The 60,000 selected SNPs, evenly spaced on the goat genome, were submitted for oligo manufacturing  and published in dbSNP along with flanking sequences and map position on goat assemblies (i.e. scaffolds and pseudo-chromosomes), sheep genome V2 and cattle UMD3.1 assembly. Ten breeds were then used to validate the SNP content and 52,295 loci could be successfully genotyped and used to generate a final cluster file. The combined strategy of using mainly whole genome Next Generation Sequencing and mapping on a contig genome assembly, complemented with Illumina design tools proved to be efficient in producing this GoatSNP50 chip. Advances in use of molecular markers are expected to accelerate goat genomic studies in coming years.The success of Genome Wide Association Studies in the discovery of sequence variation linked to complex traits in humans has increased interest in high throughput SNP genotyping assays in livestock species. Primary goals are QTL detection and genomic selection. The purpose here was design of a 50–60,000 SNP chip for goats. The success of a moderate density SNP assay depends on reliable bioinformatic SNP detection procedures, the technological success rate of the SNP design, even spacing of SNPs on the genome and selection of Minor Allele Frequencies (MAF) suitable to use in diverse breeds. Through the federation of three SNP discovery projects consolidated as the International Goat Genome Consortium, we have identified approximately twelve million high quality SNP variants in the goat genome stored in a database together with their biological and technical characteristics. These SNPs were identified within and between six breeds : Alpine, Boer, Creole, Katjang, Saanen and Savanna, comprising a total of 97 animals. Whole genome and Reduced Representation Library sequences were aligned on >10 kb scaffolds of the  Goat was one of the first domesticated animals, around 10,500 years ago in the Fertile Crescent  in 2010, whose aims were to consolidate research efforts at the international level. The Goat Genome reference sequence has been published http://goat.kiz.ac.cn/GGD/). A RH panel has also been developed Recently, the development of next generation sequencing (NGS) allowed de novo assembled goat genome were made available by BGI sequencing center (www.genomics.cn) to allow mapping of the flanking sequences of the identified SNPs. The assembly consisted of 285,379 scaffolds representing 2,665,022,236 bp. Since a Yunling female goat was sequenced to create the assembly, no representation of the Y chromosome was included in the sequence. As shown in Scaffolds of the SNP discovery projects were underway in three laboratories, before they were consolidated within the IGGC. This explains why the SNP discovery was achieved through two different pipelines, for milk and mixed breeds  in one project and meat breeds  in another project.For dairy and mixed breeds, sixteen animals  were sequenced using a HiSeq2000  and produced 80 to 220 million high quality (passing filter) 100 bp reads per animal. This resulted in a sequencing coverage of 13.5 to 26X depending on the breed, after filtration of the BAM files. Another set of 17 Dutch Saanen animals was used to construct a RRL library. Sequencing from this RRL library resulted in 120 million 76bp sequences. The sequence coverage, using those data, after BAM filtration, was 2.85X on average in the genomic regions that were covered with one read at least.SNP discovery was performed for each breed separately after which the results were combined. A minimum of two reads per allele was mandatory in the definition of a SNP not including the reference, with a minimum sequencing depth of six and a maximum depth of 100, to avoid repeated regions. These criteria have also been used with success in cattle For meat breeds, sequencing was performed on two pools of animals (20 Boer goats in pool 1 and 20 Savanna together with 24 Katjang goats in pool 2). Over 680 million sequences (106 Gbp) were obtained for each pool, resulting in an average coverage of 35X. After alignment to sheep and cattle reference genomes, SNP discovery was performed within each pool and resulted in 4,001,890 SNPs . A minimum of three reads per allele was used to define a SNP, not including the reference. Around 90% of the four million SNPs and their flanking sequences were successfully aligned with goat sequence scaffolds. The number of these discovery SNPs is reported in Additional SNPs were identified in expression data, from published ESTs and RNASeq projects (see acknowledgements section) and included in the candidates for final SNP selection. The detection procedure described in the methods section was followed by mapping on the goat scaffolds and resulted in a 7,008 SNP list.Regarding PRNP, casein and DGAT1 genes, already known as important for milk composition and disease resistance, regions orthologous to cattle were identified on goat scaffolds 881, 980 and 2304 respectively  and exteA total of 11,924,638 variants were tracked including 1,229,120 Indels and 10,695,518 discovery SNPs. For each variant, Illumina Assay Design Tool (ADT) score, Infinium type  and allele frequency was stored and used for final SNP selection.Illumina iSelect 60K designs can include both Infinium II SNPs , that require a single bead type, and Infinium I SNPs  that require two bead types for genotyping Since the average spacing of markers on a 50–60K SNP chip is about 60,000 bp, we disregarded SNPs mapping on contigs of fewer than 10,000 bp. To maximise the success rate of the beadpool, we also eliminated INDELs or SNPs that a) represented >2 alleles b) were within 20bp from another variant c) had an ADT score <0.8 or d) had an estimated MAF <0.2 in breeds of interest. This list of 537,145 SNP was ranked using increasing category number  and decreasing MAF. Class 1 SNPs were detected in ESTs and all included. The following ten classes corresponded to a decreasing number of breeds in which the SNP is observed. The first selection of 60,000 SNPs was done with spacing along the genome using an in-house algorithm (available on request). More than 97% of the SNPs were heterozygous in at least three breeds. To capture some remaining large genomic regions without SNPs, 1,000 SNPs of this first list were replaced by an alternative SNP, presenting either a higher number category or a <0.2 MAF or an <0.8 ADT score. Finally, as shown in Estimated MAFs of the selected SNP ranged from 0.05 to 0.5 with 50% of the selected SNP with a >0.420 MAF, as shown in To check the spacing procedure on a closely related assembled genome, the selected SNP flanking sequences were aligned on cattle UMD3.1 assembly. A total of 59,001 SNP flanking sequences were aligned on the cattle genome. As shown in http://www.ncbi.nlm.nih.gov/projects/SNP/snp_viewBatch.cgi?sbid=1057128 and File S1). The submission also included 60bp flanking sequences on each side of the variant site, estimated MAF value, Illumina_score, Illumina_state (successful or not) and position on goat scaffolds, CHIR_1.0, Sheep_OARV2 and Bovine_UMD3.1. Unique dbSNP Submitter SNP (ss) accession numbers were assigned for each variant, including those on the goatSNP50 chip, and they are listed in File S2. The corresponding Reference SNP (rs) numbers were assigned for dbSNP Build 136 release in December, 2012. As the goat reference genome evolves, it is possible a small fraction of rs numbers may be updated in future dbSNP release if evidence suggests a different assignment.For the 60,000 selected SNPs, the variants were submitted to dbSNP under the handle IGGC with batch ID “CAPRI-BATCH1” (www.goatgenome.org).The file containing this information is also downloadable from the IGGC website . This beadpoool was tested with 288 animals. 281 animals were successfully genotyped on 52,295 loci (87.15% of the submitted SNPs and 98% of the synthesized SNPs) that had a minimum average call rate of 99.83% . These gThe results of MAF >5% are indicated in The GoatSNP50 chip described in this paper has been validated as a useful tool for a variety of goat breeds, with >78% SNPs segregating in seven breeds, including Angora and Skopelos, that had not been used for SNP discovery. This chip has already been successfully used for genetic diversity studies on Boer, Cashmere and Rangelang goats via the quality checking process by Illumina. This results in a random failure that is unrelated to the sequence and design of the probes themselves.Technology failures during probe synthesis resulted in 11% loss of SNPs that were submitted to design resulting in 89% conversion of the pre-manufacturing content which was slightly below expected conversion of working assays based upon the weighted average ADT design score of.95 leading to the expectation that 95% of the assays would convert to working assays. The reason for the failure of a subset of the probes to appear in the final post-manufacturing content is independent of the likelihood of a sequence to convert to a working design due to the random nature of the assembly of beads into wells and the minimum redundancy threshold in the Infinium manufacturing process for each probe. If a probe was manufactured successfully, but the number of beads representing that probe were not represented in a minimum redundancy of over 5x , then it was removed In addition, because we did not have goat genome input into the ADT tool, we were not able to do a check on probe design based upon expected repetitive, low diversity or otherwise known problematic regions of the genome. As a result, any scores that come from the design as “species: Other” will be slightly inflated compared to whether we had the genome check integrated into the design process.50–60K SNP chips are primarily used for finding association between markers and phenotypes. To achieve this well, both a uniform distribution of the SNPs on the genome and the complete coverage of the genome are required. A large excess of SNPs is required to be able to evenly space the SNPs on the genome. Groenen and co-authors The combined strategy of using mainly whole genome NGS and mapping on a contig genome assembly, coupled with Illumina designing tools proved to be efficient when creating the GoatSNP50 