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Either your web browser doesn't support Javascript or it is currently turned off. In the latter case, please turn on Javascript support in your web browser and reload this page. Genes , 13 Jul , 8 7 DOI: Following the discovery in the late s that hard tissues such as bones and teeth preserve genetic information, the field of ancient DNA analysis has typically concentrated upon these substrates. The onset of high-throughput sequencing, combined with optimized DNA recovery methods, has enabled the analysis of a myriad of ancient species and specimens worldwide, dating back to the Middle Pleistocene.

We conclude that there is a pressing need for more refined models of DNA preservation and bespoke tools for DNA extraction and analysis to authenticate and maximize the utility of the data obtained. With such tools in place the potential for neglected or underexploited substrates to provide a unique insight into phylogenetics, microbial evolution and evolutionary processes will be realized.

The demonstration that mitochondrial DNA mtDNA could be isolated and cloned from museum specimens influenced the choice of ancient samples for the next decade. Early aDNA research focused on soft tissue [ 2 , 3 , 4 , 5 , 6 , 7 , 8 ], however, the first successful extraction of aDNA from archaeological bones by three independent laboratories in — revolutionized the field [ 9 , 10 , 11 ].

For the last 30 years, bone and teeth have been the most frequently studied substrates in paleogenetic research. Most paleontological hard tissues, however, contain very low proportions of endogenous DNA [ 12 ], and thus the search for methods to maximize the utility of endogenous DNA data from bony specimens has been extensive, including rapid column-based extractions [ 13 , 14 , 15 ], and the use of alternative skeletal elements e.

With improved accessibility to highly decayed and fragmentary genetic information, facilitated by the NGS revolution, aDNA is increasingly being extracted from what are now considered novel or alternative substrates Figure 1.

The relative proportion of studies discussed in this review papers from to May targeting various alternative substrates for the recovery of ancient DNA. NHC: natural history collections. Although all biological materials have the potential to preserve DNA, relatively few will resist decay over time, unless protected within an inorganic matrix or deliberately preserved through human intervention.

Here, we review the potential for DNA preservation within three broad classes of materials: 1 archaeological artifacts and ecofacts; 2 calcified or mineralized substrates; and 3 biological and cultural archives. Archaeological excavations worldwide recover millions of artifacts drawn from all time periods in all stages of decay—yet the only materials routinely analyzed genetically are hard tissues.

Bones and teeth are mostly targeted due to scientific focus on human and, to a lesser extent, animal evolution, while the extraction of DNA from other hard tissues like preserved antler, or antler artifacts has facilitated research into cervid population genetics and object manufacture [ 21 , 22 ].

Researchers are now extracting DNA from an array of artifacts i. Although DNA preservation is variable, these items provide unique access to evolutionary pathways, taxonomy and phylogeny that may be simply unobtainable from the analysis of hard tissue.

A range of early polymerase chain reaction PCR based approaches e. Seeds tend to preserve in archaeological sites only when charred, desiccated, frozen, or deposited in anoxic conditions. Charred seeds, making up the vast majority of recovered archaeobotanical materials, have variable degrees of DNA preservation, depending, in part, on the extent of charring, as well as their age and depositional environment [ 36 , 37 , 38 ].

For example, in Bunning et al. Likewise, Nistelberger et al. Variation in DNA preservation is also observed within desiccated remains. For example, in their analysis of five year-old barley grains from a single site in Israel, Mascher et al. Successful DNA recovery has also been achieved using other archaeological plant tissues, including maize cobs [ 32 , 39 , 40 ], fruit stones [ 41 ], pollen [ 42 , 43 , 44 ], grains and seeds [ 45 , 46 , 47 , 48 ], rind [ 37 ], and chaff [ 49 , 50 ].

Using more advanced DNA recovery and bioinformatic techniques such as whole genome sequencing, single nucleotide polymorphism SNP capture, exome analyses, RNA analysis, and methylation patterns, these archaeobotanical projects are demonstrating the vast potential of ancient plant remains to address significant debates around the origins, movement and adaptation of domestic crops [ 32 , 37 ], reconstruction of paleo-environments [ 57 ] and models of DNA decay within plant remains [ 34 , 36 ].

Leather, hair, baleen, claws and feathers are all composed of layers of collagen or keratin, which frequently decompose when deposited underground. In some extraordinary contexts, e. Although reports on DNA extraction from archaeological leathers are few, mtDNA within this substrate seems to be particularly resilient, with previous studies recovering 70— bp fragments from medieval [ 58 ] and Neolithic [ 59 ] leather; although the tanning process may be particularly damaging for nuclear DNA [ 58 , 60 ].

