Diet adaptation in dog reflects spread of prehistoric agriculture.

Adaptations allowing dogs to thrive on a diet rich in starch, including a significant AMY2B copy number gain, constituted a crucial step in the evolution of the dog from the wolf. It is however not clear whether this change was associated with the initial domestication, or represents a secondary shift related to the subsequent development of agriculture. Previous efforts to study this process were based on geographically limited data sets and low-resolution methods, and it is therefore not known to what extent the diet adaptations are universal among dogs and whether there are regional differences associated with alternative human subsistence strategies. Here we use droplet PCR to investigate worldwide AMY2B copy number diversity among indigenous as well as breed dogs and wolves to elucidate how a change in dog diet was associated with the domestication process and subsequent shifts in human subsistence. We find that AMY2B copy numbers are bimodally distributed with high copy numbers (median 2nAMY2B=11) in a majority of dogs but no, or few, duplications (median 2nAMY2B=3) in a small group of dogs originating mostly in Australia and the Arctic. We show that this pattern correlates geographically to the spread of prehistoric agriculture and conclude that the diet change may not have been associated with initial domestication but rather the subsequent development and spread of agriculture to most, but not all regions of the globe.

Language mapping in healthy volunteers and brain tumor patients with a novel navigated TMS system: evidence of tumor-induced plasticity.

OBJECTIVE: This article explores the feasibility of a novel repetitive navigated transcranial magnetic stimulation (rnTMS) system and compares language mapping results obtained by rnTMS in healthy volunteers and brain tumor patients. METHODS: Fifteen right-handed healthy volunteers and 50 right-handed consecutive patients with left-sided gliomas were examined with a picture-naming task combined with time-locked rnTMS (5-10 Hz and 80-120% resting motor threshold) applied over both hemispheres. Induced errors were classified into four psycholinguistic types and assigned to their respective cortical areas according to the coil position during stimulation. RESULTS: In healthy volunteers, language disturbances were almost exclusively induced in the left hemisphere. In patients errors were more frequent and induced at a comparative rate over both hemispheres. Predominantly dysarthric errors were induced in volunteers, whereas semantic errors were most frequent in the patient group. CONCLUSION: The right hemisphere's increased sensitivity to rnTMS suggests reorganization in language representation in brain tumor patients. SIGNIFICANCE: rnTMS is a novel technology for exploring cortical language representation. This study proves the feasibility and safety of rnTMS in patients with brain tumor.

MHC variability supports dog domestication from a large number of wolves: high diversity in Asia.

The process of dog domestication is still somewhat unresolved. Earlier studies indicate that domestic dogs from all over the world have a common origin in Asia. So far, major histocompatibility complex (MHC) diversity has not been studied in detail in Asian dogs, although high levels of genetic diversity are expected at the domestication locality. We sequenced the second exon of the canine MHC gene DLA-DRB1 from 128 Asian dogs and compared our data with a previously published large data set of MHC alleles, mostly from European dogs. Our results show that Asian dogs have a higher MHC diversity than European dogs. We also estimated that there is only a small probability that new alleles have arisen by mutation since domestication. Based on the assumption that all of the currently known 102 DLA-DRB1 alleles come from the founding wolf population, we simulated the number of founding wolf individuals. Our simulations indicate an effective population size of at least 500 founding wolves, suggesting that the founding wolf population was large or that backcrossing has taken place.

Origins of domestic dog in southern East Asia is supported by analysis of Y-chromosome DNA.