chip. We hope that with this tool goat genomics studies can now advance rapidly.DNA samples from this study came from three Institutes in France, Netherlands and Malaysia. Neither sperm collection nor blood sampling was performed specifically for this study. Sperm collection was performed on bucks by Capgenes, which obtained the authorization from DGAL  FR CC 860. Sperm collection was made by Artificial Insemination stations, and we used extra doses from this collection. Blood samples were taken from commercial farms. Animals did not belong to any experimental design but were sampled by veterinarians and/or under Veterinarian supervision for routine veterinary care; extra samples were requested when blood sampling occurred.High quality genomic DNA was extracted from sperm of seven Alpine, three Creole and six French Saanen bucks. Briefly, the content of three straws from the same animal was pelleted  after dilution in 500 µL of 10mM TrisHCl, 10mM EDTA, 150mM NaCl pH = 7.5 buffer and rinsed in the same buffer. Disulphide bonds of protamines were reduced with DTT and then proteins digested through the addition of 200 µL of 10mM Tris-HCl (pH 7.5), EDTA 10mM, NaCl 100mM, SDS, 2%, 0.1M DTT, incubation 1.5h at 65°C and then addition of 20 µL of 10mg/mL proteinase K and further incubation at 37°C overnight with agitation. The next day, two extractions with an equal volume of Tris-HCl (pH = 8) saturated phenol, followed by two chloroform extractions were performed and the final supernatant was precipitated with sodium acetate and ethanol. After centrifugation , the DNA pellet was dissolved in 500 µL of Tris-HCl 10mM, EDTA 0.1mM, pH = 7.4. DNA was extracted out of sperm from 17 Dutch Saanen bucks using a similar protocol. Genomic DNA was extracted from 300 µL of blood samples of 20 Boer goats, 20 Savanna goats and 24 Katjang goats individually using Promega Wizard Genomic DNA Purification System. DNA quantity and purity were measured using the Nanodrop ND1000. Possible degradation was inspected on an agarose gel and only high quality undegraded DNA samples were used to prepare the DNA pools.AluI or HhaI . The digested pools in Orange loading dye (Fermentas) were size-fractionated on precast 10% polyacrylamide in 1xTBE with the Criterion™ Cell (BioRad). After staining, the target fragment size range of 110–130 bp was sliced out of the gel. The gel slice was sheared by nesting a 0.5ml eppendorf tube (with a hole in the bottom formed with a needle) containing the gel slice inside a 2ml eppendorf tube, and centrifuged at 14000 rpm for 2 minutes. The sheared gel pieces were covered with 300ul DNA recovery buffer , vortexed, and eluted at 4°C overnight, followed by 15 minutes incubation at 65°C. The DNA was recovered using the Montage DNA gel extraction devices (Millipore) and resuspended in DNA hydration solution (Gentra Systems).For detection of SNPs on Dutch Saanen bucks, restricted representation libraries (RRL) were constructed as described by Kraus et al Each of the 16 DNAs from Alpine, Creole or Saanen goat was sequenced independently on an Illumina HiSeq2000 machine using one lane for each animal. 3µg of DNA was used for each animal to construct libraries following manufacturer’s protocol (Illumina TruSeq DNA sample prep). Then, libraries were quantified and sequenced on one lane for each on an Illumina HiSeq2000 sequencer using standard Illumina protocol (TruSeq SBS kits v3), with 100 bp paired-end reads and ∼250 bp insert length.Two genomic DNA pools were made for meat goats: pool 1 from 20 Boer goats and pool 2 from 20 Savanna goats and 24 Katjang goats. Each DNA sample was quantified three times (using Hoechst dye and fluorometer) and equal amount of genomic DNA from each sample based on the average concentration of the three readings were used to make DNA pools. The pools were whole genome shotgun sequenced on an Illumina Genome Analyzer IIx machine using the standard Illumina protocol, with 78 bp paired-end reads and ∼300 bp insert length.AluI and HhaI) were combined and prepared using the Illumina Sample Preparation kit The genomic RRLs of the 17 Dutch Saanen bucks , minimum number of reads for each allele = 2. SNP MAFs were estimated by counting the number of reads of each allele for the whole dataset .http://www.cbcb.umd.edu/research/bos_taurus_assembly.shtml and http://www.livestockgenomics.csiro.au/sheep/websites, using MosaikAlignMosaiksamtools , and DNA LandMarks  in-house algorithm. The quality filtering for SNP discovery were a SNP quality of at least 20 and minimum three reads of each allele. Approximately 300 bp of consensus flanking sequence of aligned goat reads were extracted together with the variant and the MAF was calculated as above. These sequences were aligned on the goat scaffolds using BWA .Data from the two pools of meat goats were aligned on cattle UMD3.1 and sheep Oarv2∶0 reference genomes, available respectively on de novo assembled and several stringent filters were applied to resulting contigs to predict SNPs, as described in SNPs were also detected in RNASeq projects (see acknowledgements section) conducted either on gonads (GENIDOV programme) or mammary gland (LGS programme). Sequencing data generated by 454/Roche platform were http://www.ensembl.org/, release 64) and were blasted Finally, previously published SNP in genes of interest  were added. Briefly, the genomic sequence of cattle CSN1S1, CSN2, CSN1S2, CSN3, PRP and DGAT1 genes were retrieved from ensemble genome browser  and the technical quality of the SNP for use on Illumina BeadChips  were assessed.For all the detected SNPs, 120 bp of flanking sequences were extracted. Using Categories were defined to characterize the variant:1: SNP detected in an EST.2: two alleles detected in the five considered breeds.3: two alleles detected in Alpine and Saanen and Creole and (Boer or Katjang/Savanna).4: two alleles detected in two of the three milk and mixed breeds  and in Boer and Katjang/Savanna.5: two alleles detected in Alpine and Saanen and Creole.6: two alleles detected in three out of the five breeds.10: two alleles detected in each of the two milk breeds (Saanen and Alpine).11: two alleles detected in one milk breed (Saanen or Alpine) and one meat breed (Creole or Boer or Katjang/Savanna).12: two alleles detected in at least two meat breeds (Creole and Boer or Katjang/Savanna).13: two alleles detected in one milk breed (Saanen or Alpine).20: other SNPs.90: INDELs.For all the detected variants, the following characteristics of the detected SNP were stored in an in-house database:Goat scaffold nameGoat scaffold positionBase in the goat reference genome and in all the analyzed breeds2 by 60 bp of flanking sequenceEstimated MAF (Minimum Allele Frequency): approximated here by counting the number of reads for each alleleIllumina Assay Design Tool (ADT), using the closest to goat available genome as a reference: cattle; ranging from 0 to 1 and evaluating the probability of success of the SNP assayIllumina SNP score: as determined by Illumina SNP type: I (A/T or G/C) requiring two two probes or II (other SNPs) requiring only one probeDistance to the previous and the next SNP (bp)The following criteria were used to select a first list of SNPs:Illumina score >0.8, Infinium II SNP. This list was ranked using increasing category and decreasing MAF.scaffold >10,000 bp, biallelic, minimum 20bp each side without another variant, MAF >0.2, An in-house algorithm was used to select a SNP list maximizing both the rank criteria presented before and the genome coverage. The algorithm uses the rank sorted SNPs and the scaffold length array as inputs. During the process it fills an array storing starts and end positions of already covered genome locations and populates the list of selected SNPs. The algorithm checks a SNP at each cycle. For each SNP, it first checks if it is located in an already covered area. In this case, it processes the next SNP. If the SNP is located in an uncovered area, the flanking area  is added to the covered positions array and the SNP is added to selected SNP list. Several flanking area sizes were tested. The one which was chosen: 28,500 produced a list of close to 59,000 SNPs.The remaining 1,000 SNP were manually chosen to cover large genomic regions which did not include highly ranked SNPs.BWA .SNP flanking sequences were aligned on cattle UMD3.1 assembly using The final 60,000 selected SNP list was submitted to Illumina.To assess the quality of the SNP chip, a set of 288 DNAs  were used. It included most of the animals that had been used for SNP discovery and the animal used for reference genome sequence. Additional animals were added to represent genetic diversity of international goat breeds. This breed set included 53 Alpine, 26 Angora, 30 Boer, 38 Creole, 16 Jinlan, 15 Katjang, 59 Saanen, 20 Savanna, 27 Skopelos and 1 Yunling goat, with four trios. Genotyping of these 288 DNAs was used to assess the performance of the successfully synthesized SNP assays and generate cluster files.Table S1PRNP, casein and DGAT1 orthologous regions on cattle and goat genomes. For each gene, chromosome number and location are given on cattle UMD3.1 assembly and scaffold number and location are given on goat scaffolds.(XLSX)Click here for additional data file.Table S2SNPs in PRNP, casein and DGAT1 genes. For each SNP, exon number, flanking sequence and variant, scaffold name, position on the scaffold and ss number in dbSNP are indicated.(XLSX)Click here for additional data file.File S160,000 SNP chip design. This “CAPRI-BATCH1” file contains information on the 60,000 selected SNPs as it was submitted to dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_viewBatch.cgi?sbid=1057128). The submission includes 60bp flanking sequences on each side of the variant site, MAF value, Illumina_score, Illumina_state (successful or not) and position of the variant on goat scaffolds (Chinese_assembly_v1 column) and goat CHIR_1.0 assembly, (Chinese_assembly_v2_pseudo column), Sheep_OARV2 and Bovine_UMD3.1.(GZ)Click here for additional data file.File S260,000 SNP ss names. This file contains the correspondence between SNP name in File S1 and NCBI_ss# name for the 60,000 selected SNPs.(GZ)Click here for additional data file.


