Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries

Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062–147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217–73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples.     

White rot Basidiomycetes isolated from Chiloé National Park in Los Lagos region, Chile

Wood decomposition is an important component in forest ecosystems but information about the diversity of fungi causing decay is lacking. This is especially true for the temperate rain forests in Chile. These investigations show results of a biodiversity study of white-rot fungi in wood obtained from Chiloé National Park in Los Lagos region, Chile. Culturing from white-rotted wood followed by sequencing of the complete internal transcribed spacer region of the ribosomal DNA (rDNA) or partial large subunit region of the rDNA, identified 12 different species in the Basidiomycota. All of these fungi were characterized as white rot fungi and were identified with a BLAST match of 97 % or greater to sequences in the GenBank database. Fungi obtained were species of Phlebia, Mycoacia, Hyphodontia, Bjerkandera, Phanerochaete, Stereum, Trametes, and Ceriporiopsis. This report identifies for the first time in Chile the species Ceriporiopsis subvermispora, Hyphodontia radula, Phlebia radiata, Phanerochaete affinis, Peniophora cinerea, Stereum gausapatum, Phlebia setulosa and Phanerochaete sordida. Scanning electron microscopy was used to characterize the type of decay caused by the fungi that were isolated and a combination of selective lignin degraders and simultaneous white rot fungi were found. Fungi that cause a selective degradation of lignin are of interest for bioprocessing technologies that require modification or degradation of lignin without cellulose removal.     

Interaction of a peptide corresponding to the loop domain of the S2 SARS-CoV virus protein with model membranes

The severe acute respiratory syndrome coronavirus (SARS-CoV) envelope spike (S) glycoprotein is responsible for the fusion between the membranes of the virus and the target cell. In the case of the S2 domain of protein S, it has been found a highly hydrophobic and interfacial domain flanked by the heptad repeat 1 and 2 regions; significantly, different peptides pertaining to this domain have shown a significant leakage effect and an important plaque formation inhibition, which, similarly to HIV-1 gp41, support the role of this region in the fusion process. Therefore, we have carried out a study of the binding and interaction with model membranes of a peptide corresponding to segment 1073–1095 of the SARS-CoV S glycoprotein, peptide SARS_L in the presence of different membrane model systems, as well as the structural changes taking place in both the lipid and the peptide induced by the binding of the peptide to the membrane. Our results show that SARS_L strongly partitions into phospholipid membranes and organizes differently in lipid environments, displaying membrane activity modulated by the lipid composition of the membrane. These data would support its role in SARS-CoV mediated membrane fusion and suggest that the region where this peptide resides could be involved in the merging of the viral and target cell membranes.     

Seasonal variation in leaf traits between congeneric savanna and forest trees in Central Brazil: implications for forest expansion into savanna

The ecology of forest and savanna trees species will largely determine the structure and dynamics of the forest–savanna boundaries, but little is known about the constraints to leaf trait variation imposed by selective forces and evolutionary history during the process of savanna invasion by forest species. We compared seasonal patterns in leaf traits related to leaf structure, carbon assimilation, water, and nutrient relations in 10 congeneric species pairs, each containing one savanna species and one forest species. All individuals were growing in dystrophic oxisols in a fire-protected savanna of Central Brazil. We tested the hypothesis that forest species would be more constrained by seasonal drought and nutrient-poor soils than their savanna congeners. We also hypothesized that habitat, rather than phylogeny, would explain more of the interspecific variance in leaf traits of the studied species. We found that throughout the year forest trees had higher specific leaf area (SLA) but lower integrated water use efficiency than savanna trees. Forest and savanna species maintained similar values of predawn and midday leaf water potential along the year. Lower values were measured in the dry season. However, this was achieved by a stronger regulation of stomatal conductance and of CO₂ assimilation on an area basis (A _area) in forest trees, particularly toward the end of the dry season. Relative to savanna trees, forest trees maintained similar (P, K, Ca, and Mg) or slightly higher (N) leaf nutrient concentrations. For the majority of traits, more variance was explained by phylogeny, than by habitat of origin, with the exception of SLA, leaf N concentration, and A _area, which were apparently subjected to different selective pressures in the savanna and forest environments. In conclusion, water shortage during extended droughts would be more limiting for forest trees than nutrient-poor soils.     

