Optimal Ancient DNA Yields from the Inner Ear Part of the Human Petrous Bone

The invention and development of next or second generation sequencing methods has resulted in a dramatic transformation of ancient DNA research and allowed shotgun sequencing of entire genomes from fossil specimens. However, although there are exceptions, most fossil specimens contain only low (~ 1% or less) percentages of endogenous DNA. The only skeletal element for which a systematically higher endogenous DNA content compared to other skeletal elements has been shown is the petrous part of the temporal bone. In this study we investigate whether (a) different parts of the petrous bone of archaeological human specimens give different percentages of endogenous DNA yields, (b) there are significant differences in average DNA read lengths, damage patterns and total DNA concentration, and (c) it is possible to obtain endogenous ancient DNA from petrous bones from hot environments. We carried out intra-petrous comparisons for ten petrous bones from specimens from Holocene archaeological contexts across Eurasia dated between 10,000-1,800 calibrated years before present (cal. BP). We obtained shotgun DNA sequences from three distinct areas within the petrous: a spongy part of trabecular bone (part A), the dense part of cortical bone encircling the osseous inner ear, or otic capsule (part B), and the dense part within the otic capsule (part C). Our results confirm that dense bone parts of the petrous bone can provide high endogenous aDNA yields and indicate that endogenous DNA fractions for part C can exceed those obtained for part B by up to 65-fold and those from part A by up to 177-fold, while total endogenous DNA concentrations are up to 126-fold and 109-fold higher for these comparisons. Our results also show that while endogenous yields from part C were lower than 1% for samples from hot (both arid and humid) parts, the DNA damage patterns indicate that at least some of the reads originate from ancient DNA molecules, potentially enabling ancient DNA analyses of samples from hot regions that are otherwise not amenable to ancient DNA analyses.     

Amplification of DNA of Xanthomonas axonopodis pv. citri from historic citrus canker herbarium specimens

Herbaria are important resources for the study of the origins and dispersal of plant pathogens, particularly bacterial plant pathogens that incite local lesions in which large numbers of pathogen genomes are concentrated. Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus bacterial canker disease, is a notable example of such a pathogen. The appearance of novel strains of the pathogen in Florida and elsewhere make it increasingly important to understand the relationships among strains of this pathogen. USDA-ARS at Beltsville, Maryland maintains approximately 700 herbarium specimens with citrus canker disease lesions up to 90 years old, originally collected from all over the world, and so is an important resource for phytogeographic studies of this bacterium. Unfortunately, DNA in herbarium specimens is degraded and may contain high levels of inhibitors of PCR. In this study, we compared a total of 23 DNA isolation techniques in combination with 31 novel primer pairs in order to develop an efficient protocol for the analysis of Xac DNA in herbarium specimens. We identified the most reliable extraction method, identified in terms of successful amplification by our panel of 31 primer pairs. We also identified the most robust primer pairs, identified as successful in the largest number of extracts prepared by different methods. We amplified Xac genomic sequences up to 542 bp long from herbarium samples up to 89 years old. Primers varied in effectiveness, with some primer pairs amplifying Xac DNA from a 1/10,000 dilution of extract from a single lesion from a citrus canker herbarium specimen. Our methodology will be useful to identify pathogens and perform molecular analyses of bacterial and possibly fungal genomes from herbarium specimens.     

Redefining bacterial populations: a post-genomic reformation

Sexual reproduction and recombination are essential for the survival of most eukaryotic populations. Until recently, the impact of these processes on the structure of bacterial populations has been largely overlooked. The advent of large-scale whole-genome sequencing and the concomitant development of molecular tools, such as microarray technology, facilitate the sensitive detection of recombination events in bacteria. These techniques are revealing that bacterial populations are comprised of isolates that show a surprisingly wide spectrum of genetic diversity at the DNA level. Our new awareness of this genetic diversity is increasing our understanding of population structures and of how these affect host pathogen relationships.     