Felis silvestris catus, particularly within its breeds. A custom illumina GoldenGate Assay consisting of 1536 single nucleotide polymorphisms (SNPs) equally divided over ten 1 Mb chromosomal regions was developed, and genotyped across 18 globally recognized cat breeds and two distinct random bred populations. The pair-wise LD descriptive measure (r2) was calculated between the SNPs in each region and within each population independently. LD decay was estimated by determining the non-linear least-squares of all pair-wise estimates as a function of distance using established models. The point of 50% decay of 2r was used to compare the extent of LD between breeds. The longest extent of LD was observed in the Burmese breed, where the distance at which 2r ≈ 0.25 was ∼380 kb, comparable to several horse and dog breeds. The shortest extent of LD was found in the Siberian breed, with an 2r ≈ 0.25 at approximately 17 kb, comparable to random bred cats and human populations. A comprehensive haplotype analysis was also conducted. The haplotype structure of each region within each breed mirrored the LD estimates. The LD of cat breeds largely reflects the breeds’ population history and breeding strategies. Understanding LD in diverse populations will contribute to an efficient use of the newly developed SNP array for the cat in the design of genome-wide association studies, as well as to the interpretation of results for the fine mapping of disease and phenotypic traits.Domestic cats have a unique breeding history and can be used as models for human hereditary and infectious diseases. In the current era of genome-wide association studies, insights regarding linkage disequilibrium (LD) are essential for efficient association studies. The objective of this study is to investigate the extent of LD in the domestic cat,  Felis silvestris catus, escaped active and intentional breed development until the 19th century Felis silvestris lybica ssp. A variety of agricultural species was domesticated during the Neolithic revolution, over the course of which specialized breeds emerged. However, the domestic cat, Prionailurus bengalensisThe development of cat breeds has followed four major strategies. The primary and historically most common strategy has been the manipulation of a subset of cats from a natural population. Persian, Turkish Angora, Maine Coon, Norwegian Forest Cat, and Siberian breeds are each likely to have arisen from specific feral random bred cat populations that were segregating for the longhair mutations priori knowledge of the extent of LD has proven to be instrumental in the design of GWA studies and fine-mapping of genetic diseases Linkage disequilibrium (LD) is the non-random association of alleles at different loci on chromosomes in a gamete. A The alternative strategies to develop cat breeds create inherent difficulties in estimating LD. The natural decay of LD via recombination and gene conversion is suppressed in the development of domesticated breeds. LD within breeds is maintained by population genetic forces, such as selection, variation in migration substructure, bottlenecks, and inbreeding Pedigreed and random bred cats samples, used in this study, were collected during the period 1994–2011 and stored as archival of DNA samples. The samples were collected by laboratory personnel at cat shows and field trips, or sent by collaborators, breeders, and cat owners from various countries. All samples were collected via buccal (cheek) swabs with the exception of those from collaborators including Birman, Burmese (F), Maine Coon, and Norwegian Forest Cat, which were collected as whole blood. The samples used in this study were selected from the DNA archive and meet selection criteria discussed below.Eighteen globally recognized cat breeds, 8–20 cats per breed, were selected based on their worldwide popularity, USA population size, population genetic distinctiveness, and breed history Two random bred populations were selected based on their genetic distinctness and geographical isolation http://genome.ucsc.edu/), using algorithms therein. Approximately 150 SNPs were distributed over each 1 Mb region of each chromosome with varying spacing (SNPs (N = 1536) were selected over ten different 1 Mb chromosomal regions  . The chr spacing . SNP den spacing . SNPs th spacing .SNP genotyping was performed at the Texas Biomedical Research Institute . Array data was analyzed using the illumina GenomeStudio software (version 1.7.4) to obtain genotypes. SNP genotype clusters were manually evaluated and SNPs that (i) failed genotyping, (ii) were poorly clustered, or (iii) were monomorphic, were removed from downstream analysis .st across breeds, a supervised analysis using ADMIXTURE 1.22 was conducted, with each of the 23 populations set as a specific K st matrix was visualized using R in the form of an unrooted neighbor-joining tree as per McEvoy et al. APE package and the Neighbor-Joining Tree Estimation function (nj) based on methods of Saitou and Nei To ascertain Fis were calculated for each population in each of the ten chromosomal regions. Tajima’s D was calculated using the pegas package in R Using the total number of SNPs (n = 1463), observed and expected heterozygosities, and Fgenetics package in R SNP genotype analysis and LD pair-wise calculations were performed using the 2r) was calculated between each pair of SNPs on the same chromosome 2r for each chromosome, separately, and as a summary of the combined data of autosomal chromosomes (denoted Auto) and all chromosomes (denoted All).The squared correlation coefficient . The Korat (D) and Western random bred populations attained statistical significance on this measure. However, the inadequate sample size for these two populations warrants caution against accepting the t-test results as definitive, and requires further investigation. Control replicates (n = 6) showed no allele-calling inconsistencies and a per SNP no call rate of 0.005%.Well-recognized breeds with a comprehensive historical and demographic record were selected for analysis . SamplesAll cats analyzed in the study have genotype call rates above 75% for the SNP data. Initial genotype cluster analysis of the 1536 SNPs that passed design showed that 73 SNPs (5%) failed in the assay . Overallst. The neighbor-joining tree illustrated a clear separation between eastern and western populations  exhibited low genetic distance values, as expected. The Japanese Bobtail appeared more closely related to western populations than to eastern populations.The populations’ phylogenetic relationship was studied by examining pair-wise Fulations . WesternObserved and expected heterozygosities varied across populations, with mean values of 0.251 and 0.242, respectively. Similarly, the mean inbreeding coefficient ranged between −0.055 and −0.012 . Tajima’The effect of MAF cut-off choice on the extents of LD was evaluated by estimated LD using two MAF, 0.05 and 0.1. Variation resulting from choice of MAF was apparent . The Man2r value was analyzed for each chromosome separately and for combined chromosomes. The chromosome specific LD decay varied among populations but the relative order of population decay rate was not consistent across chromosomes . The LD on the X chromosome extended further than any autosome. Using Pearson’s test of correlation between the mean extent of LD across populations and the molecular genomic features of each region, no correlation has been found between the extent of LD in a region and the GC content (p = 0.63), number of gene (p = 0.28), or number of simple repeat elements (p = 0.21).To determine if significant inter-chromosomal variations in LD decay were present in the cat, the LD decay was determined for each chromosome in each population. The extent of LD in all ten chromosomal regions separately and combined were summarized . Three cnt of LD . The extA comprehensive haplotype analysis was conducted for the populations under study. The haplotype blocks and the number of haplotypes within each block were compared across all populations in each chromosomal region . The lenAs an example, chromosomal region A1 and C2 were chosen for a detailed comparison across breeds . In regiAs a measure of haplotype diversity, the total number of haplotype blocks and the total number of haplotypes in each region was compared across populations . The meaCat genetic resources such as linkage maps 2r value to the one used for the cat (2r ≈ 0.25), most horse breeds reach the same level of LD at <100 kb when using a similar study design 2r ≈ 0.25 at ∼400 kb In cat breeds, LD variation spans the range between horses and dogs, with no marked extremes. LD was shortest in the Siberian breed at ∼17 kb, and most extended in the domestic Burmese at ∼380 Kb. At a comparable 2r value reached 0.2. The majority of dog breeds had LD extending geater than 200 kb, with only the Labrador Retriever and Saint Bernard reaching the same LD level as cats in <100 kb. The highest LD in dog breeds was found in the Mastiff, which extends over 5 Mb. Comparatively, the highest LD in cats was found in the domestic Burmese breed, extending approximately less than one tenth of the Mastiff LD distance.Similarly, in a study of LD in wild and domestic dogs, Gray et al. This study has focused on the most popular and genetically distinct cat breeds, many of which have several known inherited diseases The Burmese breed, both populations domestic (D) and foreign (F), displayed the largest extent of LD and the fewest and longest haplotype blocks among cat breeds. Although only a few cats were known to be the foundation of the breed, other cats were used for the breed expansion, such as Siamese. The Burmese quickly became one of the most popular breeds in the cat fancy, which contributed to a rapid population expansion. This population expansion is evident in Tajima’s D estimates for the breed, as it is in most breeds. A negative value, and in particular values below −2, are often indicative of either strong selective sweeps in a