrionalesdrsAB tree that was interpreted in dsrAB gene, is in fact due to an incorrect placement of T. commune in the 16S rRNA tree.Acidobacteria and the Nitrospirae (bootstrap = 89%). The strongly-supported Nitrospirae clade (bootstrap = 95%) contains Thermodesulfovibrio yellowstonii, Candidatus Nitrospira defluvii, and Candidatus Poribacteria. These two clades (Acidobacteria+Nitrospira) are, with moderate support (bootstrap = 55%), sister to the delta-proteobacteria. The inclusion of Candidatus Poribacteria in the Nitrospirae clade is in conflict with its placement in the 16S rRNA tree. In the 16S rRNA tree, Poribacteria is at the base of the Planctomycetes (bootstrap = 43%), and that clade is sister to the Verrucomicrobia+ Chlamydia clade (bootstrap = 54%). This “Poribacteria” genome was sequenced using a single-cell whole genome amplification (WGA) approach Delftia, which is a common beta-proteobacterial reagent contaminant) from other genomes in their sequence. Their evidence for lack of contamination was 1) a single copy of the 16SrRNA gene, 2) single copies of 29 of 55 known single-copy genes In the concatenated ML tree, there is strong support for the sister relationship of the Chloroflexi. In the concatenated ML tree, they are not sister taxa. TM7c is placed with weak support (bootstrap = 38%) as sister to Anaerolina thermophilia, within the Chloroflexi. The other candidate division TM7 genomosp. GTL1 isolate is placed with very weak support within a clade containing three insect symbionts, basal to the gamma+beta-proteobacterial clade. This placement is possibly the result of an LBA artifact. While the true placement of these two genomes is unclear based on the concatenated ML tree, it is noteworthy that they do not have the strongly-supported sister relationship that we see in the 16S rRNA tree.There are two TM7 genomes that were sequenced with the WGA approach. Using the 16S rRNA sequence from the WGA data, these two are sister taxa with bootstrap support of 100%, and they are placed, with weak support, as sister to the supertree approach. A review of available methods  led us to believe that the best strategy, given our data, was to use Bayesian Concordance Analysis (as implemented in BUCKy) with RAxML bootstrap replicates as input. We were interested in how a tree produced by BUCKy in this way compared to a ML analysis of a concatenated alignment. Our analyses including both bacteria and archaea revealed obvious long branch attraction artifacts within the bacteria. We therefore opted to focus all of our tree (method) comparisons on trees that contained only bacterial taxa, in which the long branch attraction artifact was ameliorated.We were interested in generating a single, fully resolved phylogenetic tree to be used in comparative analyses. We chose 24 highly-conserved, single-copy genes to be used for phylogenetic analysis. We wanted to use an approach that avoids some of the  assumptions about how genes evolve, especially in microbes. In particular, we wanted to use an approach that does not assume that every gene shares a single phylogenetic history, i.e., a For the Bayesian Concordance Analysis, we first attempted to use Bayesian phylogenetic inference, but current methods were unable to mix and converge on our data after many months of CPU time. Instead, we opted to employ a maximum likelihood approach using RAxML. We used two approaches to combine the data from the 24 phylogenetic marker genes. The first approach was to concatenate alignments of all 24 genes and run a single ML analysis, with bootstrap replicates. The second approach was to generate many bootstrap replicates from an alignment of each gene, and then combine the information from the single gene trees using BUCKy. We also performed ML analysis of an alignment of 16S rRNA sequences for each of the taxa included in the other two trees. This 16S rRNA tree was used as a standard to which to compare the phylogenetic placement of particular taxa in our concatenated ML and BUCKy trees.The concatenated ML tree and the BUCKy tree were more similar to each other than either was to the 16S rRNA tree. There are differences between the arrangements of taxa in all three trees, but most of the major bacterial groupings (phylum-level) were shared among all three. The ML tree based on the concatenated alignment was better-supported overall than the 16S rRNA tree  and the As has been observed in previous, similar simulations, a Bayesian approach to the reconstruction of phylogenies of this size is currently computationally infeasible From among the trees we generated during this process, we would choose the concatenated ML tree as our working representation of the relationships among bacterial genomes for 4 reasons: 1) it produced a fully resolved tree, which is essential for many of the downstream analyses that we intend to do; 2) it provides an estimate of branch lengths, which is not only generally informative, but also essential for many downstream analyses; 3) it is accompanied by support values that are meaningful, if for no other reason, than the community at large is accustomed to an intuitive interpretation of bootstrap support values, where as the concordance factors produced by BUCKy, when this large number of taxa are analyzed, are currently difficult to interpret, ; and 4) because it is much simpler and less time-consuming to run than any of the other methods we tested here.www.figshare.com).All alignments and trees produced in this study are freely available via Figshare . Phylogenetic tree of 761 bacterial taxa, inferred from a concatenated, partitioned alignment of 24 genes using RAxML. The branches of phyla with at least 5 representatives are colored, other lineages are all drawn with black lines. Support values are calculated from 100 rapid bootstrap replicates. This is the same tree as (PDF)Click here for additional data file.Figure S2Maximum likelihood tree of bacteria from 16S rRNA genes . Phylogenetic tree of 761 bacterial taxa, inferred from an alignment of the 16S rRNA gene using RAxML. The branches of phyla with at least 5 representatives are colored, other lineages are all drawn with black lines. Support values are calculated from 100 bootstrap replicates. This is the same tree as (PDF)Click here for additional data file.Figure S3FastTree of all bacteria and archaea analyzed (2966 taxa) from 38 concatenated genes. Phylogenetic tree of 2966 bacterial and archaeal taxa, inferred from a concatenated alignment of 38 marker genes  using FastTree.