Bacterial pathogen evolution: breaking news

The immense social and economic impact of bacterial pathogens, from drug-resistant infections in hospitals to the devastation of agricultural resources, has resulted in major investment to understand the causes and consequences of pathogen evolution. Recent genome sequencing projects have provided insight into the evolution of bacterial genome structures; revealing the impact of mobile DNA on genome restructuring and pathogenicity. Sequencing of multiple genomes of related strains has enabled the delineation of pathogen evolution and facilitated the tracking of bacterial pathogens globally. Other recent theoretical and empirical studies have shown that pathogen evolution is significantly influenced by ecological factors, such as the distribution of hosts within the environment and the effects of co-infection. We suggest that the time is ripe for experimentalists to use genomics in conjunction with evolutionary ecology experiments to further understanding of how bacterial pathogens evolve.     

Development and validation of a modified broad-range 16S rDNA PCR for diagnostic purposes in clinical microbiology

Broad-range PCR followed by sequencing identifies bacterial pathogens, even in challenging settings such as patients receiving antibiotics or infected with fastidious or non-cultivable organisms. The major problem with broad-range PCR is the risk of sample contamination. Risk is present at every step of the procedure, starting from sample collection. Contaminating bacterial DNA may be present not only in laboratory reagents but also at the surface of plastic consumables and containers used for specimen drawing and transport to the diagnostic laboratory. Contaminating DNA is amplified efficiently, leading to false-positive results. Thus, high specificity depends on eliminating such spurious targets, an awkward problem given the abundance of such targets and a highly sensitive method that detects very small numbers of molecules. Several investigators have reported strategies for eliminating the amplification of contaminating DNA sequences. So far, none of these methods has been entirely effective and reproducible. Here we describe a method that uses Exonuclease III (ExoIII) to disable contaminating sequences from acting as templates, while maintaining the high sensitivity of PCR for pathogen DNA. We use this assay in 144 clinical specimens from normally sterile sites, identifying pathogens from 24 (17%). Conventional methods identified pathogens in only four of these specimens, all of which were positive for the same pathogen by PCR. Compared with conventional methods, broad-range PCR with ExoIII pre-treatment of reagents substantially improves the diagnostic yield of bacterial pathogen identification from normally sterile sites.     

DNA decay rate in papyri and human remains from Egyptian archaeological sites

The writing sheets made with strips from the stem (caulis) of papyri (Cyperus papyrus) are one of the most ingenious products of ancient technology. We extracted DNA from samples of modern papyri varying in age from 0-100 years BP and from ancient specimens from Egypt, with an age-span from 1,300-3,200 years BP. The copy number of the plant chloroplast DNA in the sheets was determined using a competitive PCR system designed on the basis of a short (90 bp) tract of the chloroplast's ribulose bisphosphate carboxylase large subunit (rbcL) gene sequence. The results allowed us to establish that the DNA half-life in papyri is about 19-24 years. This means that the last DNA fragments will vanish within no more than 532-672 years from the sheets being manufactured. In a parallel investigation, we checked the archaeological specimens for the presence of residual DNA and determined the extent of racemization of aspartic (Asp) acid in both modern and ancient specimens, as a previous report (Poinar et al. [1996], Science 272:864-866) showed that racemization of aspartic acid and DNA decay are linked. The results confirmed the complete loss of authentic DNA, even in the less ancient (8th century AD) papyri. On the other hand, when the regression for Asp racemization rates in papyri was compared with that for human and animal remains from Egyptian archaeological sites, it proved, quite surprisingly, that the regressions are virtually identical. Our study provides an indirect argument against the reliability of claims about the recovery of authentic DNA from Egyptian mummies and bone remains.     

Subcellular alterations that lead to diarrhea during bacterial pathogenesis

Pathogenic microorganisms routinely exploit host cellular functions for their benefit. These alterations often enhance the survival and/or dissemination of the pathogen. However, these effects on the host can be quite debilitating. Consequently, an in-depth understanding of the molecular mechanisms employed by pathogens to manipulate their hosts is crucial. One of the common host phenotypes elicited by enteric pathogens is the generation of diarrhea. Here, we overview the current advances in understanding strategies used by bacterial pathogens to cause diarrheal diseases and discuss how the coordination of various subcellular events can influence disease progression.     

