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The human pan-tissue epigenetic clock is widely used for estimating age across the entire lifespan, but it does not lend itself well to estimating gestational age (GA) based on placental DNAm methylation (DNAm) data. We replicate previous findings demonstrating a strong correlation between GA and genome-wide DNAm changes. Using substantially more DNAm arrays (n=1,102 in the training set) than a previous study, we present three new placental epigenetic clocks: 1) a robust placental clock (RPC) which is unaffected by common pregnancy complications (e.g., gestational diabetes, preeclampsia), and 2) a control placental clock (CPC) constructed using placental samples from pregnancies without known placental pathology, and 3) a refined RPC for uncomplicated term pregnancies. These placental clocks are highly accurate estimators of GA based on placental tissue; e.g., predicted GA based on RPC is highly correlated with actual GA (r>0.95 in test data, median error less than one week). We show that epigenetic clocks derived from cord blood or other tissues do not accurately estimate GA in placental samples. While fundamentally different from Horvath's pan-tissue epigenetic clock, placental clocks closely track fetal age during development and may have interesting applications.

Whole genome sequencing (WGS) of thousands of microbial genomes has provided considerable insight into evolutionary mechanisms in the microbial world. While substantially fewer eukaryotic genomes are available for analyses the number is rapidly increasing. This mini-review summarizes broadly evolutionary dynamics of base composition in the different domains of life from the perspective of prokaryotes. Common and different evolutionary mechanisms influencing genomic base composition in eukaryotes and prokaryotes are discussed. The conclusion from the data currently available suggests that while there are similarities there are also striking differences in how genomic base composition has evolved within prokaryotes and eukaryotes. For instance, homologous recombination appears to increase GC content locally in eukaryotes due to a non-selective process termed GC-biased gene conversion (gBGC). For prokaryotes on the other hand, increase in genomic GC content seems to be driven by the environment and selection. We find that similar phenomena observed for some organisms in each respective domain may be caused by very different mechanisms: while gBGC and recombination rates appear to explain the negative correlation between GC3 (GC content based on the third codon nucleotides) and genome size in some eukaryotes uptake of AT rich DNA sequences is the main reason for a similar negative correlation observed in prokaryotes. We provide further examples that indicate that base composition in prokaryotes and eukaryotes have evolved under very different constraints.

The spread of antibiotic resistance within and between different bacterial populations is a major health problem on a global scale. The identification of genetic transformation in genomic data from Neisseria meningitidis, the meningococcus (Mc), and other bacteria is problematic, since similar or even identical alleles may be involved. A particular challenge in naturally transformable bacteria generally is to distinguish between common ancestry and true recombined sites in sampled genome sequences. Furthermore, the identification of recombination following experimental transformation of homologous alleles requires identifiable differences between donor and recipient, which in itself influences the propensity for homologous recombination (HR). This study identifies the distribution of HR events following intraspecies and interspecies Mc transformations of rpoB alleles encoding rifampicin resistance by whole-genome DNA sequencing and single nucleotide variant analysis. The HR events analysed were confined to the genomic region surrounding the single nucleotide genetic marker used for selection. An exponential length distribution of these recombined events was found, ranging from a few nucleotides to about 72 kb stretches. The lengths of imported sequences were on average found to be longer following experimental transformation of the recipient with genomic DNA from an intraspecies versus an interspecies donor (P<0.001). The recombination events were generally observed to be mosaic, with donor sequences interspersed with recipient sequence. Here, we present four models to explain these observations, by fragmentation of the transformed DNA, by interruptions of the recombination mechanism, by secondary recombination of endogenous self-DNA, or by repair/replication mechanisms.

Background: Gastrointestinal symptoms and changes in colonic transit time (CTT) are common in the population., Purpose: To evaluate consecutive patients who had been examined for CTT, along with completion of a diary about laxative and drug use, lifestyle factors, and gastrointestinal symptoms, to identify possible associations with longer or prolonged CTT., Material and Methods: A total of 610 consecutive patients had undergone the radiopaque marker method with an abdominal X-ray for clinical purposes. The patients had completed a diary regarding medical treatment, lifestyle factors, stool habits, and their perceived constipation and abdominal pain during the examination period. The associations between CTT and laxative use, lifestyle factors, stool habits, and symptoms were calculated by logistic regression., Results: Women had longer CTT (2.5 [1.6-3.9] vs. 1.7 [1.1-3.0] days, P  < 0.001), lower weekly stool frequency (6 [3-10] vs. 8 [5-12], P  = 0.001), and perceived more constipation ( P  = 0.025) and abdominal pain ( P  = 0.001) than men. High coffee consumption ( P  = 0.045), bulk-forming ( P  = 0.007) and osmotic ( P  = 0.001) laxatives, and lower stool frequency, shaped stool, and perceived constipation ( P for trend < 0.001) were associated with longer CTT. In total, 382 patients (63%) were treated with drugs affecting motility. In the 228 patients without drug treatment, longer CTT was associated with female sex and smoking, and lower frequency of symptoms and prolonged CTT were observed compared to patients using drugs. Tea, alcohol, and abdominal pain did not associate with CTT., Conclusions: Female sex, coffee, smoking, drug use, infrequent stools, shaped stool, and perception of constipation are associated with longer or prolonged CTT.

On the basis of population genomic and phylogeographic analyses of 1669 Mycobacterium tuberculosis lineage 4 (L4) genomes, we find that dispersal of L4 has been completely dominated by historical migrations out of Europe. We demonstrate an intimate temporal relationship between European colonial expansion into Africa and the Americas and the spread of L4 tuberculosis (TB). Markedly, in the age of antibiotics, mutations conferring antimicrobial resistance overwhelmingly emerged locally (at the level of nations), with minimal cross-border transmission of resistance. The latter finding was found to reflect the relatively recent emergence of these mutations, as a similar degree of local restriction was observed for susceptible variants emerging on comparable time scales. The restricted international transmission of drug-resistant TB suggests that containment efforts at the level of individual countries could be successful.

Background: The purpose of the present study was to examine the GC content of substituted bases (sbGC) in the core genomes of 35 bacterial species. Each species, or core genome, constituted genomes from at least 10 strains. We also wanted to explore whether sbGC for each strain was associated with the corresponding species' core genome GC content (cgGC). We present a simple mathematical model that estimates sbGC from cgGC. The model assumes only that the estimated sbGC is a function of cgGC proportional to fixed AT→GC (α) and GC → AT (β) mutation rates. Non-linear regression was used to estimate parameters α and β from the empirical data described above., Results: We found that sbGC for each strain showed a non-linear association with the corresponding cgGC with a bias towards higher GC content for most core genomes (66.3% of the strains), assuming as a null-hypothesis that sbGC should be approximately equal to cgGC. The most GC rich core genomes (i.e. approximately %GC > 60), on the other hand, exhibited slightly less GC-biased sbGC than expected. The best fitted regression model indicates that GC → AT mutation rates β = (1.91 ± 0.13) p < 0.001 are approximately (1.91/0.79) = 2.42 times as high, on average, as AT→GC α = (- 0.79 ± 0.25) p < 0.001 mutation rates. Whether the observed sbGC GC-bias for all but the most GC-rich prokaryotic species is due to selection, compensating for the GC → AT mutation bias, and/or selective neutral processes is currently debated. Residual standard error was found to be σ = 0.076 indicating estimated errors of sbGC to be approximately within ±15.2% GC (95% confidence interval) for the strains of all species in the study., Conclusion: Not only did our mathematical model give reasonable estimates of sbGC it also provides further support to previous observations that mutation rates in prokaryotes exhibit a universal GC → AT bias that appears to be remarkably consistent between taxa.