Acute creatine administration improves mitochondrial membrane potential and protects against pentylenetetrazol-induced seizures

A growing body of evidence indicates that creatine (Cr) exerts beneficial effects on a variety of pathologies where energy metabolism and oxidative stress play an etiological role. However, the benefits of Cr treatment for epileptics are still shrouded in controversy. In the present study, we found that acute Cr treatment (300 mg/kg, p.o.) prevented the increase in electroencephalographic wave amplitude typically elicited by PTZ (30, 45 or 60 mg/kg, i.p.). Cr treatment also increased the latency periods of first myoclonic jerks, lengthened the latency periods of the generalized tonic–clonic seizures and reduced the time spent in the generalized tonic–clonic seizures induced by PTZ (60 mg/kg). Administration of PTZ (all doses) decreased Na⁺, K⁺-ATPase activity as well as adenosine triphosphate (ATP) and adenosine diphosphate levels in the cerebral cortex, but Cr treatment prevented these effects. Cr administration also prevented increases in xanthine oxidase activity, adenosine monophosphate levels, adenosine levels, inosine levels and uric acid levels that normally occur after PTZ treatment (60 mg/kg, i.p.). We also showed that Cr treatment increased the total Cr (Cr + PCr) content, creatine kinase activity and the mitochondrial membrane potential (ΔΨ) in the cerebral cortex. In addition, Cr prevented PTZ-induced mitochondrial dysfunction characterized by decreasing ΔΨ, increasing thiobarbituric acid-reactive substance levels and increasing protein carbonylation. These experimental findings reinforce the idea that mitochondrial dysfunction plays a critical role in models of epileptic seizures and suggest that buffering brain energy levels through Cr treatment may be a promising therapeutic approach for the treatment of this neurological disease.     

The effectiveness of post-contact defenses in a prey with no pre-contact detection

Most empirical and theoretical papers on prey–predator interactions are for animals with long-range detection, animals that can detect and react to predators long before these touch the prey. Heavy-bodied and chemically defended harvestmen (Arachnida, Opiliones) are an exception to this general pattern and rely on contact to detect arthropod predators. We examined the interactions between the Brazilian wandering spider Ctenus ornatus with harvestmen (Mischonyx cuspidatus) or control prey (Gryllus sp. and M. cuspidatus immature, both with soft integuments). Considering a prey–predator system in which fleeing from or reacting to a predator at a distance is not possible, we predicted both a high survival value of near-range defense mechanisms and that mortality would be higher in the absence of such defense mechanisms. We also expected the predator to behave differently when interacting with harvestmen or with a control prey without such defense mechanisms. Our results from laboratory experiments partially matched our predictions: First of all, histological sections showed that the integument of adult harvestmen is thicker than that of immature harvestmen and that of crickets. Adult harvestmen were less preyed upon than the control prey; the heavy armature increases the survival rate but the secretions from the scent glands do not. The predator did behave differently when attacking harvestmen compared to crickets. Despite the large size difference between predator and harvestmen, the protection provided by the armature allowed some of the harvestmen to survive encounters without pre-contact detection, thus greatly reducing the reliance on long-range detection to survive encounters with predators. Harvestmen call for theoretical and empirical work on prey–predator interactions that take into account the possibility that prey may not detect the predator before contact is established.     

Exergy-based accounting for land as a natural resource in life cycle assessment

Purpose

In life cycle assessment (LCA), literature suggests accounting for land as a resource either by what it delivers (e.g., biomass content) or the time and space needed to produce biomass (land occupation), in order to avoid double-counting. This paper proposes and implements a new framework to calculate exergy-based spatial explicit characterization factors (CF) for land as a resource, which deals with both biomass and area occupied on the global scale.

Methods

We created a schematic overview of the Earth, dividing it into two systems (human-made and natural), making it possible to account for what is actually extracted from nature, i.e., the biomass content was set as the elementary flow to be accounted at natural systems and the land occupation (through the potential natural net primary production) was set as the elementary flow at human-made systems. Through exergy, we were able to create CF for land resources for these two different systems. The relevancy of the new CF was tested for a number of biobased products.