(SVG)Click here for additional data file.Figure S4Maximum likelihood tree of bacteria and archaea from concatenated alignment. Phylogenetic tree of 841 bacterial and archaeal taxa, inferred from an alignment of 24 marker genes using RAxML. Support values are calculated from 100 bootstrap replicates.(PDF)Click here for additional data file.Figure S5BUCKy tree . Primary concordance (“BUCKy”) tree constructed using Bayesian Concordance Analysis of RAxML bootstrap replicates for each of the 24 phylogenetic marker genes. Values at the nodes are concordance factors. The branches of phyla with at least 5 representatives are colored, other lineages are all drawn with black lines. This is the same tree as (PDF)Click here for additional data file.Figure S650% majority rule consensus computed from the 24 best ML single-gene trees generated by RAxML.(PDF)Click here for additional data file.Figure S7Concatenated Maximum Likelihood tree. Phylogenetic tree inferred from a concatenated, partitioned alignment of 24 genes using RAxML. The branches of phyla with at least 5 representatives are colored, other lineages are all drawn with black lines. Support values are calculated from 100 rapid bootstrap replicates. This representation is a radial cladogram, in which branch length is not proportional to time, and some branches may be elongated so that the names of the taxa appear on the circumference of the circle.(EPS)Click here for additional data file.Figure S816S rRNA Maximum Likelihood tree. Phylogenetic tree inferred from an alignment of the 16S rRNA gene using RAxML. The branches of phyla with at least 5 representatives are colored, other lineages are all drawn with black lines. Support values are calculated from 100 bootstrap replicates. This representation is a radial cladogram, in which branch length is not proportional to time, and some branches may be elongated so that the names of the taxa appear on the circumference of the circle.(EPS)Click here for additional data file.Figure S9BUCKy tree. Primary concordance (“BUCKy”) tree constructed using Bayesian Concordance Analysis of RAxML bootstrap replicates for each of the 24 phylogenetic marker genes. Values at the nodes are concordance factors. The branches of phyla with at least 5 representatives are colored, other lineages are all drawn with black lines. This representation is a radial cladogram, in which branch length is not proportional to time, and some branches may be elongated so that the names of the taxa appear on the circumference of the circle.(EPS)Click here for additional data file.Figure S10A majority-rule consensus tree calculated from the bootstrap replicates of one of the 24 genes. Majority-rule consensus tree computed from the bootstrap replicates for the 30S ribosomal protein S3. This gene has an alignment length of 180 sites, which is the average length for the 24 marker genes used in this study. This representation is a radial cladogram, in which branch length is not proportional to time, and some branches may be elongated so that the taxa appear on the circumference of the circle.(EPS)Click here for additional data file.Table S1Organisms used in this study. List of organisms used in this study, with (when available) NCBI accession numbers and IMG taxon ID numbers.(XLS)Click here for additional data file.Datafile S1Maximum likelihood tree of bacteria from concatenated alignment (Newick format). Phylogenetic tree of 761 bacterial taxa, inferred from a concatenated, partitioned alignment of 24 genes using RAxML. Support values are calculated from 100 rapid bootstrap replicates. This is the same tree as (TXT)Click here for additional data file.Datafile S2Maximum likelihood tree of bacteria from 16S rRNA genes (Newick format). Phylogenetic tree of 761 bacterial taxa, inferred from an alignment of the 16S rRNA gene using RAxML. Support values are calculated from 100 bootstrap replicates. This is the same tree as (TXT)Click here for additional data file.Datafile S3FastTree of all bacteria and archaea analyzed (2966 taxa) from 38 concatenated genes (Newick format). Phylogenetic tree of 2966 bacterial and archaeal taxa, inferred from a concatenated alignment of 38 marker genes  using FastTree. This Newick format is suitable for use in most tree-viewing programs.(TXT)Click here for additional data file.Datafile S4Maximum likelihood tree of bacteria and archaea from concatenated alignment (Newick format). Phylogenetic tree of 841 bacterial and archaeal taxa, inferred from an alignment of 24 marker genes using RAxML. Support values are calculated from 100 bootstrap replicates. This Newick format is suitable for use in most tree-viewing programs.(TXT)Click here for additional data file.Datafile S5BUCKy tree (Newick format). Primary concordance (“BUCKy”) tree constructed using Bayesian Concordance Analysis of RAxML bootstrap replicates for each of the 24 phylogenetic marker genes. Values at the nodes are concordance factors. This is the same tree as (TXT)Click here for additional data file.Datafile S650% majority rule consensus computed from the 24 best ML single-gene trees generated by RAxML (Newick format). This is the same tree as (TXT)Click here for additional data file.Datafile S7Perl script that can be used  to convert RAaML bootstrap replicates into a format that can be used as input for BUCKy.(TXT)Click here for additional data file.