region, or rapid population expansion from a bottleneck. Because there was no discernible genomic variation in this statistic, it seems that one should assume that the values are being generated by genome-wide demographic processes. In particular, selection of a small number of founding individuals, and the rapid population growth through breeding, for some of these lineages, seems the most likely source of the recurrent high negative values across the populations.The domestic Burmese population suffers from an autosomal recessive craniofacial defect The large extent of LD, long haplotype blocks, deviated Tajima’s D, high levels of inbreeding, and reduced heterozygosity in utero when homozygous, while heterozygotes have variable expression Four breeds, Siberian, Manx, Turkish Angora and Japanese Bobtail, had LD and haplotype structure comparable to random bred domestic cat populations. The Manx breed is defined by a single physical characteristic, the lack of a tail. The tailless trait is dominant and lethal Similarly, breeds such as Siberians, a new breed, and Turkish Angoras, tend to resemble the random bred street cats of their populations of origin, Russia and Turkey, respectively The Persian was one of the founding western-derived breeds for the cat fancy, and was presented at the first cat show in the United Kingdom in 1871 (though they were known as Angoras at the time). Historically, the most popular breed worldwide, Persian cats have a massive variety of colorations and patterns as well as the largest breeding population in the cat fancy Siamese is an ancient eastern-derived breed and also one of the foundation breeds for the cat fancy. This large and historically popular breed shows one of the highest levels of LD among the studied cat breeds. Acknowledging the large census population size of the breed, the high LD levels may be attributed to the breed pointed coat color trait that defines the breed Contrary to other breeds with fixed traits, both domestic and foreign Korat populations exhibit moderate LD levels. The expectation would be that the Korat would show high levels of LD due to its small census population size and single blue color presentation. The Korat is putatively an ancient lineage, but was only recently recognized as a breed in 1966. Although its census population size is small, breeders actively introduce random bred cats into their breeding stocks from the breed’s country of origin, Thailand, thereby reducing inbreeding depression and maintaining high levels of heterozygosity in accordance with previous genetic studies Inter-chromosomal LD variation was detected in the combined random bred population data set and in all populations. This pattern of variation likely reflects regional genomic variation in recombination rates, but exhibited no correlation with the size of the chromosome. Similarly, no correlation has been observed between the GC content of a region, gene number, or simple repeat number and the extent of LD. The X chromosomal region exhibited the highest extent of LD across all populations. This is attributed to the differences in effective population size of the X chromosome and the autosomes. The regional genomic variation suggests that the combined autosomal data is more representative of the breeds’ LD. Regional and chromosomal differences in recombination rates could be warrant future investigation.The MAF cut-off choice for the inclusion of SNPs in an LD analysis has profound effects on the results. Increasing the MAF from 0.05 to 0.1 inflated the estimate of the extent of LD by 40%. While this inflation in LD estimate was observed in the majority of the studied populations, Manx breed deviated from the trend and exhibited little inflation . This ca2r≥0.8, which are considered GWAS - informative SNPs, allow the prediction of how successful GWA studies are likely to be in specific breeds. Domestic Burmese showed fractions of GWAS - useful SNPs ≥10% even in 200–400 Kb class distance. The eastern breeds, Burmese, Birman, and Siamese, show fractions of GWAS-informative SNPs ≥10% even at a class distance 100–200 Kb. At the class distance 40–60 Kb, which is equivalent to the density of the current 63K SNP array, Egyptian Mau, foreign Korat, Maine Coon, Norwegian Forest Cat, Ocicat, and Persian cats have GWAS-informative SNPs fraction ≥10%.Understanding the extent of LD in the domestic cat is important for the successful design and analysis of genome-wide association studies. The fraction of SNP pairs with Eastern breeds are likely to generate successful GWA studies, particularly the domestic Burmese, where GWA studies are liable to be fruitful even with few cases and controls. Western breeds such as Turkish Angora, Cornish Rex, Japanese Bobtail, Manx, Russian Blue, Siberian, and random bred cats show smaller fractions of informative SNPs in distances equivalent to the current 63K array. GWA studies in such breeds may require larger numbers of cases and control or an array with higher density.GWA studies focused upon breed defining phenotypic traits may be especially successful using the current DNA array. Positive selection pressure extends the LD from the locus of interest, producing an extended haplotype block around the causative genomic region. In such cases, SNPs that are at distance from the causative region might be in strong LD with the causal locus, signaling an association between the trait and that genomic region. Unfortunately, the drawback of a large window of informative tag markers is the necessity to explore a wide genomic region surrounding an associated SNP for a phenotypic trait. Great caution in experimental design should be applied for GWA studies involving disease since regions are likely not to be under selection. The LD estimates reported here can enhance the design of future GWA studies, especially in sample size and study design assessment, and the search for candidate genes.Figure S1Position of the 1 Mb regions and spacing of the SNPs in 10 chromosomal regions of domestic cat. a) Position of the 1 Mb regions in relation to centromere, mid-arm, and telomere. Black blocks represent centromere, except Chr. F2, which is an acrocentric chromosome, and red vertical lines represent the chosen 1 Mb region. b) Spacing of ∼ 150 SNPs in each of 10 chromosomal regions  of 1 Mb length. SNPs are positioned to be denser at one end of each 1 Mb chromosomal region.(TIFF)Click here for additional data file.Figure S2Variation in Tajima’s D estimates between domestic cat populations. a) Tajima’s D estimate for each breed/population in each chromosomal region (see legend). b) Mean Tajima’s D estimate of values obtained for individual populations (a). c) Mean Tajima’s D estimate of values obtained for individual chromosomes. Error bars in (b) and (c) represent the standard deviation.(TIFF)Click here for additional data file.Figure S3Effect of minor allele frequency choice on LD decay in domestic cat. a) LD decay of Manx breed (n = 20). b) LD decay of Birman breed (n = 20). c) LD decay of Egyptian Mau Breed (n = 9). d) LD decay of Chartreux breed (n = 8). Regardless to the sample size, the LD decay appears to be inflated when using MAF of 0.1 due to the reduction in the number of pair-wise estimates of LD measure that are modeled to get a decay graph. Decay lines that do not show the point of 50% decay (solid circle or triangle) indicate that the 50% decay point is not reached at 1000 Kb. Decay lines that show abnormally an increase instead of decrease of 2r as a function of distance such as in (b) is a result of small number of pair-wise comparisons.(TIFF)Click here for additional data file.Figure S4Extent of LD in cat populations. Bar plots represent the extent of LD for each population in each chromosomal region as well as the combined autosomal chromosomes and all chromosomes. The extent of LD represents the point of 50% decay of 2r initial value. Instances where the extent of LD reaches 1000 Kb indicate that the decay of 2r does not reach 50% of its initial value.(TIFF)Click here for additional data file.Figure S5Haplotype structure and diversity of selected 10 chromosomal regions in domestic cat populations. Haplotype analysis of chromosome regions (A1-X). Breeds/populations are represented on the y axis and the position of haplotype blocks are shown on the x axis. Haplotype blocks are represented by horizontal rectangles. The frequency of individual haplotypes within a block is proportional to the height of the color. Major haplotype is represented by black color, intermediate haplotypes with frequency >0.1 are represented by blue, light blue, red, green and yellow. Pink color represents the sum of the frequencies of all haploypes with a frequency <0.1. Empty regions between blocks indicate lack of any informative markers (MAF ≥0.05) and can be considered monomorphic across all individuals within a population.(TIFF)Click here for additional data file.Figure S6Haplotype diversity of cat populations. a) Haplotype diversity measured by the number of haplotype block defined in each chromosomal region. b) Haplotype diversity measured by the total number of haplotypes found in each chromosomal region. Legend corresponds to the ten chromosomal regions.(TIFF)Click here for additional data file.Table S1Genomic and general summary of the 1Mb regions in cat LD analysis.(DOC)Click here for additional data file.Table S2GoldenGate assay SNP location, sequence, and genotypes of discovery cats.(XLS)Click here for additional data file.Table S3Population information and summary statistics.(DOC)Click here for additional data file.Table S4Distance (Kb) for achievement of 50% decay of the LD measure. The (>1000) indicates that the LD measure did not reach 50% of its initial value at 1000 Kb.(DOC)Click here for additional data file.Table S5Background means of cat LD estimates (2r) using MAF = 0.1.(DOC)Click here for additional data file.Table S6Fractions of pairs of SNPs with an 2r value ≥0.8 at various distance classes.(DOC)Click here for additional data file.