We report here the draft genome sequence of a Streptococcus species belonging to the S. mitis group. While a clear species identification cannot be made for the isolate, it appears that its most recent common ancestor is the species S. pseudopneumoniae ., (Copyright © 2018 Kirkeleite et al.)

Brucellosis is a rarely encountered infection in Norway. The aim of this study was to explore all Brucella melitensis isolates collected in Norway from 1999 to 2016 in relation to origin of infection and antimicrobial resistance patterns. A total of 23 isolates were analysed by whole-genome sequencing and compared with selected sequences of B. melitensis available from NCBI. Additionally, SNP analysis in antibiotic resistance determining genes was performed. The majority belonged to the East Mediterranean clade (genotype II), while the remaining isolates belonged to the African clade (genotype III). These results indicate that human brucellosis in Norway is related to travels or migration from the Middle East, Asia or Africa, in accordance with results from Germany, Denmark and Sweden. Antibiotic susceptibility patterns were determined by broth microdilution method and/or gradient strip method. All isolates were susceptible for all tested antibiotics, except for rifampicin where phenotypical results indicated resistance or intermediate resistance in all isolates based on broth microdilution method, and in four isolates based on gradient strip testing. In contrast, screening of the rpoB gene did not reveal any mutations in the previously described rpoB "hot spot" regions related to rifampicin resistance, indicating overestimation of resistance based on phenotypical results.

In the African meningitis belt, a region of sub-Saharan Africa comprising 22 countries from Senegal in the west to Ethiopia in the east, large epidemics of serogroup A meningococcal meningitis have occurred periodically. After gradual introduction from 2010 of mass vaccination with a monovalent meningococcal A conjugate vaccine, serogroup A epidemics have been eliminated. Starting in 2013, the northwestern part of Nigeria has been affected by yearly outbreaks of meningitis caused by a novel strain of serogroup C Neisseria meningitidis (NmC). In 2015, the strain spread to the neighboring country Niger, where it caused a severe epidemic. Following a relative calm in 2016, the largest ever recorded epidemic of NmC broke out in Nigeria in 2017. Here, we describe the recent evolution of this new outbreak strain and show how the acquisition of capsule genes and virulence factors by a strain previously circulating asymptomatically in the African population led to the emergence of a virulent pathogen. This study illustrates the power of long-read whole-genome sequencing, combined with Illumina sequencing, for high-resolution epidemiological investigations., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Based on serological evidence and viral isolation, Zika virus (ZIKV) has circulated for many years relatively benignly in a sylvatic cycle in Africa and an urban cycle in South East Asia (SEA). With the recent availability of limited but novel Indian ZIKV sequences to add to the plethora of SEA sequences, we traced the phylogenetic history and spatio-temporal dispersal pattern of ZIKV in Asia prior to its explosive emergence in the Pacific region and the Americas. These analyses demonstrated that the introduction and dispersal of ZIKV on the Pacific islands were preceded by an extended period of relatively silent transmission in SEA, enabling the virus to expand geographically and evolve adaptively before its unanticipated introduction to immunologically naive populations on the Pacific islands and in the Americas. Our findings reveal new features of the evolution and dispersal of this intriguing virus and may benefit future disease control strategies.

[This corrects the article DOI: 10.1371/journal.pone.0187618.].

Horizontal transfer of antibiotic resistance determinants contributes to dissemination of antibiotic resistance. Such transfer of resistance genes within the human gut has been documented in some in vivo studies. The present study investigated seven bla CTX-M-1 -carrying Escherichia coli isolates from three consecutive faecal samples collected from one cystic fibrosis patient in a nine-months period, by analysing whole genome sequencing data. The analyses showed that the seven E. coli isolates represented three genetically diverse strains. All isolates contained bla CTX-M-1 -carrying Incl1 plasmids that shared a common 101 kb backbone differing by only four SNPs. The plasmids harboured by the three different E. coli strains varied within limited regions suggestive of recombination events, according to the phylogenetic topology of the genomes of the isolates harbouring them. The findings strongly suggest that horizontal transfer of a bla CTX-M-1 -carrying plasmid had occurred within the patient´s gut. The study illustrates the within-host diversity of faecally carried resistant E. coli isolates and highlights the value of collecting multiple bacterial colonies from longitudinally collected samples to assess faecal carriage of resistant enterobacteria. The clustering of the plasmids with the corresponding E. coli strains carrying them indicates that the plasmids appear to have adapted to their respective E. coli hosts.

We prospectively studied the consequences of extensive antibiotic treatment on faecal carriage of antibiotic-resistant enterobacteria in a cohort of children with cystic fibrosis (CF) and a cohort of children with cancer compared to healthy children with no or low antibiotic exposure. The study was conducted in Norway in a low resistance prevalence setting. Sixty longitudinally collected faecal samples from children with CF (n = 32), 88 samples from children with cancer (n = 45) and 127 samples from healthy children (n = 70) were examined. A direct MIC-gradient strip method was used to detect resistant Enterobacteriaceae by applying Etest strips directly onto agar-plates swabbed with faecal samples. Whole genome sequencing (WGS) data were analysed to identify resistance mechanisms in 28 multidrug-resistant Escherichia coli isolates. The prevalence of resistance to third-generation cephalosporins, gentamicin and ciprofloxacin was low in all the study groups. At inclusion the prevalence of ampicillin-resistant E. coli and trimethoprim-sulfamethoxazole-resistant E. coli in the CF group compared to healthy controls was 58.6% vs. 28.4% (p = 0.005) and 48.3% vs. 14.9% (p = 0.001), respectively, with a similar prevalence at the end of the study. The prevalence of resistant enterobacteria was not significantly different in the children with cancer compared to the healthy children, not even at the end of the study when the children with cancer had been treated with repeated courses of broad-spectrum antibiotics. Children with cancer were mainly treated with intravenous antibiotics, while the CF group mainly received peroral treatment. Our observations indicate that the mode of administration of antibiotics and the general level of antimicrobial resistance in the community may have an impact on emergence of resistance in intestinal enterobacteria during antibiotic treatment. The WGS analyses detected acquired resistance genes and/or chromosomal mutations that explained the observed phenotypic resistance in all 28 multidrug-resistant E. coli isolates examined.