Saccharomyces cerevisiae normally selects bud sites (and hence axes of cell polarization) in one of two distinct patterns, the axial pattern of haploid cells and the bipolar pattern of diploid cells. These patterns depend on distinct sets of cortical-marker proteins that transmit positional information through a common signaling pathway based on a Ras-type GTPase. It has been reported previously that various proteins of the endocytic pathway may be involved in determining the bipolar pattern but not the axial pattern. To explore this question systematically, we constructed and analyzed congenic haploid and diploid deletion mutants for 14 genes encoding proteins that are involved in endocytosis. The mutants displayed a wide range of severities in their overall endocytosis defects, as judged by their growth rates and abilities to take up the lipophilic dye FM 4–64. Consistent with the previous reports, none of the mutants displayed a significant defect in axial budding, but they displayed defects in bipolar budding that were roughly correlated with the severities of their overall endocytosis defects. Both the details of the mutant budding patterns and direct examination of GFP-tagged marker proteins suggested that both initial formation and maintenance of the normally persistent bipolar marks depend on endocytosis, as well as polarized exocytosis, in actively growing cells. Interestingly, maintenance of the bipolar marks in non-growing cells did not appear to require normal levels of endocytosis. In some cases, there was a striking lack of correlation between the overall severities of the general-endocytosis defect and the bud-site selection defect, suggesting that various endocytosis proteins may differ in their importance for the uptake of various plasma-membrane targets.The yeast  Saccharomyces cerevisiae provides an outstanding model for studies of the mechanisms of cell polarization a or Matα) use the axial pattern, in which each cell's first bud is made adjacent to the birth scar that marks its site of separation from its mother, and each subsequent bud is made adjacent to the immediately preceding division site. In contrast, normal diploid cells (Mata/α) use the bipolar pattern, in which the first bud is almost always formed at the pole distal to the birth scar, whereas subsequent buds can be formed at either pole. These two patterns depend on a common signaling pathway based on the Ras-like GTPae Rsr1/Bud1 Cell polarization is an important characteristic of most cell types and involves both the selection of a polarization axis and the subsequent asymmetric organization of the cytoskeleton, intracellular organelles, and plasma-membrane components along this axis It has also been reported that mutants defective in several actin-patch-associated proteins have defects in bipolar budding The apparent involvement of endocytosis proteins in bipolar budding suggested that the observed persistence of the bipolar markers might not be a static state but rather one involving a dynamic interplay between polarized exocytosis and endocytosis. To explore this possibility, we constructed and analyzed a set of haploid and homozygous-diploid deletion mutants for 14 endocytosis-related genes. The results help to clarify the role of endocytosis in the generation and maintenance of the bipolar-budding markers and also suggest that various endocytosis proteins may differ in their importance for the uptake of various plasma-membrane targets.bud9Δ::HIS3 strain YHH614 BUD8 or AXL2 had been deleted as described below for the endocytosis genes. The other mutant yeast strains used in this study (a and Matα haploid deletion strains were mated to obtain the homozygous diploids. Plasmids used in this study are described in Escherichia coli strain DH12S (Life Technologies) was used as a host for plasmids. Except where noted, yeast cells were grown at ∼24°C in synthetic complete (SC) medium with 2% glucose as carbon source; specific nutrients were omitted as needed to maintain plasmids Strain KNY408 was constructed by several crosses starting with is study  were con600 = 0.3–0.6). The OD600 was recorded, the culture was diluted 2-fold with fresh medium, and the time required to grow back to the original OD600 was determined.To determine doubling times in liquid medium, cells were grown to exponential phase , and the remaining cells were then collected by centrifugation (as above), released in 4 ml of fresh YM-P medium at ∼24°C, and examined by fluorescence microscopy after 5 min. Only one or two strains were included in each experiment to ensure that all samples were treated similarly.Cells were grown in YM-P medium to exponential phase and collected by centrifugation at ∼1,200 To assess the overall budding patterns of haploid cells, cells with three bud scars were scored for whether those bud scars were or were not all clustered at the birth-scar-proximal pole. To assess the overall budding patterns of diploid cells, cells with ≥5 bud scars were classified into three groups: bipolar ; random ; and unipolar . To score the positions of first, second, and third buds on diploid cells, cells with a growing bud and the appropriate numbers of bud scars  were scored for the position of the growing bud relative to the birth scar.rvs167Δ strain appeared to grow significantly better on solid medium, for unknown reasons.) We used the relative growth rates in liquid medium to organize the data presentation in To investigate the role of endocytosis in bud-site selection, we generated a set of haploid and homozygous-diploid deletion mutants for 14 endocytosis-related genes in a common strain background . We firsede1Δ and myo5Δ . Mutant and