Ancient Human Genomes and Environmental DNA from the Cement Attaching 2,000-Year-Old Head Lice Nits.

Over the past few decades, there has been a growing demand for genome analysis of ancient human remains. Destructive sampling is increasingly difficult to obtain for ethical reasons, and standard methods of breaking the skull to access the petrous bone or sampling remaining teeth are often forbidden for curatorial reasons. However, most ancient humans carried head lice and their eggs abound in historical hair specimens. Here we show that host DNA is protected by the cement that glues head lice nits to the hair of ancient Argentinian mummies, 1,500-2,000 years old. The genetic affinities deciphered from genome-wide analyses of this DNA inform that this population migrated from north-west Amazonia to the Andes of central-west Argentina; a result confirmed using the mitochondria of the host lice. The cement preserves ancient environmental DNA of the skin, including the earliest recorded case of Merkel cell polyomavirus. We found that the percentage of human DNA obtained from nit cement equals human DNA obtained from the tooth, yield 2-fold compared with a petrous bone, and 4-fold to a bloodmeal of adult lice a millennium younger. In metric studies of sheaths, the length of the cement negatively correlates with the age of the specimens, whereas hair linear distance between nit and scalp informs about the environmental conditions at the time before death. Ectoparasitic lice sheaths can offer an alternative, nondestructive source of high-quality ancient DNA from a variety of host taxa where bones and teeth are not available and reveal complementary details of their history.

Bog Microtopography and the Climatic Sensitivity of Testate Amoeba Communities: Implications for Transfer Function-Based Paleo-Water Table Reconstructions.

Although the use of sub-fossil testate amoebae as a proxy for raised bog hydrology in Holocene paleoecological studies is well-established, some detailed aspects of species-environment relationships remain under-researched. One such issue is the effect of bog surface microtopography on the climatic sensitivity of testate amoeba communities. Although it has been suggested that some microforms-especially hummocks-may be less sensitive to climatic forcing than others, this has rarely been objectively tested. To investigate this, subfossil testate amoebae assemblages have been examined in a series of shallow cores collected along a hummock-lawn-hollow transect from a bog in central Ireland and the resulting reconstructed water table records, dated using 210Pb, have been compared with instrumental weather data. Testate amoebae communities in the hollow microform were found to be significantly less diverse than those in the hummock and lawn, and both the hummock and lawn showed statistically significant correlations with instrumental temperature and precipitation data. Therefore, whilst the suggestion that paleoecological investigations should target intermediate bog microforms remains sound, the notion that hummock-based testate amoebae hydrological data are climatically-insensitive is challenged.

Interplay of Amino Acid Residues at Positions 28 and 31 in NS5A Defines Resistance Pathways in HCV GT2.

Hepatitis C virus (HCV) genotype (GT) 2 represents approximately 9% of all viral infections globally. While treatment outcomes for GT2-infected patients have improved substantially with direct-acting antiviral agents (DAAs) compared to interferon-α, the presence of polymorphisms in NS5A can impact efficacy of NS5A inhibitor-containing regimens. Thus, pathways of NS5A resistance were explored in GT2 subtypes using elbasvir, an NS5A inhibitor with broad genotype activity. Resistance selection studies, resistance analysis in NS5A-inhibitor treated virologic failures, antiviral activities in replicons bearing a panel of GT2 subtype sequences and amino acid substitutions introduced by site-directed mutagenesis were performed to define determinants of inhibitor susceptibility. Elbasvir showed differential antiviral activity in replicons bearing GT2 sequences. The EC50 values for replicons bearing reference NS5A sequences for GT2a and GT2b were 0.003 and 3.4 nanomolar (nM) respectively. Studies with recombinant replicons demonstrated crosstalk between amino acid positions 28 and 31. The combination of phenylalanine and methionine at positions 28 and 31 respectively, conferred the highest potency reduction for elbasvir in GT2a and GT2b. This combination was observed in failures from the C-SCAPE trial. Addition of grazoprevir, an NS3/4A protease inhibitor, to elbasvir more effectively suppressed the emergence of resistance in GT2 at modest inhibitor concentrations (3X EC90). Ruzasvir, a potent, pan-genotype NS5A inhibitor successfully inhibited replicons bearing GT2 resistance-associated substitutions (RASs) at positions 28 and 31. The studies demonstrate crosstalk between amino acids at positions 28 and 31 in NS5A modulate inhibitor potency and may impact treatment outcomes in some HCV GT2-infected patients.

Unraveling the structural basis of grazoprevir potency against clinically relevant substitutions in hepatitis C virus NS3/4A protease from genotype 1a.