Furthermore, internodal length changed only slightly with increasing fiber diameter in regenerating nerves; therefore, the regression relation between fiber diameter and internodal length was not a sensitive index of recovery. MCV and mean fiber diameter were the most sensitive indices of functional recovery during sciatic nerve regeneration.We analyzed the relationship between motor nerve conduction velocity (MCV) and morphological changes in regenerating nerve fibers at different times after sciatic nerve transection to identify reliable indices of functional recovery. Thirty rats were divided into five equal groups, one control group and four groups subjected to sciatic nerve transection and immediate suturing, followed by regeneration for 50, 100, 150, and 200 days, respectively. MCV was measured in each group, followed by morphometric analyses of fibers of the common peroneal nerve. MCV increased progressively with time after nerve transection, although it remained lower than the control velocity. Mean fiber diameter (axon plus myelin sheath) also increased with time after nerve transection. Recovery of mean fiber diameter was well correlated with MCV, even though regenerating nerves likely contained many small nonconducting fibers. In contrast, the change in the mean diameter of regenerating axons and relative myelin thickness ( Numerous experimental studies have investigated morphological parameters that may affect conduction velocity in myelinated nerve fibers. These parameters include fiber diameter, axon diameter, myelin thickness, and internodal length . Among tThe purpose of this study is to analyze the relationship between motor nerve conduction velocity (MCV) and morphological changes in individual fibers, including fiber diameter, myelin thickness, and internodal length, during regeneration of peripheral nerves. The most reliable indices of regeneration were determined by regression analysis at different time points following sciatic nerve transection. We found that MCV and mean fiber diameter were the most reliable indices of functional recovery during regeneration.Thirty male Sprague–Dawley rats, weighing approximately 600–700 g, were used for this study, including six control rats and four groups of six rats each subjected to sciatic nerve transection, suturing, and recovery for 50, 100, 150, or 200 days, respectively. Rats in the nerve transection groups were anesthetized by face mask inhalation of 5% halothane. The left sciatic nerve was exposed through a lateral incision in the mid-thigh. The nerve was transected sharply with micro-scissors 2-cm distal to the sciatic notch and immediately repaired with 10–0 nylon epineural sutures  under a dissection microscope . The muscle and skin were closed with 3–0 absorbable sutures . Our institutional review board approved this study, and every effort was made to reduce the number of animals used and their suffering.At 50, 100, 150, and 200 days after initial surgery, the rats were anesthetized and the sciatic nerve was exposed from the trochanteric notch to the common peroneal nerve. MCV was measured as described below. Rats were then sacrificed and the common peroneal nerve was removed for morphological analysis.The rat was wrapped in a bubble packing sheet and aluminum foil to maintain body temperature above 37°C. A Medelec Sapphire II electromyography unit  was used for stimulation and recording of compound motor action potentials (CMAPs). Two bipolar electrodes were used for stimulation; one was placed proximally on the sciatic nerve near the obturator foramen and the other on the common peroneal nerve just distal to the division; this way, the distance between the electrodes was maximum. CMAPs were recorded from the extensor digitorum longus using two 6-mm recording disc electrodes; the active electrode was applied to the muscle belly through the skin and the reference electrode was applied to the muscle tendon. The nerve was stimulated by a supramaximal 50 μsec constant current.L/T, where L (m) is the distance between the two stimulus electrodes and T (sec) is the difference in delay between CMAPs evoked by the proximal and distal stimulating electrodes.MCV was calculated by the conventional method: MCV (m/sec) =4 for 2 h, dehydrated, and embedded in Araldite. Semi-thin sections were prepared and stained with toluidine blue. These slices were used for the measurement of fiber and axon diameters.After completion of the motor nerve conduction studies, a 15-mm segment of the common peroneal nerve from the sciatic nerve bifurcation to the muscular insertion was excised. The proximal one-third of the segment was processed for preparation of semi-thin transverse slices (1 μm). Briefly, the nerve segment was fixed in 2.5% cacodylate buffered glutaraldehyde  at 4°C for 1 h and cut into 1-mm transverse sections. Sections were placed in the same fixative solution for an additional 12 h, postfixed in 1% cacodylate buffered OsO4 for 24 h. After washing in distilled water, the nerve was mounted in 50% glycerol solution under a stereomicroscope , and the individual fibers were gently teased apart using a fine needle.For measurement of internodal length, the distal two-thirds of the segment was fixed in 8% cacodylate buffered formalin (pH 7.2) for 48 h and fixed in 1% cacodylate buffered OsOMorphometric measurements were performed using a VIDS III image analysis system  connected to a microscope. The image was viewed and digitized on a display screen, and the morpho-logical parameters were measured using a cursor and digitizing table . The datt-tests. Relationships between the morphometric parameters and time after nerve transection were tested by best fit analyses using linear or logarithmic equations, and the correlation coefficients (r) were tested. P < 0.05 was considered statistically significant.MVC was compared between groups using a nonparametric Kruskal–Wallis one-way analysis of variance by ranks test. Morphometric parameters were expressed as mean ± standard error of the mean, and pairwise differences were tested using Tukey's honestly significant difference and Student's The mean MCV in the control group was 74.2 m/sec; this was significantly higher than that measured in the four rat groups allowed to recover for 50, 100, 150, and 200 days . The meag-ratio  between each group. The mean g-ratio of the control group was larger than that of any transection group. The mean value of the g-ratio was highest in the 50-day group, followed in descending order by the 150-, 100-, and 200-day groups. Therefore, only MCV and mean fiber diameter demonstrated a consistent relationship with recovery time.In all nerve transection groups, both mean fiber diameter and axon diameter were significantly smaller than those in the control group. Mean fiber diameter increased with time between 50 and 200 days after transection. The mean fiber diameter of the 50-day regeneration group was significantly smaller than that measured in the other groups . The meag-ratio revealed a significant correlation within each group (g-ratio to axon diameter relation was best fit by the following logarithmic equation:Scatter plots of axon diameter against ch group . At eachx1 is the axon diameter and y1 is the g-ratio.where r1) ranged from 0.735 to 0.910. Time after transection was associated with a rightward shift in these plots, indicating more numerous axons with large diameters and higher g-ratios. In each transection group, axon diameters were as large as 9 μm. At 50 days following transection, however, most axons where smaller than 6 μm, and the g-ratio increased steeply with increasing diameter (g-ratio). Many axons in the 150- and 200-day groups exhibited these characteristics, while few such axons were found in the control sciatic nerves  and internodal length (y2) was expressed as a linear regression line by the following formula: y2=A2×x2+B2, where x2 is fiber diameter and y2 is internodal length. As shown in x3) and the ratio of the quotient internodal length to fiber diameter (IL/FD) (y3), with a logarithmic regression curve as follows: y3=E× log(x3) +F. Indeed, although the logarithmic relation in normal nerves was weak (r3= 0.384), there were strong logarithmic correlations at each time point during nerve regeneration (coefficients ranging from 0.628 to 0.745). When the regression curves for regenerating fibers were superimposed, the regression curves at each time point after transection were overlapping, indicating that this fiber diameter to IL/FD relationship is also a poor index of functional recovery.We then investigated the quantitative relationship between fiber diameter and internodal length . Followig-ratio decreased after transection, they were not well correlated with time or MCV recovery.New nerve repair techniques should only be introduced into general clinical practice if they can be conclusively proved efficient in improving the results obtained from previous techniques. To reach this goal, evaluation methods that provide an objective measure of recovery are required. Animal models also provide objective measures of functional recovery in a manner not presently obtainable in clinical studies. Morphological and electrophysiological measures reflect the inherent variability in the rate of nerve regeneration, myelination, and functional recovery; therefore, a combination of electrophysiological and morphometric measures may yield the best indication of recovery, especially over multiple time points. We demonstrated that recovery of MCV and mean fiber diameter were well correlated with time after sciatic nerve transection. Although mean myelin thickness, axonal diameter, and Conventional MCV measurements tend to reflect primarily upon the faster conducting fibers and provide little information about the conduction properties of the entire population of regenerating fibers . The preMany of the fibers with small diameters may in fact be nonconducting and degenerating. As the nerve fibers regenerate distally and reach the appropriate target organ, fiber diameter increases and the myelin sheath grows . If sprog-ratio) to maximize conduction velocity  were observed at 100 and 200 days after nerve transection. These fibers with low g-ratios may be those that failed to reach their target organ, with ensuing collapse of the myelin sheath around a shrinking axon  . This quThus, we concluded that MCV and mean fiber diameter were the most reliable indices of functional recovery during sciatic nerve regeneration. Furthermore, the regression relation between fiber diameter and internodal length was not a sensitive index of recovery.