wild-type cells that had been labeled on ice to inhibit endocytosis showed a uniform plasma-membrane labeling . The mutnd myo5Δ ; moderatnd vrp1Δ ; and sev rvs161Δ . As expeabp1, cap1, ede1, end3, las17, rvs161, rvs167, sac6, sla1, sla2, swa2, and vrp1 mutants To investigate the possible roles of endocytosis in the generation and/or maintenance of the spatial markers that guide bud-site selection, we examined the effects of the 14 deletion mutations on the budding patterns of both haploid and diploid cells. All of the haploid mutants showed seemingly normal axial budding , as repovrp1Δ , all of the mutants showed little difference from wild type in their ability to position the daughter cell's first bud at the birth-scar-distal pole  with the least severe endocytosis defects. Even the mutants with more severe endocytosis defects showed effective use of the proximal pole in the second cell cycle  and the severity of the bud-site selection defect suggests that the various proteins of the canonical endocytosis pathway


Background  The relationship between handedness and digital dermatoglyphic patterns has never been investigated in the Sinhalese population. The goal of this study is to establish the above mentioned relationship, which would positively aid personal identification. Findings One hundred forty Sinhalese students (70 right-handed and 70 left-handed) were studied for their digital dermatoglyphic pattern distribution. The results show that a statistically significant correlation exists for; digit 5  of the right hand in female, digit 1  and digit 2  of the left hand in females, digit 3  and digit 4  of the right hand in males, digit 4  of the left hand in males.Conclusions  Statistically significant differences in handedness and digital dermatoglyphic patterns were evident among Sinhalese people. Further study with a larger sample size is recommended. Because they are unique for each individual and are strongly influenced by genetics, they also perform a significant role in anthropology, human genetics, ethnology and medicine. They are characterized by alternating strips of raised friction ridges and grooves present in a variety of patterns2. These patterns start to develop between the 5th and 6th week of intrauterine life, and are fully formed by the 21st week3. These patterns do not change throughout postnatal life and their development is determined by several genes4.Fingerprints  are a unique form of evidence that greatly contribute towards personal identification in forensic science5. Determination of the handedness of both the assailant and the victim are important in various aspects of forensic science, including personal identification6. Hence, establishing the relationship between handedness and digital dermatoglyphics will aid forensic identification.Handedness (i.e. hand dominance) is defined as the uneven distribution of fine motor skills between the left and right hand13 have investigated whether there is a correlation between handedness and digital dermatoglyphics. In 1940 Cummins discovered a slight association in the sex differences of asymmetrical occurrence of dermatoglyphic patterns8. Cromwell and Rife in 1942 found that left-handers are characterized by slightly less bimanual asymmetry than right-handers among on Caucasian school children in southwestern Ohio9. In 1943 Rife found associations characteristic of autosomal linkage between the whorl frequencies on the fingers and handedness among descended from northern European stock10. In 1994 Coren reported an increased number of arches, fewer whorls in left-handers as compared to the right-handers among Canadians11. Cho in 2010 found significant difference of dermatoglyphics patterns on digit 3, 4 and 5 among Koreans12. None have investigated this association in a Sinhalese population (an Indo-Aryan ethnic group who are native to the island of Sri Lanka14). The main goal of the current study is to determine the relationship between handedness and digital dermatoglyphics in a sample of Sinhalese population.To date, scarce amount of studies15. This required participants to demonstrate 10 unimanual tasks . These tasks are common to Sri Lankans and they were advised to state the degree of preference for the hand used in each case as either strong (two points) or weak (one point). The handedness measure was calculated by subtracting the score for the left hand from the score for the right hand, dividing by the sum of both, and multiplying it by 100, providing an absolute range from -100 (completely left-handed) to +100 (completely right-handed). We recruited 70 predominant right-handers and 70 predominant left-handers after evaluating handedness.The study was conducted at the Department of Forensic Medicine, Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka. Ethical clearance for this study was obtained from the Ethical Clearance Committee of the institute. Total of hundred forty Sinhalese students  who gave informed written consent were included in the study. Ages of females ranged between 21 and 28 years (mean ± s.d. = 24.40 ± 1.82 years) and males ranged from 22 and 28 years (mean ± s.d. = 24.67 ± 1.92 years). Firstly, handedness was assessed using the Edinburgh Handedness Inventory2. The subject was asked to roll their finger from the radial side to the ulnar side on an ink pad and then transfer their fingerprints in the same manner onto the allocated area of a double sheet of plain A4 paper . Descriptive statistics were used to express the data. Correlations between handedness and digital dermatoglyphics were evaluated by a two-sided Fisher’s exact test. P values less than 0.05 were considered statistically significant.In