First historical genome of a crop bacterial pathogen from herbarium specimen: Insights into citrus canker emergence

Over the past decade, ancient genomics has been used in the study of various pathogens. In this context, herbarium specimens provide a precious source of dated and preserved DNA material, enabling a better understanding of plant disease emergences and pathogen evolutionary history. We report here the first historical genome of a crop bacterial pathogen, Xanthomonas citri pv. citri (Xci), obtained from an infected herbarium specimen dating back to 1937. Comparing the 1937 genome within a large set of modern genomes, we reconstructed their phylogenetic relationships and estimated evolutionary parameters using Bayesian tip-calibration inferences. The arrival of Xci in the South West Indian Ocean islands was dated to the 19^th century, probably linked to human migrations following slavery abolishment. We also assessed the metagenomic community of the herbarium specimen, showed its authenticity using DNA damage patterns, and investigated its genomic features including functional SNPs and gene content, with a focus on virulence factors.     

Technical note: Removal of metal ion inhibition encountered during DNA extraction and amplification of copper‐preserved archaeological bone using size exclusion chromatography

A novel technique for the removal of metal ions inhibiting DNA extraction and PCR of archaeological bone extracts is presented using size exclusion chromatography. Two case studies, involving copper inhibition, demonstrate the effective removal of metal ion inhibition. Light microscopy, SEM, elemental analysis, and genetic analysis were used to demonstrate the effective removal of metal ions from samples that previously exhibited molecular inhibition. This research identifies that copper can cause inhibition of DNA polymerase during DNA amplification. The use of size exclusion chromatography as an additional purification step before DNA amplification from degraded bone samples successfully removes metal ions and other inhibitors, for the analysis of archaeological bone. The biochemistry of inhibition is explored through chemical and enzymatic extraction methodology on archaeological material. We demonstrate a simple purification technique that provides a high yield of purified DNA (>95%) that can be used to address most types of inhibition commonly associated with the analysis of degraded archaeological and forensic samples. We present a new opportunity for the molecular analysis of archaeological samples preserved in the presence of metal ions, such as copper, which have previously yielded no DNA results. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.     

Bacterial colonization and extinction on marine aggregates: stochastic model of species presence and abundance

Organic aggregates provide a favorable habitat for aquatic microbes, are efficiently filtered by shellfish, and may play a major role in the dynamics of aquatic pathogens. Quantifying this role requires understanding how pathogen abundance in the water and aggregate size interact to determine the presence and abundance of pathogen cells on individual aggregates. We build upon current understanding of the dynamics of bacteria and bacterial grazers on aggregates to develop a model for the dynamics of a bacterial pathogen species. The model accounts for the importance of stochasticity and the balance between colonization and extinction. Simulation results suggest that while colonization increases linearly with background density and aggregate size, extinction rates are expected to be nonlinear on small aggregates in a low background density of the pathogen. Under these conditions, we predict lower probabilities of pathogen presence and reduced abundance on aggregates compared with predictions based solely on colonization. These results suggest that the importance of aggregates to the dynamics of aquatic bacterial pathogens may be dependent on the interaction between aggregate size and background pathogen density, and that these interactions are strongly influenced by ecological interactions and pathogen traits. The model provides testable predictions and can be a useful tool for exploring how species‐specific differences in pathogen traits may alter the effect of aggregates on disease transmission.     

Prehistoric Introduction of Domestic Pigs onto the Okinawa Islands: Ancient Mitochondrial DNA Evidence

Ancient DNAs of Sus scrofa specimens excavated from archaeological sites on the Okinawa islands were examined to clarify the genetic relationships among prehistoric Sus scrofa, modern wild boars and domestic pigs inhabiting the Ryukyu archipelago, the Japanese islands, and the Asian continent. We extracted remain DNA from 161 bone specimens excavated from 12 archaeological sites on the Okinawa islands and successfully amplified mitochondrial DNA control region fragments from 33 of 161 specimens. Pairwise difference between prehistoric and modern S. scrofa nucleotide sequences showed that haplotypes of the East Asian domestic pig lineage were found from archaeological specimens together with Ryukyu wild boars native to the Ryukyu archipelago. Phylogenetic analysis of 14 ancient sequences (11 haplotypes; 574 bp) indicated that S. scrofa specimens from two Yayoi-Heian sites (Kitahara and Ara shellmiddens) and two Recent Times sites (Wakuta Kiln and Kiyuna sites) are grouped with modern East Asian domestic pigs. Sus scrofa specimens from Shimizu shellmidden (Yayoi-Heian Period) were very closely related to modern Sus scrofa riukiuanus but had a unique nucleotide insertion, indicating that the population is genetically distinct from the lineage of modern Ryukyu wild boars. This genetic evidence suggests that domestic pigs from the Asian continent were introduced to the Okinawa islands in the early Yayoi-Heian period (1700–2000 BP), or earlier.     