Pre-pregnancy maternal obesity is associated with adverse offspring outcomes at birth and later in life. Individual studies have shown that epigenetic modifications such as DNA methylation could contribute. Within the Pregnancy and Childhood Epigenetics (PACE) Consortium, we meta-analysed the association between pre-pregnancy maternal BMI and methylation at over 450,000 sites in newborn blood DNA, across 19 cohorts (9,340 mother-newborn pairs). We attempted to infer causality by comparing the effects of maternal versus paternal BMI and incorporating genetic variation. In four additional cohorts (1,817 mother-child pairs), we meta-analysed the association between maternal BMI at the start of pregnancy and blood methylation in adolescents. In newborns, maternal BMI was associated with small (<0.2% per BMI unit (1 kg/m2), P < 1.06 × 10-7) methylation variation at 9,044 sites throughout the genome. Adjustment for estimated cell proportions greatly attenuated the number of significant CpGs to 104, including 86 sites common to the unadjusted model. At 72/86 sites, the direction of the association was the same in newborns and adolescents, suggesting persistence of signals. However, we found evidence for acausal intrauterine effect of maternal BMI on newborn methylation at just 8/86 sites. In conclusion, this well-powered analysis identified robust associations between maternal adiposity and variations in newborn blood DNA methylation, but these small effects may be better explained by genetic or lifestyle factors than a causal intrauterine mechanism. This highlights the need for large-scale collaborative approaches and the application of causal inference techniques in epigenetic epidemiology., (© The Author 2017. Published by Oxford University Press.)

Infrared thermography (IRT) is increasingly applied as a noninvasive technique for measuring surface body temperature alterations related to e.g. stress, emotions and leg pathologies in avian species. As a basis for the validation of IRT as a future tool for veterinary diagnostics such as detection and/or prediction of subclinical footpad pathologies in broiler chickens, this study explored effects of manual restraint at two different ages on footpad temperatures. Head region temperatures were applied as additional measures of emotional arousal and stress. The study demonstrated that footpad temperatures dropped during 10min of restraint (p<0.001, -0.45°C 95% CI (-0.49, -0.41) per min), whilst head region temperatures (e.g. nostril, wattle, eye, and average head temperature) rose (p=0.004, 0.76°C 95% CI (0.39, 1.15) per 10min), which is consistent with body temperature alterations during emotional arousal and stress, termed stress-induces hyperthermia. Temperature differed between 30 and 36 d (p<0.001, 1.71°C 95% CI (1.04, 2.38) per week), but it is impossible to draw conclusions whether this effect was caused by age or by conditioning. Furthermore, sequential sampling order affected temperature (p=0.04, 0.13°C 95% CI (0.01, 0.25)). In conclusion, one needs to take into account the duration of handling and restraint during the assessment of footpad temperatures, as well as the chickens age, previous experience and sequential sampling order, when using IRT technology as a future noninvasive tool to study temperature alterations associated with subclinical footpad pathologies in broiler chickens., (Copyright © 2017 Elsevier Inc. All rights reserved.)

RNA viruses are abundant infectious agents and present in all domains of life. Arthropods, including ticks, are well known as vectors of many viruses of concern for human and animal health. Despite their obvious importance, the extent and structure of viral diversity in ticks is still poorly understood, particularly in Europe. Using a bulk RNA-sequencing approach that captures the complete transcriptome, we analysed the virome of the most common tick in Europe - Ixodes ricinus. In total, RNA sequencing was performed on six libraries consisting of 33 I. ricinus nymphs and adults sampled in Norway. Despite the small number of animals surveyed, our virus identification pipeline revealed nine diverse and novel viral species, phylogenetically positioned within four different viral groups - bunyaviruses, luteoviruses, mononegavirales and partitiviruses - and sometimes characterized by extensive genetic diversity including a potentially novel genus of bunyaviruses. This work sheds new light on the virus diversity in I. ricinus, expands our knowledge of potential host/vector-associations and tick-transmitted viruses within several viral groups, and pushes the latitudinal limit where it is likely to find tick-associated viruses. Notably, our phylogenetic analysis revealed the presence of tick-specific virus clades that span multiple continents, highlighting the role of ticks as important virus reservoirs.

Background: The core genome consists of genes shared by the vast majority of a species and is therefore assumed to have been subjected to substantially stronger purifying selection than the more mobile elements of the genome, also known as the accessory genome. Here we examine intragenic base composition differences in core genomes and corresponding accessory genomes in 36 species, represented by the genomes of 731 bacterial strains, to assess the impact of selective forces on base composition in microbes. We also explore, in turn, how these results compare with findings for whole genome intragenic regions., Results: We found that GC content in coding regions is significantly higher in core genomes than accessory genomes and whole genomes. Likewise, GC content variation within coding regions was significantly lower in core genomes than in accessory genomes and whole genomes. Relative entropy in coding regions, measured as the difference between observed and expected trinucleotide frequencies estimated from mononucleotide frequencies, was significantly higher in the core genomes than in accessory and whole genomes. Relative entropy was positively associated with coding region GC content within the accessory genomes, but not within the corresponding coding regions of core or whole genomes., Conclusion: The higher intragenic GC content and relative entropy, as well as the lower GC content variation, observed in the core genomes is most likely associated with selective constraints. It is unclear whether the positive association between GC content and relative entropy in the more mobile accessory genomes constitutes signatures of selection or selective neutral processes.

The "Beijing" Mycobacterium tuberculosis (Mtb) lineage 2 (L2) is spreading globally and has been associated with accelerated disease progression and increased antibiotic resistance. Here we performed a phylodynamic reconstruction of one of the L2 sublineages, the central Asian clade (CAC), which has recently spread to western Europe. We find that recent historical events have contributed to the evolution and dispersal of the CAC. Our timing estimates indicate that the clade was likely introduced to Afghanistan during the 1979-1989 Soviet-Afghan war and spread further after population displacement in the wake of the American invasion in 2001. We also find that drug resistance mutations accumulated on a massive scale in Mtb isolates from former Soviet republics after the fall of the Soviet Union, a pattern that was not observed in CAC isolates from Afghanistan. Our results underscore the detrimental effects of political instability and population displacement on tuberculosis control and demonstrate the power of phylodynamic methods in exploring bacterial evolution in space and time., Competing Interests: The authors declare no conflict of interest.

Genome-wide association studies (GWAS) have become indispensable in human medicine and genomics, but very few have been carried out on bacteria. Here we introduce Scoary, an ultra-fast, easy-to-use, and widely applicable software tool that scores the components of the pan-genome for associations to observed phenotypic traits while accounting for population stratification, with minimal assumptions about evolutionary processes. We call our approach pan-GWAS to distinguish it from traditional, single nucleotide polymorphism (SNP)-based GWAS. Scoary is implemented in Python and is available under an open source GPLv3 license at https://github.com/AdmiralenOla/Scoary .

