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.     

Small molecules interacting with α-synuclein: antiaggregating and cytoprotective properties

Curcumin, a dietary polyphenol, has shown a potential to act on the symptoms of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases, as a consequence of its antioxidant, anti-inflammatory and anti-protein aggregation properties. Unfortunately, curcumin undergoes rapid degradation at physiological pH into ferulic acid, vanillin and dehydrozingerone, making it an unlikely drug candidate. Here, we evaluated the ability of some curcumin by-products: dehydrozingerone (1), its O-methyl derivative (2), zingerone (3), and their biphenyl analogues (4–6) to interact with α-synuclein (AS), using CD and fluorescence spectroscopy. In addition, the antioxidant properties and the cytoprotective effects in rat pheochromocytoma (PC12) cells prior to intoxication with H₂O₂, MPP+ and MnCl₂ were examined while the Congo red assay was used to evaluate the ability of these compounds to prevent aggregation of AS. We found that the biphenyl zingerone analogue (6) interacts with high affinity with AS and also displays the best antioxidant properties while the biphenyl analogues of dehydrozingerone (4) and of O-methyl-dehydrozingerone (5) are able to partially inhibit the aggregation process of AS, suggesting the potential role of a hydroxylated biphenyl scaffold in the design of AS aggregation inhibitors.     

Dynamic changes in microbiota and mycobiota during spontaneous ‘Vino Santo Trentino’ fermentation

Vino Santo is a sweet wine produced from late harvesting and pressing of Nosiola grapes in a small, well‐defined geographical area in the Italian Alps. We used metagenomics to characterize the dynamics of microbial communities in the products of three wineries, resulting from spontaneous fermentation with almost the same timing and procedure. Comparing fermentation dynamics and grape microbial composition, we show a rapid increase in a small number of wine yeast species, with a parallel decrease in complexity. Despite the application of similar protocols, slight changes in the procedures led to significant differences in the microbiota in the three cases of fermentation: (i) fungal content of the must varied significantly in the different wineries, (ii) Pichia membranifaciens persisted in only one of the wineries, (iii) one fermentation was characterized by the balanced presence of Saccharomyces cerevisiae and Hanseniaspora osmophila during the later phases. We suggest the existence of a highly winery‐specific ‘microbial‐terroir’ contributing significantly to the final product rather than a regional ‘terroir’. Analysis of changes in abundance during fermentation showed evident correlations between different species, suggesting that fermentation is the result of a continuum of interaction between different species and physical–chemical parameters.     

Environmental implications of the use of agro‐industrial residues for biorefineries: application of a deterministic model for indirect land‐use changes

Biorefining agro‐industrial biomass residues for bioenergy production represents an opportunity for both sustainable energy supply and greenhouse gas (GHG) emissions mitigation. Yet, is bioenergy the most sustainable use for these residues? To assess the importance of the alternative use of these residues, a consequential life cycle assessment (LCA) of 32 energy‐focused biorefinery scenarios was performed based on eight selected agro‐industrial residues and four conversion pathways (two involving bioethanol and two biogas). To specifically address indirect land‐use changes (iLUC) induced by the competing feed/food sector, a deterministic iLUC model, addressing global impacts, was developed. A dedicated biochemical model was developed to establish detailed mass, energy, and substance balances for each biomass conversion pathway, as input to the LCA. The results demonstrated that, even for residual biomass, environmental savings from fossil fuel displacement can be completely outbalanced by iLUC, depending on the feed value of the biomass residue. This was the case of industrial residues (e.g. whey and beet molasses) in most of the scenarios assessed. Overall, the GHGs from iLUC impacts were quantified to 4.1 t CO₂‐eq.ha^?1_demanded yr^?1 corresponding to 1.2–1.4 t CO₂‐eq. t^?1 dry biomass diverted from feed to energy market. Only, bioenergy from straw and wild grass was shown to perform better than the alternative use, as no competition with the feed sector was involved. Biogas for heat and power production was the best performing pathway, in a short‐term context. Focusing on transport fuels, bioethanol was generally preferable to biomethane considering conventional biogas upgrading technologies. Based on the results, agro‐industrial residues cannot be considered burden‐free simply because they are a residual biomass and careful accounting of alternative utilization is a prerequisite to assess the sustainability of a given use. In this endeavor, the iLUC factors and biochemical model proposed herein can be used as templates and directly applied to any bioenergy consequential study involving demand for arable land.     