Kais a very important molecular property, and there is a strong interest in the development of reliable and fast methods for p Kaprediction. We have evaluated the p Kaprediction capabilities of QSPR models based on empirical atomic charges calculated by the Electronegativity Equalization Method (EEM). Specifically, we collected 18 EEM parameter sets created for 8 different quantum mechanical (QM) charge calculation schemes. Afterwards, we prepared a training set of 74 substituted phenols. Additionally, for each molecule we generated its dissociated form by removing the phenolic hydrogen. For all the molecules in the training set, we then calculated EEM charges using the 18 parameter sets, and the QM charges using the 8 above mentioned charge calculation schemes. For each type of QM and EEM charges, we created one QSPR model employing charges from the non-dissociated molecules (three descriptor QSPR models), and one QSPR model based on charges from both dissociated and non-dissociated molecules (QSPR models with five descriptors). Afterwards, we calculated the quality criteria and evaluated all the QSPR models obtained. We found that QSPR models employing the EEM charges proved as a good approach for the prediction of p Ka. As expected, QM QSPR models provided more accurate p Kapredictions than the EEM QSPR models but the differences were not significant. Furthermore, a big advantage of the EEM QSPR models is that their descriptors  can be calculated markedly faster than the QM charge descriptors. Moreover, we found that the EEM QSPR models are not so strongly influenced by the selection of the charge calculation approach as the QM QSPR models. The robustness of the EEM QSPR models was subsequently confirmed by cross-validation. The applicability of EEM QSPR models for other chemical classes was illustrated by a case study focused on carboxylic acids. In summary, EEM QSPR models constitute a fast and accurate p Kaprediction approach that can be used in virtual screening.The acid dissociation constant p The online version of this article (doi:10.1186/1758-2946-5-18) contains supplementary material, which is available to authorized users. Kais an important molecular property, and its values are of interest in pharmaceutical, chemical, biological and environmental research. The p Kavalues have found application in many areas, such as the evaluation and optimization of candidate drug molecules [Kadependent. For these reasons, p Kavalues are important for virtual screening. Therefore, both the research community and pharmaceutical companies are interested in the development of reliable and above all fast methods for p Kaprediction.The acid dissociation constant p olecules –3, ADME olecules , 5, pharolecules , understolecules , 8, etc.Kaprediction have been developed [Kavalues remain one of the most challenging physicochemical properties to predict. A promising approach for p Kaprediction is to use QSPR models which employ partial atomic charges as descriptors [Several approaches for p eveloped –11, nameeveloped , 13, dateveloped , ab initeveloped , 16, ANNeveloped  or QSPR eveloped –20. Howecriptors –24.Kaetc.), and many different methods have been developed for their calculation. The charge calculation methods can be divided into two main groups, namely quantum mechanical (QM) approaches and empirical approaches.The partial atomic charges cannot be determined experimentally and they are also not quantum mechanical observables. For this reason, the rules for determining partial atomic charges depend on their application  and basis set , and then partition this wave function among the atoms . This partitioning can be done using an orbital-based population analysis, such as MPA  , 26, LöwEmpirical approaches determine partial atomic charges without calculating a quantum mechanical wave function for the given molecule. Therefore they are markedly faster than QM approaches. One of the first empirical approaches developed, CHARGE , performKausing QSPR models which employ QM atomic charges was described in several studies [Kaprediction, as the QSPR models based on QM atomic charges are able to calculate p Kawith high accuracy. The weak point of QM charges is that their calculation is very slow, as the computational complexity is at least θ (E4), where E is the number of electrons in the molecule. Therefore, p Kaprediction by QSPR models based on QM charges cannot be applied in virtual screening, as it is not feasible to compute QM atomic charges for hundreds of thousands of compounds in a reasonable time. This issue can be avoided if empirical charges are used instead of QM charges. A few studies were published, which give QSPR models for predicting p Kausing topological empirical charges as descriptors  [The prediction of p  studies –24, whiccharges) , 42, 43.The geometrical charges seem to be more promissing descriptors, because they are able to take into consideration the influence of the molecule’s conformation on the atomic charges. The conformation of the atoms surrounding the dissociating hydrogens strongly influences the dissociation process, and also the atomic charges.Kaprediction by QSPR. This approach calculates charges using the following equation system: The EEM method is a geometrical empirical charge calculation approach which can be useful for p qiis the charge of atom i; Ri,jis the distance between atoms i and j; Q is the total charge of the molecule; N is the number of atoms in the molecule; Ai, Biand κ are empirical parameters. These parameters are obtained by a parameterization process, which uses QM atomic charges to calculate a set of parameters for which EEM best reproduces these QM charges. EEM is very popular, and despite the fact that it was developed more than twenty years ago, new parameterizations [θ (N3), where N is the number of atoms in the molecule.where izations , 44–50 aizations , 51, 52 Kaprediction using QSPR models which employ EEM charges. Specifically, we created and evaluated QSPR models based on EEM charges computed using 18 EEM parameter sets. We also compared these QSPR models with corresponding QSPR models which employ QM charges computed by the same charge calculation schemes used for EEM parameterization.Therefore, in the present study, we focus on p In our study, we used all EEM parameters published till now. Specifically, we found 18 different EEM parameters sets, published in 8 different articles , 44–50. The EEM charges were calculated by the program EEM SOLVER  using eaWe calculated QM atomic charges for all the combinations of QM theory level, basis set and population analysis for which we have EEM parameters  and QSPR models with five descriptors (5d QSPR models).Kaprediction in our previous study [qH), the charge on the oxygen atom from the phenolic OH group (qO), and the charge on the carbon atom binding the phenolic OH group (qC1). These descriptors were used to establish the QSPR models by the general equation: The 3d QSPR models used those descriptors which proved to be the most relevant for p us study . TherefopH, pO, pC1 and p are parameters of the QSPR model . The 5d QSPR models employ the above mentioned descriptors qH, qOand qC1 and additionally also the charge on the phenoxide O- from the dissociated molecule (qOD), and the charge on the carbon atom binding this oxygen (qC1D). Using the charges from the dissociated molecules for p Kaprediction was inspired by the work of Dixon et al. [where n et al. . The equp′ are parameters of the QSPR model.where qH), the charge on the hydrogen bound oxygen atom from the COOH group (qO), and the charge on the carbon atom binding the COOH group (qC1). Additionally, also the charge of the second carboxyl oxygen (qO2) is included. These 4d QSPR models are represented by the equation: The descriptors were again atomic charges and, similarly as for phenols, two types of QSPR models were developed and evaluated. Specifically, QSPR models with four descriptors (4d QSPR models) and QSPR models with seven descriptors (7d QSPR models). The 4d QSPR models used similar descriptors as the 3d models for phenols - the atomic charge of the hydrogen atom from the COOH group  and the charge on the carboxylic carbon atom (qC1D). The equation of the 7d QSPR models is therefore: where p′ are parameters of the QSPR model.where The parameterization of the QSPR models was done by multiple linear regression (MLR) using the software tool QSPR Designer .We prepared one 3d QSPR model and one 5d QSPR model using atomic charges calculated by each of the above mentioned 18 EEM parameter sets. These models are denoted 3d or 5d EEM QSPR models. Additionally, we created one 3d and one 5d QSPR model using atomic charges calculated by each of the corresponding 8 QM charge calculation approaches (denoted as 3d or 5d QM QSPR models). The data set of 74 phenol molecules was used for the parameterization of the QSPR models, and the obtained models were validated for all molecules in the data set.The parameterization of the 3d EEM QSPR models showed that several molecules in the data set perform as outliers. For this reason, we created also EEM QSPR models without outliers . These models are denoted 3d EEM QSPR WO models. We classified as outliers 10% of the molecules from our data set, which had the highest Cook’s square distance. Therefore the 3d EEM QSPR WO models were parameterized using 67 molecules, and their validation was also done on the data set excluding outliers.Kaand the p Kacalculated by each model, was evaluated using the squared Pearson correlation coefficient (R2), root mean square error (RMSE), and average absolute p Kaerror (s) and Fisher’s statistics of the regression (F).The quality of the QSPR models, i.e. the correlation between experimental p R2, RMSE, s and F) for all the QSPR models analyzed. All these models are statistically significant at p = 0.01. Since our data sets contained 74 and 67 molecules, respectively, the appropriate F value to consider was that for 60 samples. Thus, the 3d QSPR models are statistically significant (at p= 0.01) when F > 4.126 and the 5d QSPR models when F > 3.339. Figure R2 of all QSPR models for ease of visual comparison, and Tables Kavalues are contained in the .The quality of 3d EEM QSPR models can be markedly increased by removing the outliers. In this case, the models have average Kavalues may also be a reason for low performance in some cases. A table containing information about outlier molecules is given in the , namely atomic charges originating from the non-dissociated molecule. Nonetheless, in our study we found that using two additional charge descriptors from the dissociated molecule can markedly improve the predictive power of the EEM QSPR models. Tables d, namelyR2 values from Table R2 = 0.913, while 5d QM QSPR models have average R2 = 0.951. We also note that adding more descriptors to a QM QSPR model brings less improvement than adding more descriptors to an EEM QSPR model.Another important question is how accurate the EEM QSPR models are in comparison with QM QSPR models. Table Kaprediction capability from EEM atomic charges, as was done for QM QSPR models by Gross et al. [EEM parameters are available only for a relatively small number of theory levels (HF and B3LYP) and basis sets (STO-3G and 6–31G*). Therefore we can not perform such a deep analysis of theory level and basis set influence on p s et al.  or Svobos et al. . We can s et al. , and it Ka with accuracy comparable to MPA and NPA. In the case of EEM QSPR models, we did indeed find that MPA provided the best models, but most of the other population analyses gave comparable results. This confirms previous observations that the EEM approach is not able to faithfully mimic MK charges [Kamore accurately than the corresponding QM QSPR models.Eleven EEM parameter sets were published for B3LYP/6–31G* with six different population analyses  were previously published by Gross and Seybold [Kaprediction based on EEM charges. Therefore, we can not provide a comparison between EEM QSPR models, but we can compare against QSPR models based on QM charges only. It is seen therein that our 3d QM QSPR models show markedly higher R2 and F values than the models published by Gross and Seybold and Kreye and Seybold (even if some of these models employ higher basis sets) and comparable R2 and F values as models published by Svobodova and Geidl. Moreover, our 5d QM QSPR models outperform the models from Svobodova and Geidl. Our best EEM QSPR models  provide even better results than QM QSPR models from Gross and Seybold and Kreye and Seybold. These EEM QSPR models are not as accurate as the QM QSPR models published by Svobodova and Geidl or those developed in this work, but the loss of accuracy is not too high .QM QSPR models for p  Seybold , Kreye a Seybold  and Svob Seybold . Table 5Kaprediction. Nonetheless, the robustness of these models should be proved. Therefore, all the 5d EEM QSPR models  were tested by cross-validation. For comparison, also the cross-validation of all 5d QM QSPR models  was done. The k-fold cross-validation procedure was used [k = 5. Specifically, the set of phenol molecules was divided into five parts (each contained 20% of the molecules). The division was done randomly, and included stratification by p Kavalue. Afterwards, five cross validation steps were performed. In the first step, the first part was selected as a test set, and the remaining four parts were taken together as the training set. The test and training sets for the other steps were prepared in a similar manner, by subsequently considering one part as a test set, while the remaining parts served as a training set. For each step, the QSPR model was parameterized on the training set. Afterwards, the p Kavalues of the respective test molecules were calculated via this model, and compared with experimental p Kavalues. The results are summarized in the  provide accurate results as well. The source of the EEM parameters also did not affect the quality of the EEM QSPR models significantly.As expected, the QM QSPR models provided more accurate p Kaprediction for the test, training and complete set were comparable. The applicability of EEM QSPR models for other chemical classes was tested in a case study focused on carboxylic acids. This case study showed that EEM QSPR models are indeed applicable for p Kaprediction also for carboxylic acids. Namely, 5 from 12 of these models were able to predict p Kawith R2 > 0.9, while the best EEM QSPR model reached R2 = 0.925.The robustness of EEM QSPR models was successfully confirmed by cross-validation. Specifically, the accuracy of p Ka. Their main advantages are that they are accurate, and can predict p Kavalues very quickly, since the atomic charge descriptors used in the QSPR model can be obtained much faster by EEM than by QM. Additionally, the quality of EEM QSPR models is less dependent on the type of atomic charges used  than in the case of QM QSPR models. Accordingly, EEM QSPR models constitute a p Kaprediction approach which is very suitable for virtual screening.Therefore, EEM QSPR models constitute a very promising approach for the prediction of p The concept of the study originated from JK and was reviewed and extended by RA, while the design was put together by RSV and SG and reviewed by JK and RA. SG and CMI collected and prepared the input data. SG, OS, DS and TB performed the acquisition and post-processing of data. The data were analyzed and interpreted by RSV, SG, CMI and JK. The manuscript was written by RSV and SG in cooperation with JK and CMI, and reviewed by all authors.Radka Svobodová Vařeková and Stanislav Geidl wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.Kavalues. (XLS 36 KB)Additional file 1: Table S1a: The list of the phenol molecules, including their names, NCS numbers, CAS numbers and experimental p Additional file 2: Molecules. The SDF files with the structures of the molecules and also their dissociated forms. (ZIP 310 KB)Kavalues. (XLS 36 KB)Additional file 3: Table S1b: The list of the carboxylic acid molecules, including their names, NCS numbers, CAS numbers and experimental p Additional file 4: Table S2: The parameters of all the QSPR models for phenols. (XLS 46 KB)Kavalues for all QSPR models (for phenols). (XLS 421 KB)Additional file 5: Table S6: The table containing charge descriptors for all charge calculation approaches and predicted p Additional file 6: Table S3: The information about outlier molecules for phenols. (XLS 46 KB)Additional file 7: Table S4: The table of cross-validation results for phenols. (XLS 174 KB)Additional file 8: Table S5: The quality and statistical criteria of QSPR models for carboxylic acids. (XLS 130 KB)Authors’ original file for figure 1Authors’ original file for figure 2Authors’ original file for figure 3Below are the links to the authors’ original submitted files for images.