Global mitochondrial DNA (mtDNA) data indicates that the dog originates from domestication of wolf in Asia South of Yangtze River (ASY), with minor genetic contributions from dog-wolf hybridisation elsewhere. Archaeological data and autosomal single nucleotide polymorphism data have instead suggested that dogs originate from Europe and/or South West Asia but, because these datasets lack data from ASY, evidence pointing to ASY may have been overlooked. Analyses of additional markers for global datasets, including ASY, are therefore necessary to test if mtDNA phylogeography reflects the actual dog history and not merely stochastic events or selection. Here, we analyse 14,437 bp of Y-chromosome DNA sequence in 151 dogs sampled worldwide. We found 28 haplotypes distributed in five haplogroups. Two haplogroups were universally shared and included three haplotypes carried by 46% of all dogs, but two other haplogroups were primarily restricted to East Asia. Highest genetic diversity and virtually complete phylogenetic coverage was found within ASY. The 151 dogs were estimated to originate from 13-24 wolf founders, but there was no indication of post-domestication dog-wolf hybridisations. Thus, Y-chromosome and mtDNA data give strikingly similar pictures of dog phylogeography, most importantly that roughly 50% of the gene pools are shared universally but only ASY has nearly the full range of genetic diversity, such that the gene pools in all other regions may derive from ASY. This corroborates that ASY was the principal, and possibly sole region of wolf domestication, that a large number of wolves were domesticated, and that subsequent dog-wolf hybridisation contributed modestly to the dog gene pool.

Regional occurrence, high frequency but low diversity of mitochondrial DNA haplogroup d1 suggests a recent dog-wolf hybridization in Scandinavia.

The domestic dog mitochondrial DNA (mtDNA)-gene pool consists of a homogenous mix of haplogroups shared among all populations worldwide, indicating that the dog originated at a single time and place. However, one small haplogroup, subclade d1, found among North Scandinavian/Finnish spitz breeds at frequencies above 30%, has a clearly separate origin. We studied the genetic and geographical diversity for this phylogenetic group to investigate where and when it originated and whether through independent domestication of wolf or dog-wolf crossbreeding. We analysed 582 bp of the mtDNA control region for 514 dogs of breeds earlier shown to harbour d1 and possibly related northern spitz breeds. Subclade d1 occurred almost exclusively among Swedish/Finnish Sami reindeer-herding spitzes and some Swedish/Norwegian hunting spitzes, at a frequency of mostly 60-100%. Genetic diversity was low, with only four haplotypes: a central, most frequent, one surrounded by two haplotypes differing by an indel and one differing by a substitution. The substitution was found in a single lineage, as a heteroplasmic mix with the central haplotype. The data indicate that subclade d1 originated in northern Scandinavia, at most 480-3000 years ago and through dog-wolf crossbreeding rather than a separate domestication event. The high frequency of d1 suggests that the dog-wolf hybrid phenotype had a selective advantage.

BPSI2.0: a C/C++ interface program for species identification via DNA barcoding with a BP-neural network by calling the Matlab engine.

BP-Species Identification (BPSI2.0) is a computer program that performs species identification by training a Back-Propagation Neural Network. A short DNA barcoding segment is used as input for training a three-layer BP network. The trained network can assign an unknown query sequence to a known species in the user's database, and provide the corresponding subvector value of the output vector as a relative probability value.

Expressed sequence tags from the midgut and an epithelial cell line of Chironomus tentans: annotation, bioinformatic classification of unknown transcripts and analysis of expression levels.

Expressed sequence tags (ESTs) were generated from two Chironomus tentans cDNA libraries, constructed from an embryo epithelial cell line and from larva midgut tissue. 8584 5'-end ESTs were generated and assembled into 3110 tentative unique transcripts, providing the largest contribution of C. tentans sequences to public databases to date. Annotation using Blast gave 1975 (63.5%) transcripts with a significant match in the major gene/protein databases, 1170 with a best match to Anopheles gambiae and 480 to Drosophila melanogaster. 1091 transcripts (35.1%) had no match to any database. Studies of open reading frames suggest that at least 323 of these contain a coding sequence, indicating that a large proportion of the genes in C. tentans belong to previously unknown gene families.

ESTs from brain and testis of White Leghorn and red junglefowl: annotation, bioinformatic classification of unknown transcripts and analysis of expression levels.