Chlorella vulgaris as a lipid source: Cultivation on air and seawater‐simulating medium in a helicoidal photobioreactor

The freshwater microalga Chlorella vulgaris was cultured batchwise on the seawater‐simulating Schlösser medium either in a 1.1‐L‐working volume helicoidal photobioreactor (HeP) or Erlenmeyer flask (EF) as control and continuously supplying air as CO₂ source. In these systems, maximum biomass concentration reached 1.65 ± 0.17 g L^?1 and 1.25 ± 0.06 g L^?1, and maximum cell productivity 197.6 ± 20.4 mg L^?1 day^?1 and 160.8 ± 12.2 mg L^?1 day^?1, respectively. Compared to the Bold's Basal medium, commonly employed to cultivate this microorganism on a bench‐scale, the Schlösser medium ensured significant increases in all the growth parameters, namely maximum cell concentration (268% in EF and 126% in HeP), maximum biomass productivity (554% in EF and 72% in HeP), average specific growth rate (67% in EF and 42% in HeP), and maximum specific growth rate (233% in EF and 22% in HeP). The lipid fraction of biomass collected at the end of runs was analyzed in terms of both lipid content and fatty acid profile. It was found that the seawater‐simulating medium, despite of a 56–63% reduction of the overall biomass lipid content compared to the Bold's Basal one, led in HeP to significant increases in both the glycerides‐to‐total lipid ratio and polyunsaturated fatty acid content compared to the other conditions taken as an average. These results as a whole suggest that the HeP configuration could be a successful alternative to the present means to cultivate C. vulgaris as a lipid source. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:279–284, 2016     

Urinary proteomics for the study of genetic kidney diseases

Despite their low prevalence, genetic kidney diseases (GKD) still represent a serious health problem. They often lead to kidney failure and to the consequent need of dialysis or kidney transplant. To date, reliable diagnosis requires laborious genetic tests and/or a renal biopsy. Moreover, only scant and non-specific markers exist for prognostic purposes. Biomarkers assayed in an easily available and low-cost sample, such as urine, would be highly valuable. Urinary proteomics can provide clues related to their development through the identification of differentially expressed proteins codified by the affected genes, or other dis-regulated species, in total or fractionated urine, providing novel mechanistic insights. In this review, the authors summarize and discuss the results of the main proteomic investigations on GKD urine samples and in urinary extracellular vesicles.     

A new role for the P2Y-like GPR17 receptor in the modulation of multipotency of oligodendrocyte precursor cells in vitro

Oligodendrocyte precursor cells (OPCs, also called NG2 cells) are scattered throughout brain parenchyma, where they function as a reservoir to replace lost or damaged oligodendrocytes, the myelin-forming cells. The hypothesis that, under some circumstances, OPCs can actually behave as multipotent cells, thus generating astrocytes and neurons as well, has arisen from some in vitro and in vivo evidence, but the molecular pathways controlling this alternative fate of OPCs are not fully understood. Their identification would open new opportunities for neuronal replace strategies, by fostering the intrinsic ability of the brain to regenerate. Here, we show that the anti-epileptic epigenetic modulator valproic acid (VPA) can promote the generation of new neurons from NG2⁺ OPCs under neurogenic protocols in vitro, through their initial de-differentiation to a stem cell-like phenotype that then evolves to “hybrid” cell population, showing OPC morphology but expressing the neuronal marker βIII-tubulin and the GPR17 receptor, a key determinant in driving OPC transition towards myelinating oligodendrocytes. Under these conditions, the pharmacological blockade of the P2Y-like receptor GPR17 by cangrelor, a drug recently approved for human use, partially mimics the effects mediated by VPA thus accelerating cells’ neurogenic conversion. These data show a co-localization between neuronal markers and GPR17 in vitro, and suggest that, besides its involvement in oligodendrogenesis, GPR17 can drive the fate of neural precursor cells by instructing precursors towards the neuronal lineage. Being a membrane receptor, GPR17 represents an ideal “druggable” target to be exploited for innovative regenerative approaches to acute and chronic brain diseases.