The keratin structure of hair, claws and baleen is thought to protect endogenous DNA from contamination [ 61 , 62 ], and several studies have successfully retrieved endogenous DNA from human and animal hair [ 62 , 63 , 64 ] and whale baleen [ 65 , 66 ] preserved in Danish and Greenlandic archaeological contexts.

Preservation of claws in archaeological contexts is rare; however, research on natural history collections NHC indicates that DNA can be sufficiently well preserved for analysis of this substrate [ 67 , 68 ], with potentially the same success rate as ancient bone [ 69 ]. The base of feathers the calamus has been frequently exploited as a source of high-quality DNA in bird phylogenetics and conservation biology research, and the potential for feathers in NHC to preserve DNA for several hundred years has been known for more than two decades e.

In archaeological and subfossil environments, the upper shaft and the feather vane are the only part of the feathers that typically survive and were thought to be unsuitable for aDNA analysis. Using Moa feathers preserved in rock shelter sites in New Zealand, Rawlence et al. Subsequently, Speller et al. The potential for mtDNA to survive in these keratinous substrates has been well documented, however, the potential for nuclear DNA survival is less well explored [ 66 , 74 ].

Future metagenomic analyses are required to assess both the relative proportion and survival of mitochondrial versus nuclear DNA, and to test susceptibility of different keratinous substrates to exogenous contamination from the burial environment. Lithics represent some of the most abundant and durable artifacts within the archaeological record, particularly in prehistoric contexts.

As such, studies endeavoring to understand the day-to-day use of various stone artifacts have a long history, employing methods such as experimental archaeology [ 75 ]; useware analysis [ 76 ]; microwear polish examination [ 77 ]; and microscopic and chemical residue analysis [ 78 , 79 ].

Unsurprisingly, researchers attempted to extract DNA from lithics as early as the s. In , Shanks et al. The findings of these early studies have been called into question, first by the lack of authentication measures and contamination controls required to validate aDNA results [ 84 ], as well as by observations of domestic animal DNA within laboratory reagents [ 85 ]. Coupled with extensive controversy regarding the ability to extract authentic proteins from ancient lithic materials e.

The application of NGS approaches however, may resurrect this old idea in a new form, again through the analysis of another novel substrate: historic building materials. Two recent studies have investigated the potential for metagenomic analysis of ancient brick and stone work to elucidate building histories and investigate microbial factors influencing biodeterioration of built heritage [ 92 , 93 ], opening up potentially more fruitful research directions for ancient lithics.

Like lithic materials, ancient ceramics make up a large percentage of archaeological finds; however, attempts to mine this substrate for preserved DNA has lagged significantly compared to the analysis of other surface or adsorbed organic molecules, including lipids, amino acids, alkaloids, waxes, etc.

Attempts to extract DNA from ceramic objects have involved scraping or drilling the interior of the vessel, as well as non-destructive swabbing of the interior face [ 98 , 99 ]. Recently, aDNA identifications have been reported from pottery vessels recovered from environments assumed to be hostile to DNA. Foley et al. Based on their results, Foley et al. Robinson et al.

Here, DNA from three plant types—plantain, pine and grasses—were identified using generic plant primers, recovering fragments up to bp, significantly beyond the average length of DNA fragments they predicted by thermal age modeling. The latter study applied extensive contamination controls and numerous measures to validate their sequences, but, in so doing, highlighted a major challenge when utilizing atypical substrates: due to the infancy of the research, the proper authentication of results is imperative; paradoxically, there is currently little understanding of if and how DNA is expected to preserve within such substrates—an issue we discuss in greater detail below Section 3.

The predominant use of skeletal hard tissues in aDNA analysis is related to their ubiquity in the archaeological and paleontological record, but also because the inorganic fraction of hydroxyapatite in bone and teeth is thought to stabilize and preserve DNA through adsorption [ , ]. More recently, the potential for other mineralized substrates to preserve ancient biomolecules in deep time is also being realized [ ], including substrates such as archaeological dental calculus, coprolites, calcified soft tissues, invertebrate shells and ancient eggshells.

Unlike bones and teeth, these substrates may be exploited not only as a source of host genetic information, but also to provide insight into ancient microbiomes and paleoenvironments. Dental calculus tartar is a bacterial biofilm composed from dental plaque, saliva and gingival crevicular fluid, mineralized within a matrix of multiple calcium phosphates, forming a cement-like substrate on the surface of teeth [ , , ].

Although found virtually ubiquitously on archaeological human skeletons without modern dental hygiene interventions, the potential for this substrate to preserve abundant and varied ancient biomolecules has only recently been discovered.