We report the generation, assembly and annotation of expressed sequence tags (ESTs) from four chicken cDNA libraries, constructed from brain and testis tissue dissected from red junglefowl and White Leghorn. 21,285 5'-end ESTs were generated and assembled into 2,813 contigs and 9,737 singletons, giving 12,549 tentative unique transcripts. The transcripts were annotated using BLAST by matching to known chicken genes or to putative homologues in other species using the major gene/protein databases. The results for these similarity searches are available on www.sbc.su.se/~arve/chicken. 4,129 (32.9%) of the transcripts remained without a significant match to gene/protein databases, a proportion of unmatched transcripts similar to earlier non-mammalian EST studies. To estimate how many of these transcripts may represent novel genes, they were studied for the presence of coding sequence. It was shown that most of the unique chicken transcripts do not contain coding parts of genes, but it was estimated that at least 400 of the transcripts contain coding sequence, indicating that 3.2% of avian genes belong to previously unknown gene families. Further BLAST search against dbEST left 1,649 (13.1%) of the transcripts unmatched to any library. The number of completely unmatched transcripts containing coding sequence was estimated at 180, giving a measure of the number of putative novel chicken genes identified in this study. 84.3% of the identified transcripts were found only in testis tissue, which has been poorly studied in earlier chicken EST studies. Large differences in expression levels were found between the brain and testis libraries for a large number of transcripts, and among the 525 most frequently represented transcripts, there were at least 20 transcripts with significant difference in expression levels between red junglefowl and White Leghorn.

Phospholipase Czeta, the trigger of egg activation in mammals, is present in a non-mammalian species.

The activation of the egg to begin development into an embryo is triggered by a sperm-induced increase in intracellular egg Ca2+. There has been much controversy about how the sperm induces this fundamental developmental event, but recent studies suggest that, in mammals, egg activation is triggered by a testis-specific phospholipase C: PLCzeta. Since the discovery of PLCzeta, it has been unclear whether its role in triggering egg activation is common to all vertebrates, or is confined to mammals. Here, we demonstrate for the first time that PLCzeta is present in a non-mammalian vertebrate. Using genomic and cDNA databases, we have identified the cDNA encoding a PLCzeta orthologue in the domestic chicken that, like the mammalian isoforms, is a testis-specific gene. The chicken PLCzeta cDNA is 2152 bp in size and encodes an open reading frame of 639 amino acids. When injected into mouse oocytes, chicken PLCzeta cRNA triggers Ca2+ oscillations, indicating that it has functional properties similar to those of mammalian PLCzeta. Our findings suggest that PLCzeta may have a universal role in triggering egg activation in vertebrates.

Detection of sequence polymorphisms in red junglefowl and White Leghorn ESTs.

Over 16,000 high quality expressed sequence tags (ESTs) from red junglefowl (RJ) and White Leghorn (WL) brain and testis cDNA libraries were generated. Here, we have used this resource for detection of single nucleotide polymorphisms (SNPs), and also completed full-length sequencing of 46 pairs of clones, representing the same gene from both the RJ and WL libraries. From the main set of ESTs, which were assembled using Phrap, 746 putative SNPs were identified, of which 76% were transitions and 24% were transversions. A subset of SNPs was evaluated by sequence analysis of five RJ and five WL birds. Nine of 12 SNPs were verified in this limited sample, suggesting that a majority of the putative polymorphisms documented in this study represent real SNPs. During full-length sequencing of the 46 RJ/WL clones 100 SNPs were identified, which translated to a frequency of 1.90 SNPs/1000 bp. The number of transitions and transversions were 77% and 23%, respectively, and the proportion of non-synonymous vs. synonymous SNPs was 20% and 80%, respectively. Four large insertions/deletions were identified between the RJ and WL full-length sequences, and they appear to represent different splice variants.

Microemulsions for topical delivery of estradiol.

Estradiol has been widely used for the treatment of hormonal insufficiencies. Due to its extensive first pass metabolism after oral administration, transdermal administration of estradiol in gels and emulsions has been used to improve its bioavailability, prolong activity and to optimize metabolic profile. The purpose of this study was to investigate microemulsions as delivery systems for estradiol. Various o/w microemulsions were used to deliver estradiol across human abdominal skin in vitro. Trasdermal flux of estradiol was determined using Franz-type diffusion cells and the samples were analyzed by high-performance liquid chromatography (HPLC). The permeation data showed that microemulsion formulations increased estradiol flux 200-700-fold over the control, but permeability coefficients were decreased by 5-18 times. The superior transdermal flux of estradiol was due to 1500-fold improvement in solubilization of estradiol by microemulsions. The results suggest that microemulsions are potential vehicles for improved topical delivery of estradiol.