Background: Neisseria meningitidis colonizes humans and transmits mainly by asymptomatic carriage. We sought to determine the prevalence and epidemiology of meningococcal carriage in Ethiopia prior to the introduction of MenAfriVac, a serogroup A meningococcal conjugate vaccine., Methods: A cross-sectional meningococcal carriage study was conducted in Arba Minch, southern Ethiopia. A total of 7479 oropharyngeal samples were collected from 1 to 29 year old volunteers, between March and October, 2014. The swabs were cultured for N. meningitidis and Neisseria lactamica in Ethiopia. N. meningitidis isolates were confirmed and characterized by their serogroup, sequence type (ST) and PorA:FetA profile in Norway., Results: Overall carriage prevalence was 6.6 %. There was no significant difference in overall carriage between male (6.7 %) and female (6.4 %) participants. Highest carriage prevalence (10.9 %) for females was found in the 15-19 years of age, while prevalence among males was highest (11.3 %) in the 20-24 age group. Non-groupable isolates dominated (76.4 %), followed by serogroups X (14.0 %) and W (5.9 %) isolates. No serogroup A was found. Most non-groupable isolates were ST-192. Serogroup W isolates were assigned to the ST-11 clonal complex, and serogroup X isolates to the ST-181 and ST-41/44 clonal complexes. Overall carriage prevalence of N. lactamica was 28.1 %. Carriage of N. meningitidis and N. lactamica varied depending on age and geographic area, but there was no association between carriage of the two species., Conclusions: Epidemic strains of serogroups W and X were circulating in this area of Ethiopia. As no serogroup A was found among the carriage isolates the immediate impact of mass-vaccination with MenAfriVac on transmission of N. meningitidis in this population is expected to be marginal.

Folate is vital for fetal development. Periconceptional folic acid supplementation and food fortification are recommended to prevent neural tube defects. Mechanisms whereby periconceptional folate influences normal development and disease are poorly understood: epigenetics may be involved. We examine the association between maternal plasma folate during pregnancy and epigenome-wide DNA methylation using Illumina's HumanMethyl450 Beadchip in 1,988 newborns from two European cohorts. Here we report the combined covariate-adjusted results using meta-analysis and employ pathway and gene expression analyses. Four-hundred forty-three CpGs (320 genes) are significantly associated with maternal plasma folate levels during pregnancy (false discovery rate 5%); 48 are significant after Bonferroni correction. Most genes are not known for folate biology, including APC2, GRM8, SLC16A12, OPCML, PRPH, LHX1, KLK4 and PRSS21. Some relate to birth defects other than neural tube defects, neurological functions or varied aspects of embryonic development. These findings may inform how maternal folate impacts the developing epigenome and health outcomes in offspring.

Background: Recent findings indicated that a correlation between genomic % AT and genome size within strains of microbial species was predominantly associated with the uptake of foreign DNA. One species however, Chlamydia trachomatis, defied any explanation. In the present study 79 fully sequenced C. trachomatis genomes, representing ocular- (nine strains), urogenital- (36 strains) and lymphogranuloma venereum strains (LGV, 22 strains), in three pathogroups, in addition to 12 laboratory isolates, were scrutinized with the intent of elucidating the positive correlation between genomic AT content and genome size., Results: The average size difference between the strains of each pathogroup was largely explained by the incorporation of genetic fragments. These fragments were slightly more AT rich than their corresponding host genomes, but not enough to justify the difference in AT content between the strains of the smaller genomes lacking the fragments. In addition, a genetic region predominantly found in the ocular strains, which had the largest genomes, was on average more GC rich than the host genomes of the urogenital strains (58.64% AT vs. 58.69% AT), which had the second largest genomes, implying that the foreign genetic regions cannot alone explain the association between genome size and AT content in C. trachomatis. 23,492 SNPs were identified for all 79 genomes, and although the SNPs were on average slightly GC rich (~47% AT), a significant association was found between genome-wide SNP AT content, for each pathogroup, and genome size (p < 0.001, R (2) = 0.86) in the C. trachomatis strains., Conclusions: The correlation between genome size and AT content, with respect to the C. trachomatis pathogroups, was explained by the incorporation of genetic fragments unique to the ocular and/or urogenital strains into the LGV- and urogential strains in addition to the genome-wide SNP AT content differences between the three pathogroups.

Background: Several epidemiologic studies indicate that maternal gestational weight gain (GWG) influences health outcomes in offspring. Any underlying mechanisms have, however, not been established. A recent study of 88 children based on the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort examined the methylation levels at 1,505 Cytosine-Guanine methylation (CpG) loci and found several to be significantly associated with maternal weight gain between weeks 0 and 18 of gestation. Since these results could not be replicated we wanted to examine associations between 0 and 18 week GWG and genome-wide methylation levels using the Infinium HumanMethylation450 BeadChip (450K) platform on a larger sample size, i.e. 729 newborns sampled from the Norwegian Mother and Child Cohort Study (MoBa)., Results: We found no CpG loci associated with 0-18 week GWG after adjusting for the set of covariates used in the ALSPAC study (i.e. child's sex and maternal age) and for multiple testing (q > 0.9, both 1,505 and 473,731 tests). Hence, none of the CpG loci linked with the genes found significantly associated with 0-18 week GWG in the ALSPAC study were significant in our study., Conclusions: The inconsistency in the results with the ALSPAC study with regards to the 0-18 week GWG model may arise for several reasons: sampling from different populations, dissimilar methylome coverage, sample size and/or false positive findings.

Motivation: The explosion of whole-genome sequencing (WGS) as a tool in the mapping and understanding of genomes has been accompanied by an equally massive report of tools and pipelines for the analysis of DNA copy number variation (CNV). Most currently available tools are designed specifically for human genomes, with comparatively little literature devoted to CNVs in prokaryotic organisms. However, there are several idiosyncrasies in prokaryotic WGS data. This work proposes a step-by-step approach for detection and quantification of copy number variants specifically aimed at prokaryotes., Results: After aligning WGS reads to a reference genome, we count the individual reads in a sliding window and normalize these counts for bias introduced by differences in GC content. We then investigate the coverage in two fundamentally different ways: (i) Employing a Hidden Markov Model and (ii) by repeated sampling with replacement (bootstrapping) on each individual gene. The latter bypasses the complex problem of breakpoint determination. To demonstrate our method, we apply it to real and simulated WGS data and benchmark it against two popular methods for CNV detection. The proposed methodology will in some cases represent a significant jump in accuracy from other current methods., Availability and Implementation: CNOGpro is written entirely in the R programming language and is available from the CRAN repository (http://cran.r-project.org) under the GNU General Public License., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The upstream region of coding genes is important for several reasons, for instance locating transcription factor, binding sites, and start site initiation in genomic DNA. Motivated by a recently conducted study, where multivariate approach was successfully applied to coding sequence modeling, we have introduced a partial least squares (PLS) based procedure for the classification of true upstream prokaryotic sequence from background upstream sequence. The upstream sequences of conserved coding genes over genomes were considered in analysis, where conserved coding genes were found by using pan-genomics concept for each considered prokaryotic species. PLS uses position specific scoring matrix (PSSM) to study the characteristics of upstream region. Results obtained by PLS based method were compared with Gini importance of random forest (RF) and support vector machine (SVM), which is much used method for sequence classification. The upstream sequence classification performance was evaluated by using cross validation, and suggested approach identifies prokaryotic upstream region significantly better to RF (p-value < 0.01) and SVM (p-value < 0.01). Further, the proposed method also produced results that concurred with known biological characteristics of the upstream region.