this study we observed the handedness-wise digital dermatoglyphics pattern distribution of 140 individuals .Right hand.Left hand.Right hand.Left hand.The percentage of digital dermatoglyphics pattern distributions for both hands in male and female Sinhalese are shown in1. This is valuable as a means of identification. In this study, effort has been made to study the relationship between dermatoglyphic and handedness in 140 Sinhalese students.It has been affirmed that the digital dermatoglyphic pattern of the skin is unique and unchallengeable for an individualThe results showed that a statistically significant correlation exists in digit 5 of the right hand while digit 1 and digit 2 of left hand in female. In males digit 3 and digit 4 of right hand and digit 4 of left hand showed a statistically significant correlation.In the past, few studies have been conducted on different ethnic groups with the idea of establishing a relationship between handedness and dermatoglyphic pattern. Results of some studies are in line with the present study.9 observed a slightly higher frequency of whorls (1.3%) on left ring fingers (digit 4) of left-handers than of right-handers. Whorls were absent on the right ring finger of both right- and left-handers. They further observed that the incidence of arches only on digit 3 of right hands shows highly significant differences between left-handers and right-handers (P<0.001).In their study on Caucasian school children in southwestern Ohio, Cromwell and Rife (1942)11 in his study on Canadians found that left-handers were more likely to have arches and radial loops, while fewer whorls than right-handers. The correlation of handedness and digital dermatoglyphics was most marked on the left hand, which showed significant differences on four digits except digit 1. On the right hand, handedness was associated with a digital dermatoglyphics patterns only on digit 4.Coren (1994)12, in their study on Koreans, found that both hands of left handers exhibited more arch and ulnar loop types than the right-handers and less whorl and radial loop types than the right-handers. The digital dermatoglyphic pattern of digit 3, digit 4 and digit 5 of the left hand showed a statistically significant relationship between left- and right-handed people.Cho (2010)13, he found that whorls were significantly less frequent, and ulnar loops significantly more frequent in all digits for right-handed people when compared to left-handed people. The ulnar fluctuating asymmetries of digits 1 and 4 showed a highly significant relationship with handedness.In Karev's study on Bulgarian individuals16, in his study on students at Ohio State University, USA, observed that arches were more common on the left middle finger of right-handed students than left-handed students. Left-handedness has a frequency of about 10% in the general population with a slightly higher frequency in the male population compared to the female population18. In our study we analyzed dermatoglyphics pattern of 70 left hander’s  and compared it with right hander’s . Gender wise differences in digital dermatoglyphics patterns have been established for now and then19. We compared handedness wise difference of dermatoglyphics pattern in right and left hand of both male and female Sinhalese separately.Rife (1955)The major limitation of our study is the small sample size. Despite the small sample size, it exhibited a significant handedness wise difference of dermatoglyphics among Sinhalese. Additional research involve large sample are needed to further confirm current findings.The present study supports the hypothesis that handedness and digital dermatoglyphics are correlated in members of the Sinhalese population. Our results show that there is a statistically significant difference in fingerprint patterns between right- and left-handed people for digit 5 of the right hand and for digits 1 and 2 of the left hand in females, and digit 3 and digit 4 of the right hand and digit 4 of the left hand in males. The results of this study support the relationship between handedness and digital dermatoglyphics in the Sinhalese population. The results can be used as supporting evidence for personal identification. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In the Methods section, the authors say: “We recruited 50 predominant right-handers and 50 predominant left-handers after evaluating handedness” but in the Results section they say “In this study we observed the handedness-wise digital dermatoglyphics pattern distribution of 140 individuals ". The error should be correctedI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The article is clearly laid out and all the key elements are present. It was significantly improved after including previous reviewers' comments.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We would like to thank Matea Zajc Petranović for spending her valuable time to review the manuscript. The study was based on data from 100 individuals . The greatest limitation of this study is the small sample size, as authors indicate; this size would be even smaller if the sample was analyzed by sex, as should have been done. The distribution of digital patterns presents sex differences in most of the samples analyzed, and so, the results cannot be accepted because they are affected by the effect of sex differences.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. We would like to thank Professor Esperanza Gutierrez Redomero for the valuable time spent reviewing our manuscript and the important comments she has made. We have attempted to address and incorporate most of the concerns that were raised in version 2 of our article.