Enterovirus antibody levels during the first two years of life in prediabetic autoantibody-positive children.

AIMS/HYPOTHESIS: We evaluated the role of enterovirus infections in the pathogenesis of Type I (insulin-dependent) diabetes mellitus by monitoring enterovirus antibody levels in prediabetic children who turned positive for diabetes-associated autoantibodies in a prospective birth cohort study. METHODS: Serial serum samples taken during prospective observation starting at birth were analysed for IgG and IgA class antibodies against enterovirus antigens including purified coxsackievirus B4, echovirus 11, poliovirus 1 and a synthetic enterovirus peptide antigen using enzyme immunoassay. Maternal samples taken at the end of the third month of pregnancy were also studied. Analyses were done from 21 childen who developed autoantibodies and from 104 autoantibody-negative control children who were matched for the time of birth, gender and HLA susceptibility alleles. For comparison, adenovirus antibodies were also analysed from all samples collected. RESULTS: IgG class enterovirus antibody levels were high in maternal samples and in cord blood in both case and control children. After birth the IgG levels decreased reaching a nadir at the age of 6 months. No IgA class antibodies were detected at birth but started to emerge postnatally. Antibody levels did not differ between the autoantibody positive and the control children during the first 6 months of life. From 6 months to 24 months of age, the autoantibody positive children had higher IgG and IgA levels against coxsackievirus B4, echovirus 11 and the synthetic enterovirus peptide antigens than control children but poliovirus 1 and adenovirus antibodies were closely similar in the two groups. The difference between children with autoantibodies and control children was predominantly seen among boys and among those with the HLA-DQB1*0302/x genotype. CONCLUSIONS/INTERPRETATION: Our data show that children who seroconverted for diabetes-associated auto-antibodies develop stronger humoral immune responses to coxsackievirus B4, echovirus 11 and a synthetic enterovirus peptide antigen than children who remained negative for autoantibodies. Poliovirus antibodies induced by uniform vaccinations did not differ between the prediabetic and control children suggesting that the regulation of antibody responses to enteroviruses is not disturbed. Accordingly, the results imply a stronger enterovirus exposure in prediabetic children supporting the role of enteroviruses in the pathogenesis of Type I diabetes.

A novel method for forensic DNA investigations: repeat-type sequence analysis of tandemly repeated mtDNA in domestic dogs.

A highly variable and heteroplasmic tandem repeat region situated in the mitochondrial mt DNA control region (CR) in domestic dogs and wolves was studied to evaluate its suitability as a forensic genetic marker for analysis of single hairs. The tandem repeat array is composed of three 10-bp repeat types that are distributed so that a secondary DNA sequence is formed. Thus, the region presents two levels of variation: variation in the number of repeats and variation in the secondary DNA sequence of repeat types. Two analysis methods were therefore tested; fragment length analysis and analysis of the sequence of repeat types. Fragment analysis produced unique profiles that could be used to discriminate between blood samples from maternally closely related individuals. However, different hairs from one individual did not have the same fragment profile, and the method is, therefore, not suitable for analysis of single hairs. In contrast, analysis of the repeat type sequences (array types) is highly informative. When different hairs from one individual were studied, identical array types were found. The repeat-type sequence variation was studied among individuals having identical nonrepetitive CR mtDNA sequence variants. Seven, six, and two individuals, representing three different sequence variants, respectively, were analyzed. All these individuals had different array types, which implies a very high genetic variation between individuals in this region. The analysis method considerably improves the exclusion capacity of mtDNA analysis of domestic dogs compared with sequence analysis of non-repetitive DNA.

mtDNA tandem repeats in domestic dogs and wolves: mutation mechanism studied by analysis of the sequence of imperfect repeats.