Background: There are several studies describing loss of genes through reductive evolution in microbes, but how selective forces are associated with genome expansion due to horizontal gene transfer (HGT) has not received similar attention. The aim of this study was therefore to examine how selective pressures influence genome expansion in 53 fully sequenced and assembled Escherichia coli strains. We also explored potential connections between genome expansion and the attainment of virulence factors. This was performed using estimations of several genomic parameters such as AT content, genomic drift (measured using relative entropy), genome size and estimated HGT size, which were subsequently compared to analogous parameters computed from the core genome consisting of 1729 genes common to the 53 E. coli strains. Moreover, we analyzed how selective pressures (quantified using relative entropy and dN/dS), acting on the E. coli core genome, influenced lineage and phylogroup formation., Results: Hierarchical clustering of dS and dN estimations from the E. coli core genome resulted in phylogenetic trees with topologies in agreement with known E. coli taxonomy and phylogroups. High values of dS, compared to dN, indicate that the E. coli core genome has been subjected to substantial purifying selection over time; significantly more than the non-core part of the genome (p<0.001). This is further supported by a linear association between strain-wise dS and dN values (β = 26.94 ± 0.44, R2~0.98, p<0.001). The non-core part of the genome was also significantly more AT-rich (p<0.001) than the core genome and E. coli genome size correlated with estimated HGT size (p<0.001). In addition, genome size (p<0.001), AT content (p<0.001) as well as estimated HGT size (p<0.005) were all associated with the presence of virulence factors, suggesting that pathogenicity traits in E. coli are largely attained through HGT. No associations were found between selective pressures operating on the E. coli core genome, as estimated using relative entropy, and genome size (p~0.98)., Conclusions: On a larger time frame, genome expansion in E. coli, which is significantly associated with the acquisition of virulence factors, appears to be independent of selective forces operating on the core genome.

Genomic %AT has been found to correlate negatively with genome size in microbes. While microbes with large genomes are often GC rich and free living, AT-rich bacteria tend to be host associated with smaller genomes. With over 2000 fully sequenced and assembled microbial genomes available, we explored the relationship among genomic %AT, genome size, relative entropy (a measure associated with genetic drift) and fraction of genome islands (GIs) in microbial species with the genomes of more than 10 strains available. A negative correlation with genome size was found in six out of 12 phylogenetic groups and subphyla and a positive correlation in only two. At the species level, we found a trend of positive correlations between genomic %AT and genome size in eight out of 20 species, while only four showed a negative correlation. Estimated chromosomal fractions of GIs were found to correlate positively with genome size in the strains of 14 out of 18 species and genomic %AT in the strains of seven species (two correlated negatively). Although GIs explain most of the observed positive correlations between genomic %AT and size, Chlamydia trachomatis seem to be an exception; therefore, these findings needs to be further explored., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, a major pathogen of salmonid fish species worldwide. Very low levels of intra-species genetic diversity have hampered efforts to understand the transmission dynamics and recent evolutionary history of this Gram-positive bacterium. We exploited recent advances in the next-generation sequencing technology to generate genome-wide single-nucleotide polymorphism (SNP) data from 68 diverse R. salmoninarum isolates representing broad geographical and temporal ranges and different host species. Phylogenetic analysis robustly delineated two lineages (lineage 1 and lineage 2); futhermore, dating analysis estimated that the time to the most recent ancestor of all the isolates is 1239 years ago (95% credible interval (CI) 444-2720 years ago). Our data reveal the intercontinental spread of lineage 1 over the last century, concurrent with anthropogenic movement of live fish, feed and ova for aquaculture purposes and stocking of recreational fisheries, whilst lineage 2 appears to have been endemic in wild Eastern Atlantic salmonid stocks before commercial activity. The high resolution of the SNP-based analyses allowed us to separate closely related isolates linked to neighboring fish farms, indicating that they formed part of single outbreaks. We were able to demonstrate that the main lineage 1 subgroup of R. salmoninarum isolated from Norway and the UK likely represent an introduction to these areas ~40 years ago. This study demonstrates the promise of this technology for analysis of micro and medium scale evolutionary relationships in veterinary and environmental microorganisms, as well as human pathogens.

Coupled helical coils show promising mechanical behavior to be used as tubular organ constructs, e.g., in trachea or urethra. They are potentially easy to manufacture by filament winding of biocompatible and resorbable polymers, and could be tailored for suitable mechanical properties. In this study, coupled helical coils were manufactured by filament winding of melt-extruded polycaprolactone, which was reported to demonstrate desired in vivo degradation speed matching tissue regeneration rate. The tensile and bending stiffness was characterized for a set of couple helical coils with different geometric designs, with right-handed and left-handed polymer helices fused together in joints where the filaments cross. The Young's modulus of unidirectional polycaprolactone filaments was characterized, and used as input together with the structural parameters of the coupled coils in finite element simulations of tensile loading and three-point bending of the coils. A favorable comparison of the numerical and experimental results was found, which paves way for use of the proposed numerical approach in stiffness design under reversible elastic conditions of filament wound tubular constructs., (© 2013 Published by Elsevier Ltd.)

The repair of complex craniofacial bone defects is challenging and a successful result is dependent on the size of the defect, quality of the soft tissue covering the defect, and choice of reconstruction method. The objective of this study was to develop a bioactive cranial implant that could provide a permanent reconstructive solution to the patient by stimulating bone healing of the defect. In this paper the authors report on the feasibility and clinical results of using such a newly developed device for the repair of a large traumatic and therapy-resistant cranial bone defect. The patient had undergone numerous attempts at repair, in which established methods had been tried without success. A mosaic-designed device was manufactured and implanted, comprising interconnected ceramic tiles with a defined calcium phosphate composition. The clinical outcome 30 months after surgery revealed a restored cranial vault without postoperative complications. Computed tomography demonstrated signs of bone ingrowth. Examination with combined (18)F-fluoride PET and CT provided further evidence of bone healing of the cranial defect.

The Firmicutes represent a major component of the intestinal microflora. The intestinal Firmicutes are a large, diverse group of organisms, many of which are poorly characterized due to their anaerobic growth requirements. Although most Firmicutes are Gram positive, members of the class Negativicutes, including the genus Veillonella, stain Gram negative. Veillonella are among the most abundant organisms of the oral and intestinal microflora of animals and humans, in spite of being strict anaerobes. In this work, the genomes of 24 Negativicutes, including eight Veillonella spp., are compared to 20 other Firmicutes genomes; a further 101 prokaryotic genomes were included, covering 26 phyla. Thus a total of 145 prokaryotic genomes were analyzed by various methods to investigate the apparent conflict of the Veillonella Gram stain and their taxonomic position within the Firmicutes. Comparison of the genome sequences confirms that the Negativicutes are distantly related to Clostridium spp., based on 16S rRNA, complete genomic DNA sequences, and a consensus tree based on conserved proteins. The genus Veillonella is relatively homogeneous: inter-genus pair-wise comparison identifies at least 1,350 shared proteins, although less than half of these are found in any given Clostridium genome. Only 27 proteins are found conserved in all analyzed prokaryote genomes. Veillonella has distinct metabolic properties, and significant similarities to genomes of Proteobacteria are not detected, with the exception of a shared LPS biosynthesis pathway. The clade within the class Negativicutes to which the genus Veillonella belongs exhibits unique properties, most of which are in common with Gram-positives and some with Gram negatives. They are only distantly related to Clostridia, but are even less closely related to Gram-negative species. Though the Negativicutes stain Gram-negative and possess two membranes, the genome and proteome analysis presented here confirm their place within the (mainly) Gram positive phylum of the Firmicutes. Further studies are required to unveil the evolutionary history of the Veillonella and other Negativicutes.