The preservation of ancient oral bacterial DNA was first recognized through gold-labeled antibody transmission electron microscopy [ ] and subsequently through targeted PCR and 16S ribosomal RNA rRNA amplicon metagenomics [ , , ]. Shotgun metagenomic approaches have demonstrated that although calculus is dominated by bacterial and to a lesser extent fungal, archeal and viral DNA derived from the oral microbiome, minute quantities of host DNA as well as inhaled or ingested eukaryotic DNA also survive [ , , ].

Research to date has demonstrated that calculus preserves an exceptional abundance of entrapped DNA and proteins [ , , , ], and even ancient metabolites [ ]. These combined studies have provided insight into the oral ecology of ancient humans and hominids, and even allowed for microbial genome reconstruction dating back to nearly 50k years before present BP [ , ].

In some studies, calculus has been noted to be relatively resistant to exogenous bacterial colonization from the burial environment [ ]. Unlike calculus, paleofeces and coprolites have been long been recognized for their value as a source of ancient biomolecules and paleodietary information.

Paleofeces can be preserved via various mechanisms such as rapid desiccation or waterlogging, while coprolites form through the precipitation of calcium and phosphate from ingested bone especially in carnivores and scavengers or through the acquisition of preservational constituents via groundwater [ ]. Due to the specific environmental conditions required for their preservation, paleofeces and coprolites are also relatively rare archaeological finds compared with the ubiquity of dental calculus.

At the turn of the millennium, Poinar et al. These studies laid the foundation for other human and animal paleofeces and coprolite analyses using targeted PCR to identify the presence and genetic diversity of humans in the Americas [ , , ], elucidate the taxonomy and diet of extinct [ , , , ] and extant birds [ , ] as well as to taxonomically identify parasite eggs within coprolites [ , , ].

More recently, high-throughput approaches have been applied to recover more representative observations of dietary components within paleofeces and coprolites. Last year, Wood et al. Analysis of retroviral DNA has also been attempted to access paleodietary information.

In their metagenomic analysis of ancient human coprolites from the Caribbean, Rivera-Perez et al. Although the identified retroviruses corresponded with expected dietary patterns within the region, the dominance of well-characterized taxa e.

Paleofeces and coprolites, however, provide genetic information beyond dietary inclusions. Tito et al. In a follow up study using a 16S rRNA metabarcoding approach on an expanded sample set, they revealed that not all fecal deposits may preserve the signature of endogenous ecology at the time of deposition [ ]. While rapidly desiccated feces from cave environments were found to preserve the integrity of the gut ecology, others were found to have undergone self-digestion and decomposition as well as bacterial infiltration from the burial environment.

The portion of coprolite sampled for analysis can also bias the identified microbial communities. Cano et al. Paleofeces and coprolites can also reveal the presence of ancient viruses. Multiple-displacement amplification of viral genomes within a 14 th Century human coprolite revealed an ancient virome comprised predominantly of double-stranded viral DNA Although the virome was similar to that found in modern fecal matter and soil, further characterization of virome taxonomy and function was limited by the relatively high proportion of sequences of unknown origin.

Although found principally within the digestive or urinary tracts, calcified tissues and stony neoplastic tumors or exudates may form within the lungs, vascular system, tear ducts, tendons, or skin, and are occasionally recovered during archaeological excavations. Two recent studies have exemplified the wealth of genetic information that may be preserved within these serendipitous finds.

Kay et al. Although tuberculosis was initially suspected as the cause of the pathology, the study instead identified sequences matching Brucella melitensis 0.

Like the calcified nodule from Sardinia, the tumor contained an amalgam of human and microbial DNA. The human sex chromosome component of the DNA revealed a dominance of X chromosome sequences, with minute quantities of conserved Y chromosome sequences, interpreted to result from the presence of a male fetus, and suggesting that the growths developed in placental tissue [ ].

The bacterial DNA recovered from shotgun sequencing identified Staphylococcus saprophyticus and Gardnerella vaginalis in high abundance, allowing for genome reconstruction and strain level analysis. Although calcified nodules may represent a rather unusual archaeological find, these case studies display the potential of these substrates to provide insight both into host genetics and ancient health and disease. There is a long history of research around organic molecules preserved within the calcareous exoskeleton of marine invertebrates.

Previous research has focused almost exclusively on the preservation of proteins, particularly those responsible for biomineralization [ , ], and the use of these proteins for amino acid geochronology [ , , , ] and more recently, taxonomic identification [ ].

The potential for the calcium carbonate of marine invertebrate shell to preserve genetic material has only recently been explored. Likewise, Villanea et al. Very recently, Der Sarkissian et al. They recovered average fragment lengths of 43—50 bp, enabling positive taxonomic identification of various mollusk species, marine organism pathogens as well as minute quantities of mtDNA from the marine environment [ ].

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Novel Substrates as Sources of Ancient DNA: Prospects and Hurdles.

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