Previously, it was suggested a relation between the presence of apoptosis markers with cytoplasm in mammalian sperm. In this work, flow cytometry, confocal microscopy and image analysis were used to analyze the relationship between active caspase-3 and -7 and intracellular esterases expression in ejaculated or epididymal ram sperm. Sperm obtained from ejaculates from the caput, corpus, or cauda of the epididymis were treated with an inhibitor of active caspase-3 and -7 and a marker of cytoplasmic esterases. Additionally, ejaculated sperm were incubated for one, two, or three hours before evaluation for active caspases. Sperm subpopulations positive for active caspases and/or intracellular esterases were detected by flow cytometry and confocal microscopy; however, image analysis of confocal images showed that the correlation between active caspases and cytoplasmic esterases in sperm is region-specific. Lower values of Spearman correlation coefficients were found when whole sperm or head sperm was analyzed; however, a high correlation was observed for midpiece sperm. Incubation of sperm for two or three hours promoted the autoactivation of caspases. It has been suggested that the presence of apoptotic markers in sperm are related with a process of abortive apoptosis and with errors during spermiogenesis. Our results permit us suggest that the origin of the relationship between active caspases and cytoplasmic esterases is due to differentiation errors occurring during spermiogenesis because the percentages of sperm with active caspases are not different in the caput, corpus, or cauda of the epididymis. In this study we demonstrate that existing sperm subpopulations can express active caspases and intracellular esterases and that the correlation between these molecules is high in midpiece sperm. Somatic Because of this, we hypothesize that the abnormal presence of cytoplasm in sperm could be related to the presence of active caspases.Calcein is a non fluorescent membrane permeable used to detect viable cell On the other hand, active caspases are detected immediately when sperm are ejaculated; this clearly indicates that sperm with active caspases are in the male reproductive tract, although it is not know where the caspases were activated. During traverse for different regions of epididymis the sperm undergo a series of changes called maturation, one of these changes involve elimination of cytoplasmic droplet in vitro it is possible that caspases are activated during such process. In this study we analyzed a) the relationship between spermatic cytoplasm and active caspases in ejaculated sperm, b) whether the presence of sperm with active caspases change during epididymal transit, and c) whether the presence of active caspases is related with incubation time.Caspases in sperm are prone to be activated All experiments were performed using neat semen. The rams were 1.5 years-of-age and weighed 65–80 kg. Subject animals were kept at the Facultad de Medicina Veterinaria y Zootecnia under uniform nutritional conditions. Semen samples were collected from four mature mexican creole rams. Ejaculates were obtained from each ram every second day. At least five ejaculates were obtained from each animal. With the aim of minimizing the rams' stress, the technician in charge of obtaining the ejaculates was always the same. Rams were human killed according to Norma Oficial Mexicana NOM-033-ZOO-1995 and epididymides were obtained. The protocol of this work was revised by Bioethics Committee of Facultad de Medicina Veterinaria y Zootecnia the nine of October of 2009 and approval letter (s/n) was received.In order to achieve the objective of this work, three experiments were conducted. For the first, active caspases-3 and -7, and cytoplasm were detected in ejaculated sperm by flow cytometry and image analysis of confocal microscopy. For the second experiment, ejaculated sperm were incubated for one, two, or three hours before evaluating the presence of active caspases. For the third experiment, the presence of active caspases-3 and -7, and cytoplasm was evaluated by flow cytometry in sperm from different regions of the epididymis.in situ with a fluorescent inhibitor of caspases (FLICA™) using the Image-iT™ LIVE Red Caspase-3 and -7 Detection Kit  according to the manufacturer's instructions. Briefly, 10 µl of a FLICA working solution was added to 300 µl of sperm and mixed gently. The suspension was incubated for 60 minutes at 37°C and protected from light. After incubation, the samples were stained with an indicator of cytoplasm. In this study, we used calcein as an indicator of cytoplasm. Calcein-acetoxymethylester  is a non-fluorescent membrane-permeable molecule that is reduced to fluorescent calcein by intracellular eesterases de novo by adding sodium nitroprusside  at a final concentration of 400 µM during one hour de novo active caspases are detected.Active caspases-3 and -7 activity were labeled The volume of all samples was adjusted to 500 µl with TNE buffer  prior to flow cytometry analysis. A FACScan flow cytometer  equipped with an argon laser (488 nm) was used to evaluate the sperm parameters. A live gate was used in the FSC and SSC parameters to exclude aggregates and debris, thereby, restricting data acquisition to an almost pure population of sperm. List mode data of 10,000 events were collected for each sample using CellQuest 3.3 software (BDIS) in a Power Mac G4 with Mac OS 9. Percentages of sperm positives to active caspases or cytoplasmic esterases were obtained with WinList 3.0 software  in a PC with windows XP.The left epididymis was used to obtain sperm from the caput, corpus and cauda. For one animal, the right epididymis was selected because the left epididymis presented with cysts, without inflammation or other pathological signs. The caput, corpus, or cauda of the epididymis was placed in a petri case with 10 ml of PBS buffer (pH 7.4), minced with scissors, and incubated for 20 min at 35°C. Sperm were filtered with a nylon mesh of 100 µm in a sterile tube of polypropylene. Detection of active caspases and cytoplasmic esterases was done as specified previously.4, 0.3 mM sodium pyruvate, 0.3 mM K2HPO4, 1.5 IU/mL penicillin, and 1.5 mg/mL streptomycin, pH 6.4) (SIGMA) at 37°C under 5% CO2 in air during one, two or three hours before evaluated for active caspases. Detection of active caspases was done as specified above.Aliquots of ejaculated sperm were incubated in HTF medium . Micrographs of sperm coexpressing cytoplasmic and active caspases-3 and -7 were captured with EZ-C1 2.30 software (Nikon).Almost one image of sperm coexpressing active caspases and intracellular esterases was obtained for each slide . Confocal images were digitally processed to