Introduction: Genomic base composition ranges from less than 25% AT to more than 85% AT in prokaryotes. Since only a small fraction of prokaryotic genomes is not protein coding even a minor change in genomic base composition will induce profound protein changes. We examined how amino acid and codon frequencies were distributed in over 2000 microbial genomes and how these distributions were affected by base compositional changes. In addition, we wanted to know how genome-wide amino acid usage was biased in the different genomes and how changes to base composition and mutations affected this bias. To carry this out, we used a Generalized Additive Mixed-effects Model (GAMM) to explore non-linear associations and strong data dependences in closely related microbes; principal component analysis (PCA) was used to examine genomic amino acid- and codon frequencies, while the concept of relative entropy was used to analyze genomic mutation rates., Results: We found that genomic amino acid frequencies carried a stronger phylogenetic signal than codon frequencies, but that this signal was weak compared to that of genomic %AT. Further, in contrast to codon usage bias (CUB), amino acid usage bias (AAUB) was differently distributed in AT- and GC-rich genomes in the sense that AT-rich genomes did not prefer specific amino acids over others to the same extent as GC-rich genomes. AAUB was also associated with relative entropy; genomes with low AAUB contained more random mutations as a consequence of relaxed purifying selection than genomes with higher AAUB., Conclusion: Genomic base composition has a substantial effect on both amino acid- and codon frequencies in bacterial genomes. While phylogeny influenced amino acid usage more in GC-rich genomes, AT-content was driving amino acid usage in AT-rich genomes. We found the GAMM model to be an excellent tool to analyze the genomic data used in this study.

A highly acute and contagious camel disease, an epidemic wave of unknown etiology, referred to here as camel sudden death syndrome, has plagued camel population in countries in the Horn of Africa. To better understand its epidemic patterns and transmission dynamics, we used epidemiologic parameters and differential equation deterministic modeling (SEIR/D-model) to predict the outcome likelihood following an exposure of susceptible camel population. Our results showed 45.7, 17.6, and 38.6 % overall morbidity, mortality, and case fatality rates of the epidemic, respectively. Pregnant camels had the highest mortality and case fatality rates, followed by breeding males, and lactating females, implying serious socioeconomic consequences. Disease dynamics appeared to be linked to livestock trade route and animal movements. The epidemic exhibited a strong basic reproductive number (R (0)) with an average of 16 camels infected by one infectious case during the entire infectious period. The epidemic curve suggested that the critical moment of the disease development is approximately between 30 and 40 days, where both infected/exposed and infectious camels are at their highest numbers. The lag between infected/infectious curves indicates a time-shift of approximately 3-5 days from when a camel is infected and until it becomes infectious. According to this predictive model, of all animals exposed to the infection, 66.8 % (n = 868) and 33.2 % (n = 431) had recovered and died, respectively, at the end of epidemic period. Hence, if early measures are not taken, such an epidemic could cause a much more devastative effect, within short period of time than the anticipated proportion.

The present study describes effects of anticipation and consumption of a palatable reward on comb surface temperature. The purpose was to investigate temperature responses as a potential physiological indicator of positive emotional states in laying hens. A rise in body temperature in response to stimuli predictive of or during exposure to unpleasant events has been interpreted as evidence of emotions in mammals and avians. However, this phenomenon has so far only been studied during anticipation of or exposure to negative events; i.e., emotions of a negative valence. Infrared thermography was used to record potential alterations in comb surface temperature to a conditioned cue signaling a reward (mealworms) and during reward delivery. On average, comb temperature dropped 1.5 °C (95% CI: +/-1.2 °C) after exposure to CS and consumption of reward (p~0.0014) when initial comb temperature was above 30 °C. Such temperature drop indicates a peripheral vasoconstriction and has clear resemblances to emotional fever as seen during negative emotional states. Thus, we propose that a drop in peripheral temperature reflects emotional arousal more than emotional valence. Substantial temperature responses due to diet-induced thermogenesis were found, further emphasizing a cautious interpretation of altered comb temperature in studies of animal welfare., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: We wanted to compare growth differences between 13 Escherichia coli strains exposed to various concentrations of the growth inhibitor lactoferrin in two different types of broth (Syncase and Luria-Bertani (LB)). To carry this out, we present a simple statistical procedure that separates microbial growth curves that are due to natural random perturbations and growth curves that are more likely caused by biological differences.Bacterial growth was determined using optical density data (OD) recorded for triplicates at 620 nm for 18 hours for each strain. Each resulting growth curve was divided into three equally spaced intervals. We propose a procedure using linear spline regression with two knots to compute the slopes of each interval in the bacterial growth curves. These slopes are subsequently used to estimate a 95% confidence interval based on an appropriate statistical distribution. Slopes outside the confidence interval were considered as significantly different from slopes within. We also demonstrate the use of related, but more advanced methods known collectively as generalized additive models (GAMs) to model growth. In addition to impressive curve fitting capabilities with corresponding confidence intervals, GAM's allow for the computation of derivatives, i.e. growth rate estimation, with respect to each time point., Results: The results from our proposed procedure agreed well with the observed data. The results indicated that there were substantial growth differences between the E. coli strains. Most strains exhibited improved growth in the nutrient rich LB broth compared to Syncase. The inhibiting effect of lactoferrin varied between the different strains. The atypical enteropathogenic aEPEC-2 grew, on average, faster in both broths than the other strains tested while the enteroinvasive strains, EIEC-6 and EIEC-7 grew slower. The enterotoxigenic ETEC-5 strain, exhibited exceptional growth in Syncase broth, but slower growth in LB broth., Conclusions: Our results do not indicate clear growth differences between pathogroups or pathogenic versus non-pathogenic E. coli.

In 2006, a severe foodborne EHEC outbreak occured in Norway. Seventeen cases were recorded and the HUS frequency was 60%. The causative strain, Esherichia coli O103:H25, is considered to be particularly virulent. Sequencing of the outbreak strain revealed resemblance to the 2011 German outbreak strain E. coli O104:H4, both in genome and Shiga toxin 2-encoding (Stx2) phage sequence. The nucleotide identity between the Stx2 phages from the Norwegian and German outbreak strains was 90%. During the 2006 outbreak, stx(2)-positive O103:H25 E. coli was isolated from two patients. All the other outbreak associated isolates, including all food isolates, were stx-negative, and carried a different phage replacing the Stx2 phage. This phage was of similar size to the Stx2 phage, but had a distinctive early phage region and no stx gene. The sequence of the early region of this phage was not retrieved from the bacterial host genome, and the origin of the phage is unknown. The contaminated food most likely contained a mixture of E. coli O103:H25 cells with either one of the phages.