We aimed to engineer transgenic plants for the purpose of early detection of plant pathogen infection, which was accomplished by employing synthetic pathogen inducible promoters fused to reporter genes for altered phenotypes in response to the pathogen infection. Toward this end, a number of synthetic promoters consisting of inducible regulatory elements fused to a red fluorescent protein (RFP) reporter were constructed for use in phytosensing.Agrobacterium-mediated transient expression assay was evaluated, then utilized to assess the inducibility of each synthetic promoter construct in vivo. Tobacco (Nicotiana tabacum cv. Xanthi) leaves were infiltrated with Agrobacterium harboring the individual synthetic promoter-reporter constructs. The infiltrated tobacco leaves were re-infiltrated with biotic  or abiotic  agents 24 and 48 hours after initial agroinfiltration, followed by RFP measurements at relevant time points after treatment. These analyses indicated that the synthetic promoter constructs were capable of conferring the inducibility of the RFP reporter in response to appropriate phytohormones and bacterial pathogens, accordingly.For rapid analysis, an Agrobacterium-mediated transient expression is an efficient method for in vivo assays of promoter constructs in less than one week. Our results provide the opportunity to gain further insights into the versatility of the expression system as a potential tool for high-throughput in planta expression screening prior to generating stably transgenic plants for pathogen phytosensing. This system could also be utilized for temporary phytosensing; e.g., not requiring stably transgenic plants.These observations demonstrate that the  Transgenic techniques have become a powerful tool to address important biological and agricultural challenges, and there is great potential for the production of designer plants using modern biotechnology tools ,2. Many cis-acting regulatory elements (REs) corresponding to key proteins involved in defense, and pathogen-specific responses. These cis-acting regulatory elements are conserved among plant species, enabling them to be used to construct synthetic inducible promoters in heterologous expression systems  solid medium supplemented with rifampicin (50 mg/L), spectinomycin (200 mg/L), and streptinomycin (50 mg/L) at 28°C for 2 days. One single colony was inoculated in 2 ml YEP liquid medium supplemented with the above-mentioned antibiotics and grown for ~2 h at 28°C. One milliliter of this starter culture was then inoculated in 25 ml YEP liquid medium and grown for overnight at 28°C. Agrobacterium cells were collected by centrifugation for 10 min at 3000 g and resuspended in 25 ml infiltration medium . Centrifugation followed by resuspension in the infiltration medium was repeated for 2 times as above. The bacterial suspension was adjusted to a final OD600 of 0.3 for agroinfiltration.2 in each infiltrated area). After agroinfiltration, tobacco plants were covered with transparent plastic covers which were sprayed with water and maintained in a growth chamber at 22°C under 16 h light for 24 h. Three biological replicates  were used, and the experiments were repeated independently at least three times.Tobacco plants were removed from the growth chambers and placed under a white fluorescent lamp for 1 h prior to infiltration to open the stomata fully as an aid to infiltration. Infiltration was performed on near fully expanded leaves  that were still attached to the intact plant. Leaves of the same age on the same branch were used for each experimental test. Each bacterial suspension was infiltrated into leaves of three different plants from the abaxial side of the leaf with a needleless syringe. By infiltration, 100 μl of bacterial suspension was injected into each spot   medium overnight. After centrifugation, bacterial cells were resuspended in 10 mM MgCl2, followed by centrifugation and were resuspended again. Twenty-four hours after the initial agroinfiltration, each bacterial suspension was further infiltrated at the same infiltrated spots. For mock control treatments, leaves were infiltrated with 10 mM MgCl2. The numbers of bacteria was estimated in leaf disks (5 mm in diameter) taken from infiltrated areas at different time points post-infection. The discs were ground in 1 ml of 10 mM MgCl2, and serial dilutions were plated out on TSB solid medium. After incubation at 28°C for 24 h, the colonies were counted. All the experiments were repeated independently at least two times.For chemical treatments, 48 hrs after the initial agroinfiltration, 4 mM salicylic acid (SA), 4 mg/ml ethephon , or 100 μM methyl jasmonate (MeJA)  was further infiltrated to the same infiltrated spots. For mock control treatments, leaves were infiltrated with water. For bacterial pathogen treatments, pporRFP reporter was measured at time points 0 and 72 h after phytohormone treatments and at time points 0, 24, 48, and 72 h after bacterial pathogen treatments. The treated plants were visualized using an epifluorescence microscope Olympus SZX12 , and the images were captured using imaging software QCapture 2.56 . Fluorescent signal intensity was measured via scanning fluorescence spectrometry using SPEX Fluorolog II spectrophotometer . The infiltrated spots were excited at 530 nm, and emission spectra was scanned and recorded from 550 to 640 nm. Intensity was measured at 591 nm in counts per second (cps).Expression of pporRFP expression by using measurements from non-transgenic tobacco as background expression when treated with the biotic and abiotic treatments. Data normalization was then conducted as described [t-test (p < 0.05).Background subtraction was applied to each measurement of escribed . The folpporRFP expression. CNS conceived and coordinated the study, assisted with revision to the manuscript, and obtained the funding. All authors read, contributed to, and approved the final version of this manuscript.WL designed and constructed all the synthetic promoter constructs, performed a majority of the experiments, analyzed the data, and drafted the manuscript. MM participated in designing the experiments and data analysis, performed the experiments corresponding to the bacterial growth population size, and revised the manuscript. MRR assisted with growing the tobacco plants and preparation of the bacterial inoculums. MHF assisted with data collection corresponding to the fluorescent signal intensity of the


Diabetic nephropathy is one of the most significant microvascular complications in patients with type 2 diabetics. The concise mechanism of diabetic nephropathy is unknown and there is no successful treatment. The objective of study was to investigate effects of Chinese herbs (Tangshen Formula) on diabetic nephropathy in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. OLETF rats and LETO rats were divided into four groups: LETO control, OLETF diabetics, OLETF diabetics treated with Tangshen Formula, and OLETF diabetics treated with Monopril. Body weight, blood glucose, and 24 h urinary proteins were measured once every four weeks. Blood samples and kidney tissues were obtained for analyses of total cholesterol, triglyceride, whole blood viscosity, plasma viscosity, and pathohistological examination at 36 and 56 weeksrespectively. Untreated OLETF rats displayed diabetic nephropathy over the study period. Treatment of OLETF rats with Tangshen Formula attenuated the increases in blood glucose, body weight, 24 h urinary protein content, serum total cholesterol, whole blood viscosity and plasma viscosity at certain time. Treatment with Tangshen Formula also reduced glomerulosclerotic index and interstitial fibrotic index seen in OLETF rats. In conclusion, Tangshen Formula could attenuate the development of diabetic nephropathy in OLETF rat diabetic model. Diabetic nephropathy (DN) is one of the most important microvascular complications associated with type 2 diabetic patients and has emerged as a leading cause of the end-stage renal disease in developed countries , 2. DN iA useful model of the human type II diabetes mellitus is the Otsuka Long-Evans Tokushima Fatty (OLETF) rat. This model is characterized by the development of late-onset hyperglycemia, mild obesity, and the associated diabetic complications. At 22 weeks of age the OLETF rats develop overt albuminuria and at 30 weeks, OLETF showed significant proteinuria and morphological changes in the kidney such as glomerular hypertrophy and extracellular matrix expansion. At 54 weeks, OLETF rats developed advanced kidney injuries such as heavy proteinuria, diffuse glomerulosclerosis, nodular lesions and severe tubulointerstitial fibrosis, which are resemble to late stage of human DN. Thus, the OLETF rat is considered to be a useful animal model to study type 2 diabetic nephropathy .In China, traditional herbal medicines have been widely used for the treatment of diabetes and its complications for thousands of years. Recently they have received worldwide attention and are becoming promising sources of new therapeutic agents for diabetic nephropathy . One sucFour-week-old male OLETF rats and age-matched Long-Evans