Effects of Bacillus anthracis hydrophobicity and induction of host cell death on sample collection from environmental surfaces

The objective of this study is to determine whether DNA signature recovery of Bacillus anthracis strains from different environmental substrates correlates with pathogen cell surface hydrophobicity and induction of host cell death. We compared recovery of DNA signatures from a panel of B. anthracis strains collected from two environmental substrates, non-porous surfaces and soil, using real-time qPCR. We further assessed both cell surface hydrophobicity of the B. anthracis strains by contact angle measurements and host cell viability in response to B. anthracis infection in a mouse macrophage cell model system. Our studies demonstrated correlation between reduced B. anthracis sample recovery from environmental substrates and increased cell surface hydrophobicity. Surprisingly, the most hydrophilic strain, K4596, which exhibited the highest level of recovery from the environmental surfaces, induced the highest level of host cell cytotoxicity compared to more hydrophobic B. anthracis strains in the panel. Our results suggest that cell surface hydrophobicity may play a leading role in mediating pathogen adherence to environmental surfaces. These findings can contribute to the optimization of pathogen detection efforts by understanding how bacterial parameters such as hydrophobicity and induction of host cell death affect bacterial adherence to environmental surfaces.     

Microscopic,chemical and molecular methods for examining fossil preservation

Advances in technology over the past two decades have resulted in unprecedented access to data from biological specimens. These data have expanded our understanding of physical characteristics, physiological, cellular and subcellular processes, and evolutionary relationships at the molecular level and beyond. Paleontological and archaeological sciences have recently begun to apply these technologies to fossil and subfossil representatives of extinct organisms. Data derived from multidisciplinary, non-traditional techniques can be difficult to decipher, and without a basic understanding of the type of information provided by these methods, their usefulness for fossil studies may be overlooked. This review describes some of these powerful new analytical tools, the data that may be accessible through their use, advantages and limitations, and how they can be applied to fossil material to elucidate characteristics of extinct organisms and their paleoecological environments.     

DAF-16-dependent suppression of immunity during reproduction in Caenorhabditis elegans

To further understand how the nematode Caenorhabditis elegans defends itself against pathogen attack, we analyzed enhanced pathogen resistance (epr) mutants obtained from a forward genetic screen. We also examined several well-characterized sterile mutants that exhibit an Epr phenotype. We found that sterility and pathogen resistance are highly correlated and that resistance in both epr and sterile mutants is dependent on DAF-16 activity. Our data indicate that a DAF-16-dependent signaling pathway distinct from previously described pathways is involved in the activation of genes that confer resistance to bacterial pathogens. The timing of DAF-16-dependent gene activation in sterile mutants coincides with the onset of embryonic development in wild-type animals, suggesting that signals from developing embryos normally downregulate the immune response.     

Accurate determination of phenotypic information from historic thoroughbred horses by single base extension

Historic DNA data have the potential to identify phenotypic information otherwise invisible in the historical, archaeological and palaeontological record. In order to determine whether a single nucleotide polymorphism typing protocol based on single based extension (SNaPshot™) could produce reliable phenotypic data from historic samples, we genotyped three coat colour markers for a sample of historic Thoroughbred horses for which both phenotypic and correct genotypic information were known from pedigree information in the General Stud Book. Experimental results were consistent with the pedigrees in all cases. Thus we demonstrate that historic DNA techniques can produce reliable phenotypic information from museum specimens.     