obtain the relation between values of pixels in green and red channel. Images were processed with ImageJ 1.42q software All results are expressed as mean ± SEM. Normality of percentages of sperm with active caspases was corroborated graphically in different experiments by checking residuals. An ANOVA for repeated measures was performed to compare percentages of ejaculated sperm with active caspases after different incubation periods, and a one way ANOVA was conducted to analyze the percentages of sperm with active caspases in different regions of epididymides; the Tukey test was used as a post hoc test. Relationships among sperm subpopulations  or between pixels of red (active caspase-3 and -7) and green (cytoplasmic esterases) channels of confocal mycroscopy images were analyzed by Spearman correlation. All analysis were performed with the R 2.9.0 software FLICA stained active caspases in sperm, but when a nonfluorescent inhibitor of active caspases was added before FLICA, that stain diminished . Based oTo analyze the role of time of incubation on activation of caspases aliquots of ejaculated sperm were incubated for one, two, or three hours before evaluation of active caspases. The percentage of total sperm expressing active caspases increased after two (P = 0.026) or three (P = 0.045) hours of incubation .Confocal images of sperm coexpressing active caspases and cytoplasmic esterases were digitally processed to separate red and green channels and to determine threshold pixels of sperm in the blue channel to create a mask before conducting the correlation analysis . The relThe mean percentages of sperm expressing active caspases from caput, corpus or cauda of epididymis did not differ. However, the percentages of total sperm expressing active caspases in different regions of epididymis were lower than those of the ejaculates.The presence of active caspases has been demonstrated in the sperm of ovine In this work, we observed different sperm subpopulations, one of which presents concomitantly cytoplasmic esterases and active caspases. The fact that sperm subpopulations with different cellular properties such as the presence of cytoplasmic esterases and active caspases are detected in an ejaculate could indicate differences in alteration during transit through the epididymis or alteration during human management. We did not observe changes in the percentage of sperm with active caspases from the caput, corpus, or cauda of the epididymis, but the percentage of ejaculated sperm with active caspases increased after three hours of incubation. The fact that changes in the percentage of sperm with active caspases in the different regions of epididymis were not observed in this work indicates that 1) these molecules are carried in an active state from the time they leave the testis or 2) that conditions of incubation could activate caspases. This supports the suggestion of events of “abortive apoptosis” proposed by Sakkas et al. For testicular germ cell of rats, it has been demonstrated that some markers of apoptosis are restricted to residual bodies On the other hand, two studies indicated that FLICA inhibitors could associate with no identified target(s) other than cysteine of active caspases, although the expression follows the pattern of apoptosis For humans a broad range of percentages of sperm with active caspases has been observed when FLICA is used (∼15–80%) We do not know why the percentages of of sperm expressing active caspases were high in our study; an explanation to this is related with a possible low specificity of FLICA reagent, however it is discarded due to a increase of sperm with active caspases is observed when an inducer of active caspases is added to incubating sperm; conversely, the percentage of sperm with active caspases do not increase when an inhibitor of active caspases is added to incubating sperm before addition of the inducer of active caspases, as observed in our controls.In summary, we found that ejaculated sperm form a heterogeneous population that includes subpopulations one of which carries active caspases and cytoplasmic esterases. In sperm marked for active caspases and cytoplasmic esterases, there is a correlation between these two markers in the middle piece. Active caspases are activated during incubation but not during epididymis traverse. It is possible, then, that active caspases are carried in that state from the testis.


Hypothesis: Light at certain wavelengths, can cross the cell, tissue sections and organ tissues for in-vitro studies, but can also be used for in-vivo studies. The ability to guide the growth of neural extensions was proven by laser. Synapses produced with laser have the instability as main disadvantage, in the absence of high densities of neurons. The main mechanism of synapse formation is the synaptic plasticity. In this case, dendritic learning spines increase in size and are transformed in dendritic memory spines.Aim: The goal of this study was to evaluate if the creation of synapses using laser, without cell disruptions, is feasible.Methods and Results: We have stimulated neuronal guiding growth using a laser system called multipoint optical tweezers. Approaches were made between dendrites and neuronal bodies. Normal mechanism of synapses formation was stimulated by using electrical stimuli, applied by using a patch-clamp set-up. This approach revealed the transmission of electrical stimuli on both sides of the synapse, but also its unidirectional transmission, which is correlated with cell integrity.Discussion: The feasibility of achieving laser synapses was proven, and this method can be useful for the development of neural circuits, and for the modulation of the existing ones. We suggest that light is the best tool to guide neural growth and synapse formation. This method is cost efficient and very easy to perform. Our model is very good for understanding molecular mechanisms in pathological neurons that are relocated in key positions from pathological tissues or from transfected cell lines. Neuroscience is a fast growing research field interdependent with borderline research areas. Biophysics and biotechnology offer tools for studying neurons from single cell to more complex intercellular interactions. It represents a gap between researchers from different fields that requires predictability for scientists who can design a new method to investigate neural networks. If research of single cell is covered by equipment, interactions between cells are limited to self-assembling interactions or slice from brain. For the better understanding of neurons in single cell and interactions in neural network, we need to be able to design guided neural networks that can answer to specific questions. It is possible to create neural networks on predesigned patterns, but we are limited by insufficient research tools that can be provided by applied research fields. 