The mitochondrial (mt) DNA control region (CR) of dogs and wolves contains an array of imperfect 10 bp tandem repeats. This region was studied for 14 domestic dogs representing the four major phylogenetic groups of nonrepetitive CR and for 5 wolves. Three repeat types were found among these individuals, distributed so that different sequences of the repeat types were formed in different molecules. This enabled a detailed study of the arrays and of the mutation events that they undergo. Extensive heteroplasmy was observed in all individuals; 85 different array types were found in one individual, and the total number of types was estimated at 384. Among unrelated individuals, no identical molecules were found, indicating a high rate of evolution of the region. By performing a pedigree analysis, array types which had been inherited from mother to offspring and array types which were the result of somatic mutations, respectively, could be identified, showing that about 20% of the molecules within an individual had somatic mutations. By direct pairwise comparison of the mutated and the original array types, the physiognomy of the inserted or deleted elements (indels) and the approximate positions of the mutations could be determined. All mutations could be explained by replication slippage or point mutations. The majority of the indels were 1-5 repeats long, but deletions of up to 17 repeats were found. Mutations were found in all parts of the arrays, but at a higher frequency in the 5' end. Furthermore, the inherited array types within the mother-offspring pair were aligned and compared so that germ line mutations could be studied. The pattern of the germ line mutations was approximately the same as that of the somatic mutations.

Forensic evidence based on mtDNA from dog and wolf hairs.

In six forensic cases involving murder, bank robbery, theft and poaching, evidence material comprising shed hairs supposedly originating from dogs or wolves was analyzed by mitochondrial (mt) DNA sequencing. A 79 bp segment of the control region was amplified, sequenced, and compared with an established database of the domestic dog and wolf populations. In three murder cases exclusions of all eight suspects could be made. Furthermore, two of the murders could be linked to each other by a rare sequence variant, and the breed of the dog was indicated. In a theft case and a bank robbery a link could be established between the evidence material and the suspects. In a case of suspected wolf poaching, it could be established that the evidential material was of dog rather than wolf origin. We conclude that single hairs from common pets are suitable for DNA analysis and that the described method has proved to be a valuable tool for forensic investigations.

Evaluation of cytotoxicity of various ophthalmic drugs, eye drop excipients and cyclodextrins in an immortalized human corneal epithelial cell line.

PURPOSE: An immortalized human corneal epithelial cell line (HCE) was tested as a screening tool for prediction of topical ocular irritation/toxicity by pharmaceuticals METHODS: Effects of various drugs, excipients and cyclodextrins (CDs) on viability of HCE cells were evaluated using two in vitro cytotoxicity tests, 3-(4,5-dimethlthiazol-2-yl)-205-diphenyl tetrazolium bromide (MTT) dye reduction assay and propidium iodide assay. RESULTS: Mitochondrion-based MTT test was a more sensitive indicator of cytotoxicity than the plasma membrane-based propidium iodide test. The tests revealed following cytotoxic rankings for ophthalmic drugs: dipivefrin > timolol > pilocarpine approximately equal to dexamethasone; for excipients: benzalkonium chloride (BAC) > sodium edetate (NA2 EDTA)>polyvinyl alcohol (PVA) > methylparaben; and for CDs :alpha- CD > dimethyl beta-cyclodextrin (DM-beta-CD) > sulfobutyl ether beta-cyclodextrin ((SBE)7m-beta-CD approximately equal to hydroxypropyl-beta-cyclodextrin (HP-beta-CD) > lambda CD. In consideration of the in vivo clinical situation, the short exposure time (5 min) is more relevant even though toxic effects of some test substances were seen only after longer exposure time (30 and 60 min). CONCLUSIONS: Immortalized HCE cells are a promising tool for rapid cytotoxicity assays of ocular medications. The cell line is potentially useful in predicting the in vivo coreal toxicity of ocularly applied compounds.

Ethics in health care management: developing an instrument to assess humane caring.