Correlative proteomics identify the key roles of stress tolerance strategies in Acinetobacter baumannii in response to polymyxin and human macrophages

The opportunistic pathogen Acinetobacter baumannii possesses stress tolerance strategies against host innate immunity and antibiotic killing. However, how the host-pathogen-antibiotic interaction affects the overall molecular regulation of bacterial pathogenesis and host response remains unexplored. Here, we simultaneously investigate proteomic changes in A. baumannii and macrophages following infection in the absence or presence of the polymyxins. We discover that macrophages and polymyxins exhibit complementary effects to disarm several stress tolerance and survival strategies in A. baumannii, including oxidative stress resistance, copper tolerance, bacterial iron acquisition and stringent response regulation systems. Using the spoT mutant strains, we demonstrate that bacterial cells with defects in stringent response exhibit enhanced susceptibility to polymyxin killing and reduced survival in infected mice, compared to the wild-type strain. Together, our findings highlight that better understanding of host-pathogen-antibiotic interplay is critical for optimization of antibiotic use in patients and the discovery of new antimicrobial strategy to tackle multidrug-resistant bacterial infections.     

Ancient DNA from an Early Neolithic Iberian population supports a pioneer colonization by first farmers

The Neolithic transition has been widely debated particularly regarding the extent to which this revolution implied a demographic expansion from the Near East. We attempted to shed some light on this process in northeastern Iberia by combining ancient DNA (aDNA) data from Early Neolithic settlers and published DNA data from Middle Neolithic and modern samples from the same region. We successfully extracted and amplified mitochondrial DNA from 13 human specimens, found at three archaeological sites dated back to the Cardial culture in the Early Neolithic (Can Sadurní and Chaves) and to the Late Early Neolithic (Sant Pau del Camp). We found that haplogroups with a low frequency in modern populations-N* and X1-are found at higher frequencies in our Early Neolithic population (～31%). Genetic differentiation between Early and Middle Neolithic populations was significant (F(ST) ～0.13, P<10(-5)), suggesting that genetic drift played an important role at this time. To improve our understanding of the Neolithic demographic processes, we used a Bayesian coalescence-based simulation approach to identify the most likely of three demographic scenarios that might explain the genetic data. The three scenarios were chosen to reflect archaeological knowledge and previous genetic studies using similar inferential approaches. We found that models that ignore population structure, as previously used in aDNA studies, are unlikely to explain the data. Our results are compatible with a pioneer colonization of northeastern Iberia at the Early Neolithic characterized by the arrival of small genetically distinctive groups, showing cultural and genetic connections with the Near East.     

Comparative genomics of Mycoplasma: analysis of conserved essential genes and diversity of the pan-genome

Mycoplasma, the smallest self-replicating organism with a minimal metabolism and little genomic redundancy, is expected to be a close approximation to the minimal set of genes needed to sustain bacterial life. This study employs comparative evolutionary analysis of twenty Mycoplasma genomes to gain an improved understanding of essential genes. By analyzing the core genome of mycoplasmas, we finally revealed the conserved essential genes set for mycoplasma survival. Further analysis showed that the core genome set has many characteristics in common with experimentally identified essential genes. Several key genes, which are related to DNA replication and repair and can be disrupted in transposon mutagenesis studies, may be critical for bacteria survival especially over long period natural selection. Phylogenomic reconstructions based on 3,355 homologous groups allowed robust estimation of phylogenetic relatedness among mycoplasma strains. To obtain deeper insight into the relative roles of molecular evolution in pathogen adaptation to their hosts, we also analyzed the positive selection pressures on particular sites and lineages. There appears to be an approximate correlation between the divergence of species and the level of positive selection detected in corresponding lineages.     

Host genetics and pathogen species modulate infection-induced changes in social aggregation behaviour

Identifying how infection modifies host behaviours that determine social contact networks is important for understanding heterogeneity in infectious disease dynamics. Here, we investigate whether group social behaviour is modified during bacterial infection in fruit flies (Drosophila melanogaster) according to pathogen species, infectious dose, host genetic background and sex. In one experiment, we find that systemic infection with four different bacterial species results in a reduction in the mean pairwise distance within infected female flies, and that the extent of this change depends on pathogen species. However, susceptible flies did not show any evidence of avoidance in the presence of infected flies. In a separate experiment, we observed genetic- and sex-based variation in social aggregation within infected, same-sex groups, with infected female flies aggregating more closely than infected males. In general, our results confirm that bacterial infection induces changes in fruit fly behaviour across a range of pathogen species, but also highlight that these effects vary between fly genetic backgrounds and can be sex-specific. We discuss possible explanations for sex differences in social aggregation and their consequences for individual variation in pathogen transmission.