1]. In order to increase the speed of this process, it is necessary to run a huge number of tests that require an equivalent number of measurement set-ups [2]. Optical investigation methods have accuracy and are relatively non-expensive [3]. Interaction between light and living cells can be mediated by small mechanisms that are inspired from Mother Nature.Neural networks models are very important for pharmacologic industry. Drug design process needs an important step of testing on similar models in order to observe different actions of proposed active compounds [4] that cannot be challenged by any human designed computer. In order to increase resistance to nuclear radiation we can transfect factors from radio resistant tumors, or use neuron lines from scorpions. There are many well-known examples of radio resistance. Nuclear research can benefit from tools designed this way. Neural networks can give an important alternative for computing in nuclear radiation area. Living cells have a capacity to auto repair [5]. Small synaptic spins, which can be formed without environmental stimulation, represent short lifetime interneuron connection [6]. If synapse is efficient in transmitting signals, these spins can be transformed in large spins with long lifetime [7]. Researches in guided neural growth show that is possible to use light to manipulate the way of neural development. This is important for the construct predesigned neural pathways from individual cells to almost connected cells [5]. Neurons can make synapses randomly, but only frequently used synapses have a long lifetime [8]. These oscillators can be inhibited or stimulated by environmental factors, locally or externally. It is possible to create logical circuits that can be assembled in more complex biological computer. From these considerations, neural networks constructed this way, need an auto-oscillatory design for self-organizing after guided neural growth , which revealed the possibility to guide the growth of neuronal extensions with lasers.The punctual issue of this paper focuses on the construction of synapse with lasers. This approach was inspired as an element of Ehrlicher experiments published in 2002 [Dorsal root ganglia neurons culture9]. They were incubated for 1h at 37ºC in IncMix solution containing (in mM): NaCl 155; KH2PO4 1.5; HEPES 5.6; NaHEPES 4.8; glucose 5, 50 μg/ml gentamicin, 1 mg/ml collagenase  and 1 mg/ml dispase (Gibco). Neurons were dissociated by trituration and plated for 1h on Petri dishes (Corning) treated with poly-D-Lysine  and were cultured at 37ºC in DMEM and Hams’s F10 medium with 10% horse serum and 50 μg/ ml gentamicin, 5% CO2.Adult male Wistar rats (150-200 g) were anesthetized by inhalation of 100% CO2 (2 min exposure) followed by decapitation. The procedures were conducted in accordance with the Guidelines of University of Bucharest regarding the care and use of animals for experimentation. Dorsal root ganglia were removed as previously described [SolutionsThe extracellular solution contained (in mM): 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES (pH 7.4 with NaOH at 25ºC) to which glucose (5 mM) was added on the day of the experiment. The intracellular solution contained (in mM): 120 KCl, 10 NaCl, 10 BAPTA / 24.2 KOH, 10 HEPES, 1 CaCl2, 3.45 MgCl2, (pH 7.2 at 25ºC with KOH) and filtered at 0.2 μm immediately before use. All the reagents were purchased from Sigma.Electrophysiological recordingsElectrophysiological characterization of neurons in primary culture was performed by whole-cell patch-clamp technique. Borosilicate micropipettes were prepared at a resistance of 3-7 MΩ with a P-30 vertical pipette puller (Sutter). Whole-cell recordings were made with Axopatch 200B (Axon Instruments). Stimuli were controlled and digitized with pClamp 10.0 and Digidata 1440 interface (Molecular Devices). Series resistance (Rs) was compensated (40-60%) with the patch-clamp amplifier. Double patch-clamp recordings were used to evaluate synapses. Synaptic connections were detected in current-clamp mode by stimulating the presynaptic neuron depolarization by injecting a rectangular current of 100-300 pA with duration of 1 s and recording in the postsynaptic neuron the depolarizing postsynaptic potential. The protocol was repeated in reverse order, stimulating the postsynaptic neuron and recording presynaptic neuron.Neural guiding and synapse formationLaser stimulation of neuronal extensions is a common method of guiding neuronal growth. This is dependent on the wavelength, to a degree that cannot be presently evaluated. Stimulation was performed at a wavelength of 1064 nm. Laser stimulation was made by using the optical tweezers set, mounted on an AxioObserver D1 (Zeiss). Neuronal extensions were guided to the proximity of neuronal bodies, in order to optimize spatial formation of synapses.Fig. 1).It starts from a cell that already has an extension of neuronal, possibly an extension of neuronal asymmetry and laser stimulation, in order to guide neural growth , within 2 minutes from the previous image. After laser pulse stimulation, we noticed an increase in neuronal extension, in the direction of optical tweezers .To reflect the substrate adhesion of the neural guided growth, guidance is accomplished correlated with changing direction of extension growth by stimulating the X-axis (to the left), for a period of about 1 minute .An important characteristic of laser radiation is the ability to perform photoporation. This provides a solution of continuity between the extracellular and intracellular environment. Synapse formation by neuronal growth guidance may lead to mechanical tension in the membranes of neuronal extensions, with an increase in their fragility. We assessed its unidirectional transmission to demonstrate if there was a synapse or a gap type connection mediated by photoporation between the two cells , and then vice versa . The existence of chemical synapses mediated by optical guided synapse formation was confirmed by unidirectional transmission.Initially, the neuron with optical guided neuronal growth was electrically stimulated, and neuronal activity was recorded on target neuron .Laser light is a great tool, but it is not used at its full capacity in medicine. It was proved that the creation of synapses using laser, without cell disruptions, is feasible. Synapse function was evaluated by electrophysiological methods, which allowed an assessment of cellular integrity by using the unidirectional transmission of the electrical stimulus  of interest. 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