Arctic foxes, 620 that were trapped and 22 found dead on Svalbard, Norway (1996-2004), as well as 10 foxes trapped in Nenets, North-West Russia (1999), were tested for rabies virus antigen in brain tissue by standard direct fluorescent antibody test. Rabies antigen was found in two foxes from Svalbard and in three from Russia. Blood samples from 515 of the fox carcasses were screened for rabies antibodies with negative result. Our results, together with a previous screening (1980-1989, n=817) indicate that the prevalence of rabies in Svalbard has remained low or that the virus has not been enzootic in the arctic fox population since the first reported outbreak in 1980. Brain tissues from four arctic foxes (one from Svalbard, three from Russia) in which rabies virus antigen was detected were further analyzed by reverse-transcriptase polymerase chain reaction direct amplicon sequencing and phylogenetic analysis. Sequences were compared to corresponding sequences from rabies virus isolates from other arctic regions. The Svalbard isolate and two of the Russian isolates were identical (310 nucleotides), whereas the third Russian isolate differed in six nucleotide positions. However, when translated into amino acid sequences, none of these substitutions produced changes in the amino acid sequence. These findings suggest that the spread of rabies virus to Svalbard was likely due to migration of arctic foxes over sea ice from Russia to Svalbard. Furthermore, when compared to other Arctic rabies virus isolates, a high degree of homology was found, suggesting a high contact rate between arctic fox populations from different arctic regions. The high degree of homology also indicates that other, and more variable, regions of the genome than this part of the nucleoprotein gene should be used to distinguish Arctic rabies virus isolates for epidemiologic purposes.

Infectious salmon anemia (ISA) is an orthomyxoviral disease that has had devastating effects on farmed Atlantic salmon. ISA is still a disease resulting in continued loss of revenues and therefore development of effective vaccines is of great importance. Commercial vaccines against ISA are available, but the efficacy is poorly described. There is little information about vaccine-induced immune factors preventing ISA virus (ISAV) infection today. In this study we assessed the protective effects and immunogenicity of vaccines containing three different quantities of the inactivated ISAV antigen. Our findings indicated that immunization induced effective protection in Atlantic salmon with a relative percent survival (RPS) as high as 86. The level of protection was correlated to the amount of ISAV antigen in the vaccine, and fish immunized with high antigen amounts produced detectable ISAV-specific and neutralizing antibodies. While ISAV infection was detectable in non-vaccinated control fish challenged by cohabitation, no infection was detected in fish immunized with high antigen amounts. After challenge, transcriptional analysis of selected immune-related genes demonstrated activation of innate immune responses in ISAV-infected control fish, but not in vaccine protected fish. This study furthers the knowledge about vaccine efficacy and vaccine-induced immunity to ISAV challenge in Atlantic salmon., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The closely related bacterial species Bacillus cereus and Bacillus weihenstephanensis are adapted to the mesophilic and the psychrotrophic temperature range, respectively. While B. cereus strains are associated with foodborne diseases, B. weihenstephanensis strains are so far not, although similar virulence genes are found in both species. Our investigations show that both species were virulent in the insect model, Galleria mellonella, following infection via oral and haemocoel routes. However, virulence of B. weihenstephanensis was much higher at 15°C than at 37°C. Furthermore, a temperature-dependent difference between the species was seen in a cell culture cytotoxicity assay. In summary, our results demonstrate for the first time virulence of B. weihenstephanensis strains in an in vivo model. In addition, we found that G. mellonella is a useful model for studies of the psychrotolerant species of the B. cereus group, suggesting that insects might be an ecological growth niche for several members of this bacterial group., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The ratio of genomic oligonucleotide frequencies relative to the mean genomic AT/GC content has been shown to be similar for closely related species and, therefore, said to reflect a "genomic signature". The genomic signature has been found to be more similar within genomes than between closely related genomes. Furthermore, genomic signatures of closely related organisms are, in turn, more similar than more distantly related organisms. Since the genomic signature is remarkably stable within a genome, it can be extracted from only a fraction of the genomic DNA sequence. Genomic signatures, therefore, have many applications. The most notable examples include recognition of pathogenicity islands in microbial genomes and identification of hosts from arbitrary DNA sequences, the latter being of great importance in metagenomics. What shapes the genomic signature in microbial DNA has been readily discussed, but difficult to pinpoint exactly. Most attempts so far have mainly focused on correlations from in silico data. This mini-review seeks to summarize possible influences shaping the genomic signature and to survey a set of applications.

Anaerobic chlorate respiration requires electron transport from the bacterial inner membrane to the soluble periplasmic chlorate reductase. We have recently demonstrated that soluble c cytochromes function as electron carriers for chlorate reduction in Ideonella dechloratans (Smedja Bäcklund et al. 2009). In the present work, we describe a gene encoding soluble c-type cytochrome [cyt; GenBank ID: EU768872] located close to the gene cluster for chlorate reduction in I. dechloratans. The predicted amino acid sequence does not match any of the peptide masses or partial sequences obtained earlier from periplasmic c cytochromes. The gene, without the predicted signal sequence, was expressed heterologously in E. coli and the recombinant protein was purified, refolded and reconstituted with heme. The reconstituted protein shows spectral properties characteristic for c cytochromes, with an absorption maximum at 553 nm for the alpha band in the reduced state. Pyridine hemochrome analysis demonstrates the formation of covalently bound heme., (Copyright 2009 Elsevier GmbH. All rights reserved.)