Tokushima Otsuka (LETO) rats were kindly provided by Tokushima Research Institute . All rats were housed at 22 ± 3°C and 50 ± 10% humidity using a 12 h light/dark cycle. All animals were given free access to standard rat chow and water. Four groups of rats were prepared at 12 weeks of age: n = 20, LETO control),non-diabetic LETO rats given distilled water , diabetic OLETF rats given water ,diabetic OLETF rats administered Tangshen Formula . OLETF rats administered Monopril , radix rehmanniae (15 g), radix notoginseng (2 g), prepared radix et rhizome rhei with wine (8 g), fructus aurantii (6 g), fructus corni (11 g) and ramulus euonymi (12 g) . The experimental extraction procedure was identical to that used for the clinical preparation of the formula. Briefly, herbs were well mixed and soaked in distilled water for 30 min, boiled in 10 volumes of water (v/w) for 1 h, and extracted twice. The extract was filtrated and condensed to the concentration of 1 g/ml and processed to powder by spray drying. The volume administered to the animals was calculated to be 1 ml/100 g body weight . Ten rats from the LETO control group and seven rats from the other three groups were sacrificed at 36 weeks of age. The remaining rats in each group were sacrificed at 56 weeks of age. The endpoints of experiment were determined according to pathological changes during life span of OLETF rats. Rats were sacrificed by intraperitoneal injection of chloral hydrate. Blood samples were then collected from the abdominal aorta, and kidneys were removed and separated into two pieces for histopathological examination. This study was approved by the Ethics Committee of China-Japan Friendship Institute of Clinical Medicine and performed in accordance with Guiding Principles for the Care and Use of Laboratory Animals.Body weight of rats was measured at 4-week intervals. Blood was sampled from the tail vein at 4-week intervals, and blood glucose levels measured by One Touch Ultrablood glucose monitoring system . Rats were housed individually in metabolic cages  for 24-h urinary collection at 4-week intervals. Rats were sacrificed and aortic blood was collected and divided into 2 parts. One part of blood samples was collected into tubes without anticoagulant and centrifuged at 3000 g/min and 4°C for 15 min. Serum was separated and total cholesterol, triglycerides, urea nitrogen, creatinine, total protein, and albumin were measured using a CD-1600CS hematology analyzer .2EDTA. Blood viscosity was measured at 37°C with a coaxial cylinder microviscometer  at a shear rate of 0.945 s and 94.5 s followed by plasma viscosity determination after centrifugation.The other part of blood was collected into tubes containing Naμm) from each sample were cut and stained with periodic acid Schiff's stain for determination of glomerulosclerosis. The degree of glomerulosclerosis, defined as a thickening of the basement membrane and mesangial expansion, was evaluated as described previously [n1) + (2 × n2) + (3 × n3) + (4 × n4)/n0 + n1 + n2 + n3 + n4, where nx is number of glomeruli in each grade of glomerulosclerosis. The observer was blinded to all tissue samples.Sections of kidney tissue were removed and immediately fixed in 10% phosphate buffered formalin solution and embedded in paraffin. Sections  as described previously in detail .P < .05 being considered statistically significant. Data are presented as mean ± SEM unless stated otherwise. Significant differences between groups were compared using ANOVA with Body weight of OLETF rats was heavier than that of LETO rats from 12 to 36 weeks . HoweverBlood glucose levels for the LETO control rats did not change throughout the study period . Blood gUrinary protein in all OLETF rats gradually and continually increased during the entire study period compared with levels in the LETO rats that remained unchanged . Rats tr There was no statistical difference in whole blood viscosity at 0.945 s and 94.5 s among the four groups at 36 weeks of age. However, whole blood viscosity for control OLETF rats was markedly higher than those of LETO rats at 56 weeks of age . These tPlasma viscosity of control OLETF rats was markedly increased compared with the LETO rats at 36 and 56 weeks of age. Plasma viscosity of the Tangshen Formula treated OLETF rats were significantly lower than that of the untreated rats at weeks 36 and 56 . P < .01) in untreated OLETF rats at 56 weeks of age. Triglyceride levels in untreated OLETF rats were markedly increased compared to the LETO rats at 36 and 56 weeks of age (P < .01). Serum total protein and albumin of control OLETF rats were similar to LETO rats at 36 weeks of age; however, they were statistically lower at 56 weeks of age. There was no difference in urea nitrogen levels between LETO and control OLETF rats at 36 weeks of age but urea nitrogen levels of control OLETF rats were higher than those of LETO rats at 56 weeks of age. Serum creatinine levels in untreated OLETF rats were significantly lower than in LETO rats at both 36 and 56 weeks of age (P < .01) while triglyceride levels of OLETF rats were significantly decreased by Monopril at 36 weeks of age (P < .05).There was no statistical difference in total cholesterol between control OLETF rats and LETO rats at 36 weeks of age . Compares of age . This fiGlomerular lesions in OLETF rats were characterized by hyalinosis, thickening of the basement membrane, mesangial expansion and sclerotic lesions. These pathological changes at 56 weeks of age were more severe than those at 36 weeks of age. Interstitial fibrosis in OLETF rats was focal and mild at 36 weeks of age while at 56 weeks of age severe changes occurred that included protein cast, tubular dilation or atrophy, and infiltration of inflammatory cells. Treatment with Tangshen Formula and Monopril significantly suppressed glomerulosclerosis in OLETF rats. Interstitial fibrotic lesions in OLETF rats were significantly improved by treatment with Tangshen Formula at both 36 and 56 weeks of age . GlomeruIn recent years the number of people suffering from the end-stage renal disease has increased worldwide. Approximately one third of ESRD results from diabetic nephropathy (DN). Effective treatment of DN is an important challenge to nephrologists today . Animal Traditional Chinese Medicine also exhibits beneficial effects on DN and is becoming a promising therapy , 16. HowOLETF rats are widely used in many laboratories as a model of type 2 diabetes in humans. OLETF rats are characterized by late-onset hyperglycemia, a mild and chronic course of diabetes mellitus, and the associated complications of diabetes. Diabetes develops in almost all male OLETF rats by 25 weeks of age. As the disease progresses, renal complication becomes inevitable in this animal , 17. Hyperglycemia is a critical factor in the development of diabetic nephropathy. It is associated with an increase in mesangial cell proliferation and hypertrophy, as well as increased matrix production and basement membrane thickening , 19. TheP < .01), while triglyceride levels increased by 13%. The increase in cholesterol is believed to be a compensatory production of lipids in the liver stimulated by hypoalbuminemia [We observed an increase in total serum cholesterol in OLETF rats over the study period (approximately 70%, uminemia . Circulauminemia . ROS reduminemia , 28. It uminemia , 29, 30.We showed that Tangshen Formula and Monopril had beneficial effects on serum lipids. Total cholesterol was statistically lower in OLETF rats treated with Tangshen Formula at 56 weeks of age than control OLETF rats, while Monopril lowered triglyceride levels of OLETF rats at 36 weeks of age. It is not surprising that Monopril had a lipid-lowering effect since angiotensin converting enzyme inhibitors are known to lower serum total cholesterol and triglycerides in DN patients . This reChanges in blood rheology are an important factor that can lead to microvascular complications in diabetes. Hyperviscosity-related resistance to glomerular blood flow causes a compensatory elevation in glomerular hydrostatic pressure, which leads to an increase in protein permeability across the glomerular basement membrane. The increased viscosity may also lead to a cessation of glomerular blood flow and subsequent capillary thrombosis and glomerulosclerosis –38. Our The results from this study suggest that the beneficial effect of Tangshen Formula may be due to its ability to limit the development of hyperlipidemia as well as its beneficial effects on renal hemorheology in the OLETF rats. Tangshen Formula could reduce plasma viscosity at 36 weeks age, and at 56 weeks of age, Tangshen formula exhibits renoprotective effects by reduce the protein excretion and alleviated histological changes, the effects may be related to rectify the abnormality lipid metabolism, hyperglycemia and hemorheology disorder. This theory is illustrated in In conclusion, we found that Tangshen Formula attenuates the development of diabetic nephropathy in type 2 diabetic OLETF rats. Tangshen Formula may, therefore, represent a potentially new therapeutic strategy in the treatment of diabetic nephropathy in type 2 diabetes.