Results and discussion

Site-generic CF were created for land as a resource for natural systems providing goods to humans, and site-generic and site-dependent CF (at grid, region, country, and continent level) were created for land as a resource within human-made systems. This framework differed from other methods in the sense of accounting for both land occupation and biomass content but without double-counting. It is set operationally for LCA and able to account for land resources with more completeness, allowing spatial differentiation. When site-dependent CF were considered for land resources, the overall resource consumption of certain products increased up to 77 % in comparison with site-generic CF-based data.

Conclusions

This paper clearly distinguished the origin of the resource (natural or human-made systems), allowing consistent accounting for land as a resource. Site-dependent CF for human-made systems allowed spatial differentiation, which was not considered in other resource accounting life cycle impact assessment methods.     

Evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in Trypanosoma cruzi persistence

Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PP_i) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF‐hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PP_i) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP‐OE) resulted in a significant decrease in cytosolic PP_i, and short and long‐chain polyP levels. Additionally, the TcVSP‐OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PP_i and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection.     

Type-1 (CB1) Cannabinoid Receptor Promotes Neuronal Differentiation and Maturation of Neural Stem Cells

Neural stem cells (NSCs) are self-renewing cells that can differentiate into multiple neural lineages and repopulate regions of the brain after injury. We have investigated the role of endocannabinoids (eCBs), endogenous cues that modulate neuronal functions including neurogenesis, and their receptors CB₁ and CB₂ in mouse NSCs. Real-time PCR and Western blot analyses indicated that CB₁ is present at higher levels than CB₂ in NSCs. The eCB anandamide (AEA) or the CB₁-specific agonist ACEA enhanced NSC differentiation into neurons, but not astrocytes and oligodendrocytes, whereas the CB₂-specific agonist JWH133 was ineffective. Conversely, the effect of AEA was inhibited by CB₁, but not CB₂, antagonist, corroborating the specificity of the response. CB₁ activation also enhanced maturation of neurons, as indicated by morphometric analysis of neurites. CB₁ stimulation caused long-term inhibition of the ERK1/2 pathway. Consistently, pharmacological inhibition of the ERK1/2 pathway recapitulated the effects exerted by CB₁ activation on neuronal differentiation and maturation. Lastly, gene array profiling showed that CB₁ activation augmented the expression of genes involved in neuronal differentiation while decreasing that of stemness genes. These results highlight the role of CB₁ in the regulation of NSC fate and suggest that its activation may represent a pro-neuronal differentiation signal.     

Ocean Acidification Disrupts Prey Responses to Predator Cues but Not Net Prey Shell Growth in Concholepas concholepas (loco)

Background

Most research on Ocean Acidification (OA) has largely focused on the process of calcification and the physiological trade-offs employed by calcifying organisms to support the building of calcium carbonate structures. However, there is growing evidence that OA can also impact upon other key biological processes such as survival, growth and behaviour. On wave-swept rocky shores the ability of gastropods to self-right after dislodgement, and rapidly return to normal orientation, reduces the risk of predation.

Methodology/Principal Findings

The impacts of OA on this self-righting behaviour and other important parameters such as growth, survival, shell dissolution and shell deposition in Concholepas concholepas (loco) were investigated under contrasting pCO₂ levels. Although no impacts of OA on either growth or net shell calcification were found, the results did show that OA can significantly affect self-righting behaviour during the early ontogeny of this species with significantly faster righting times recorded for individuals of C. concholepas reared under increased average pCO₂ concentrations (± SE) (716±12 and 1036±14 µatm CO₂) compared to those reared at concentrations equivalent to those presently found in the surface ocean (388±8 µatm CO₂). When loco were also exposed to the predatory crab Acanthocyclus hassleri, righting times were again increased by exposure to elevated CO₂, although self-righting times were generally twice as fast as those observed in the absence of the crab.

Conclusions and Significance

These results suggest that self-righting in the early ontogeny of C. concholepas will be positively affected by pCO₂ levels expected by the end of the 21st century and beginning of the next one. However, as the rate of self-righting is an adaptive trait evolved to reduce lethal predatory attacks, our result also suggest that OA may disrupt prey responses to predators in nature.