The objective assessment of pain is difficult in animals and humans alike. Detrended fluctuation analysis (DFA) is a method which extracts "hidden" information from heart rate time series, and may offer a novel way of assessing the subjective experience associated with pain. The aim of this study was to investigate whether any fractal differences could be detected in heart rate time series of sheep due to the infliction of ischaemic pain. Heart rate variability (HRV) was recorded continuously in five ewes during treatment sequences of baseline, intervention and post-intervention for up to 60 min. Heart rate time series were subjected to a DFA, and the median of the scaling coefficients (alpha) was found to be alpha=1.10 for the baseline sequences, 1.01 for the intervention sequences and 1.00 for the post-intervention sequences. The complexity in the regulation of heartbeats decreased between baseline and intervention (p approximately 0.03) and baseline and post-intervention (p approximately 0.01), indicating reperfusion pain and nociceptive sensitization in the post-intervention sequence. Random time series based on Gaussian white noise were generated, with similar mean and variance to the HRV sequences. No difference was found between these series (p approximately 0.28), pointing to a true difference in complexity in the original data. We found no difference in the scaling coefficient alpha between the different treatments, possibly due to the small sample size or a fear induced sympathetic arousal during test day 1 confounding the results. The decrease in the scaling coefficient alpha may be due to sympathetic activation and vagal withdrawal. DFA of heart rate time series may be a useful method to evaluate the progressive shift of cardiac regulation toward sympathetic activation and vagal withdrawal produced by pain or negative emotional responses such as fear., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background: Bovine Tuberculosis (BTB) is a widespread and endemic disease of cattle in Ethiopia. Information relating to genotypic characteristics of Mycobacterium bovis strains affecting the cattle population in Ethiopia is limited. We carried out molecular characterization of M. bovis strains isolated from BTB infected cattle using the spoligotyping technique. The relationship between distribution of spoligotypes and recorded variables was also investigated. A new approach that can numerically reflect the degree of genetic polymorphism in a M. bovis population was also developed. The study was conducted from July 2006 to January 2007 in cattle slaughtered at five representative abattoirs in Ethiopia., Results: Forty-five M. bovis isolates were obtained from 406 pathologic tissue specimens collected from 337 carcasses with lesions compatible with BTB. Twelve spoligotypes were identified from 34 distinct strains; with SB1176 as a dominant spoligotype (41.2% of the isolates) followed by SB0133 (14.7%). Comparison of spoligotypes with an M. bovis global database http://www.mbovis.org revealed six new spoligotypes which were subsequently registered in the database with international identification codes of SB1517, SB1518, SB1519, SB1520, SB1521 and SB1522. The majority of strains were obtained from cattle slaughtered at Addis Ababa abattoir. On the basis of the Spoligotype Evolutionary Index, SEI (a numeric expression approach to make standardized comparison of spoligotype evolution), M. bovis isolates from Ethiopia were relatively more heterogeneous (SEI = 3.2) compared to isolates from other countries. This might be attributed to extensive livestock movement linked to trading or seasonal migration, high degree of livestock mingling, and also diversities of the country's agricultural and livestock ecosystems, in addition to lack of disease control measures that led to high infection prevalence. Multiple spoligotype infection was recorded in nine (50%) of infected carcasses and this may indicate the prevailing high degree of super infection., Conclusions: This study provided molecular evidence for the widespread distribution of M. bovis in the cattle population in Ethiopia. It also demonstrated a relatively high degree of genetic polymorphism of the isolates. Further molecular investigation of M. bovis strains in humans and other domestic animals is recommended in order to elucidate the zoonotic importance as well as reservoirs and pattern of transmission among various hosts.

Background: DNA word frequencies, normalized for genomic AT content, are remarkably stable within prokaryotic genomes and are therefore said to reflect a "genomic signature." The genomic signatures can be used to phylogenetically classify organisms from arbitrary sampled DNA. Genomic signatures can also be used to search for horizontally transferred DNA or DNA regions subjected to special selection forces. Thus, the stability of the genomic signature can be used as a measure of genomic homogeneity. The factors associated with the stability of the genomic signatures are not known, and this motivated us to investigate further. We analyzed the intra-genomic variance of genomic signatures based on AT content normalization (0(th) order Markov model) as well as genomic signatures normalized by smaller DNA words (1(st) and 2(nd) order Markov models) for 636 sequenced prokaryotic genomes. Regression models were fitted, with intra-genomic signature variance as the response variable, to a set of factors representing genomic properties such as genomic AT content, genome size, habitat, phylum, oxygen requirement, optimal growth temperature and oligonucleotide usage variance (OUV, a measure of oligonucleotide usage bias), measured as the variance between genomic tetranucleotide frequencies and Markov chain approximated tetranucleotide frequencies, as predictors., Principal Findings: Regression analysis revealed that OUV was the most important factor (p<0.001) determining intra-genomic homogeneity as measured using genomic signatures. This means that the less random the oligonucleotide usage is in the sense of higher OUV, the more homogeneous the genome is in terms of the genomic signature. The other factors influencing variance in the genomic signature (p<0.001) were genomic AT content, phylum and oxygen requirement., Conclusions: Genomic homogeneity in prokaryotes is intimately linked to genomic GC content, oligonucleotide usage bias (OUV) and aerobiosis, while oligonucleotide usage bias (OUV) is associated with genomic GC content, aerobiosis and habitat.

Background: Recently there has been an explosion in the availability of bacterial genomic sequences, making possible now an analysis of genomic signatures across more than 800 hundred different bacterial chromosomes, from a wide variety of environments.Using genomic signatures, we pair-wise compared 867 different genomic DNA sequences, taken from chromosomes and plasmids more than 100,000 base-pairs in length. Hierarchical clustering was performed on the outcome of the comparisons before a multinomial regression model was fitted. The regression model included the cluster groups as the response variable with AT content, phyla, growth temperature, selective pressure, habitat, sequence size, oxygen requirement and pathogenicity as predictors., Results: Many significant factors were associated with the genomic signature, most notably AT content. Phyla was also an important factor, although considerably less so than AT content. Small improvements to the regression model, although significant, were also obtained by factors such as sequence size, habitat, growth temperature, selective pressure measured as oligonucleotide usage variance, and oxygen requirement., Conclusion: The statistics obtained using hierarchical clustering and multinomial regression analysis indicate that the genomic signature is shaped by many factors, and this may explain the varying ability to classify prokaryotic organisms below genus level.

Background: When generating a genetically modified organism (GMO), the primary goal is to give a target organism one or several novel traits by using biotechnology techniques. A GMO will differ from its parental strain in that its pool of transcripts will be altered. Currently, there are no methods that are reliably able to determine if an organism has been genetically altered if the nature of the modification is unknown., Results: We show that the concept of computational subtraction can be used to identify transgenic cDNA sequences from genetically modified plants. Our datasets include 454-type sequences from a transgenic line of Arabidopsis thaliana and published EST datasets from commercially relevant species (rice and papaya)., Conclusion: We believe that computational subtraction represents a powerful new strategy for determining if an organism has been genetically modified as well as to define the nature of the modification. Fewer assumptions have to be made compared to methods currently in use and this is an advantage particularly when working with unknown GMOs.

Background: The genomic fractions of purine (RR) and alternating pyrimidine/purine (YR) stretches of 10 base pairs or more, have been linked to genomic AT content, the formation of different DNA helices, strand-biased gene distribution, DNA structure, and more. Although some of these factors are a consequence of the chemical properties of purines and pyrimidines, a thorough statistical examination of the distributions of YR/RR stretches in sequenced prokaryotic chromosomes has to the best of our knowledge, not been undertaken. The aim of this study is to expand upon previous research by using regression analysis to investigate how AT content, habitat, growth temperature, pathogenicity, phyla, oxygen requirement and halotolerance correlated with the distribution of RR and YR stretches in prokaryotes., Results: Our results indicate that RR and YR-stretches are differently distributed in prokaryotic phyla. RR stretches are overrepresented in all phyla except for the Actinobacteria and beta-Proteobacteria. In contrast, YR tracts are underrepresented in all phyla except for the beta-Proteobacterial group. YR-stretches are associated with phylum, pathogenicity and habitat, whilst RR-tracts are associated with phylum, AT content, oxygen requirement, growth temperature and halotolerance. All associations described were statistically significant with p < 0.001., Conclusion: Analysis of chromosomal distributions of RR/YR sequences in prokaryotes reveals a set of associations with environmental factors not observed with mono- and oligonucleotide frequencies. This implies that important information can be found in the distribution of RR/YR stretches that is more difficult to obtain from genomic mono- and oligonucleotide frequencies. The association between pathogenicity and fractions of YR stretches is assumed to be linked to recombination and horizontal transfer.