The care of patients should be professional, human and humane. This is an ethical issue. The words human (inhimillinen) and humane (ihmisläheinen) have different meanings in the Finnish language. At Kuopio University Hospital (1200 beds), in Finland, it was decided to provide patients with professional and humane caring. Ethical values differ for different groups of people. Therefore humane caring was assessed by questioning both hospital patients (n = 160) and staff (n = 196). The data were subjected to content analysis. The result was that the values obtained for both patients and staff were similar. Five descriptive categories of humane caring emerged. To discover what kind of caring was provided for patients, the categories were operationalized into 66 criteria and a questionnaire was drawn up on the basis of these criteria. The data for evaluation of the quality of caring were collected from patients (n = 1708). The data were analysed using cross-tabulations and the chi-squared test; sum variables were calculated for the five standards. The results suggest that special attention should be paid to vulnerable patient groups (e.g. the elderly and the young). One means of ethical management is to support our staff to care more humanely by discussing with them the results of this study, setting standards and criteria for humane caring and what this means in practice. It is hoped our caring will therefore be more humane.

Sequence analysis of domestic dog mitochondrial DNA for forensic use.

A method has been developed for the direct sequencing of hypervariable region 1 (HV1) of domestic dog (Canis familiaris) and wolf (Canis lupus) mitochondrial DNA (mtDNA) using single hairs as template. The method uses a robotic work-station and an automated sequencer to allow for robust routine analysis. A population data base was created in order to investigate the forensic and population-genetic informativeness of domestic dog HV1. Sequence variation, partitioning of dog breeds among sequence variants and phylogenetic relations between the variants were determined. Samples from 102 domestic dogs of 52 different breeds and two captive wolves were analyzed. Nineteen dog-sequence variants were found and the frequencies of the variants ranged from 1 to 21%. The calculated discrimination power of the region, i.e., the exclusion capacity, implied that nine out of ten disputed individuals can be excluded by this analysis. The sequence variants were found to cluster into four phylogenetic groups.

Multiple and ancient origins of the domestic dog.

Mitochondrial DNA control region sequences were analyzed from 162 wolves at 27 localities worldwide and from 140 domestic dogs representing 67 breeds. Sequences from both dogs and wolves showed considerable diversity and supported the hypothesis that wolves were the ancestors of dogs. Most dog sequences belonged to a divergent monophyletic clade sharing no sequences with wolves. The sequence divergence within this clade suggested that dogs originated more than 100,000 years before the present. Associations of dog haplotypes with other wolf lineages indicated episodes of admixture between wolves and dogs. Repeated genetic exchange between dog and wolf populations may have been an important source of variation for artificial selection.

The genetical history of an isolated population of the endangered grey wolf Canis lupus: a study of nuclear and mitochondrial polymorphisms.

The grey wolf was thought to have been exterminated in the Scandinavian peninsula when the sudden appearance of a few animals in southern Sweden was reported in 1980. These wolves founded a new Swedish population which currently numbers at least 25 individuals, one of the world's smallest populations of the species. The sudden occurrence of the founder animals caused speculation that these had not appeared by 'natural' means but rather were Swedish zoo animals deliberately released by man. To analyse if this was the case and to elucidate the genetic status of this small and isolated population, we assessed nuclear and mitochondrial (mt) genetic variability in wild and captive grey wolves, using microsatellite typing and sequence analysis of the mtDNA D-loop. The new population was found to be monomorphic for a mtDNA haplotype which also was present in the Swedish zoo population. A total of four different mtDNA haplotypes were found among all captive and wild wolves (including two animals from an occasional establishment of a few wolves in northern Sweden in the late 1970s), with a maximum sequence divergence of 3.1%. Despite the mtDNA congruence, animals from the zoo population could most likely be excluded as founders for the wild population since the latter group of animals displayed several unique microsatellite alleles (i.e. alleles not found in the zoo population). Moreover, a phylogenetic analysis of individual wolves, using microsatellite allele sharing as distance measure, placed all wild animals on a branch separated from that of the captive animals. The average degree of nuclear variability as well as allelic diversity was similar in the wild and the captive populations, respectively, but was lower than that reported for North-American populations of grey wolves. Polymorphism has declined in wild wolves born in recent years suggesting that this small population is currently suffering from a loss of genetic variability due to inbreeding. Inbreeding depression is documented in captive wolves and the long-term survival of the wild Swedish population may therefore depend on immigration of animals from Russia. This study illustrates the usefulness of microsatellites for dissecting close genetic relationships and for addressing the genetic status of individuals.