Grazoprevir is a potent pan-genotype and macrocyclic inhibitor of hepatitis C virus (HCV) NS3/4A protease and was developed for treating chronic HCV infection. In HCV genotype (GT) 1a, grazoprevir maintains potent activity against a majority of NS3 resistance-associated amino acid substitutions, including the highly prevalent and naturally occurring Q80K polymorphism that impacts simeprevir, another NS3/4A protease inhibitor. The basis for an unexpected difference in the clinical impact of some NS3 substitutions was investigated. Phenotypic analysis of resistance-associated substitutions identified in NS3 from GT1a-infected patients who failed therapy with grazoprevir (in combination with elbasvir, an inhibitor of HCV NS5A protein) showed that positions 56, 156, and 168 in NS3 were most impactful because they diminished protein-inhibitor interactions. Although an amino acid substitution from aspartic acid to alanine at position 168 (D168A) reduced the potency of grazoprevir, its combination with R155K unexpectedly nullified this effect. Molecular dynamics and free-energy surface studies indicated that Asp-168 is important in anchoring Arg-155 for ligand binding but is not critical for Lys-155 because of the inherent flexibility of its side chain. Moreover, modeling studies supported a strong direct cation-heterocycle interaction between the Lys-155 side chain of the double substitution, R155K/D168A, and the lone pair on the quinoxaline in grazoprevir. This unique interaction provides a structural basis for grazoprevir's higher potency than simeprevir, an inhibitor to which the double substitution confers a significant reduction in potency. Our findings are consistent with the detection of R155K/D168A in NS3 from virologic failures treated with simeprevir but not grazoprevir.

In Vitro Antiviral Profile of Ruzasvir, a Potent and Pangenotype Inhibitor of Hepatitis C Virus NS5A.

Inhibition of NS5A has emerged as an attractive strategy to intervene in hepatitis C virus (HCV) replication. Ruzasvir (formerly MK-8408) was developed as a novel NS5A inhibitor to improve upon the potency and barrier to resistance of early compounds. Ruzasvir inhibited HCV RNA replication with 50% effective concentrations (EC50s) of 1 to 4 pM in Huh7 or Huh7.5 cells bearing replicons for HCV genotype 1 (GT1) to GT7. The antiviral activity was modestly (10-fold) reduced in the presence of 40% normal human serum. The picomolar potency in replicon cells extended to sequences of clinical isolates available in public databases that were synthesized and tested as replicons. In GT1a, ruzasvir inhibited common NS5A resistance-associated substitutions (RASs), with the exception of M28G. De novo resistance selection studies identified pathways with certain amino acid substitutions at residues 28, 30, 31, and 93 across genotypes. Substitutions at position 93 were more common in GT1 to -4, while changes at position 31 emerged frequently in GT5 and -6. With the exception of GT4, the reintroduction of selected RASs conferred a ≥100-fold potency reduction in the antiviral activity of ruzasvir. Common RASs from other classes of direct-acting antiviral agents (DAAs) did not confer cross-resistance to ruzasvir. The interaction of ruzasvir with an NS3/4A protease inhibitor (grazoprevir) and an NS5B polymerase prodrug (uprifosbuvir) was additive to synergistic, with no evidence of antagonism or cytotoxicity. The antiviral profile of ruzasvir supported its further evaluation in human trials in combination with grazoprevir and uprifosbuvir.

Effectiveness of Elbasvir and Grazoprevir Combination, With or Without Ribavirin, for Treatment-Experienced Patients With Chronic Hepatitis C Infection.

BACKGROUND & AIMS: Patients infected with hepatitis C virus (HCV) genotype 1, 4, or 6, with or without cirrhosis, previously treated with peg-interferon and ribavirin, are a challenge to treat. We performed a phase 3 randomized controlled open-label trial to assess the effects of 12 or 16 weeks of treatment with once-daily elbasvir (an HCV NS5A inhibitor, 50 mg) and grazoprevir (an HCV NS3/4A protease inhibitor, 100 mg), in a fixed-dose combination tablet, with or without twice-daily ribavirin, in this patient population. METHODS: We analyzed data from 420 patients (35% with cirrhosis, 64% with a null or partial response to peg-interferon and ribavirin) who were randomly assigned (1:1:1:1) to groups given elbasvir and grazoprevir once daily, with or without twice-daily ribavirin, for 12 or 16 weeks, at 65 study centers in 15 countries in Europe, Asia, and Central and North America. Randomization was stratified by cirrhosis status and type of peg-interferon and ribavirin treatment failure. HCV RNA was measured using COBAS TaqMan v2.0. The primary end point was HCV RNA <15 IU/mL, 12 weeks after completion of treatment (SVR12). We aimed to determine whether the proportion of patients achieving an SVR12 in any group was greater than the reference rate (58%). RESULTS: With 12 weeks of treatment, an SVR12 was achieved by 92.4% of patients given elbasvir and grazoprevir and 94.2% of patients given elbasvir and grazoprevir with ribavirin. With 16 weeks of treatment, an SVR12 was achieved by 92.4% of patients given elbasvir and grazoprevir and 98.1% of patients given elbasvir and grazoprevir with ribavirin. Among patients treated for 12 weeks without ribavirin, virologic failure occurred in 6.8%, 0%, and 12.5% of patients with HCV genotype 1a, 1b, or 4 infection, respectively. Among patients given elbasvir and grazoprevir for 12 weeks, virologic failure occurred in 0% of patients infected with HCV genotypes 1 and 4 who relapsed after completing peg-interferon and ribavirin, and 7.5% infected with HCV genotypes 1 and 4, respectively, with a null or partial response to peg-interferon and ribavirin. Among patients treated for 16 weeks who received ribavirin, there were no incidences of virologic failure. Common adverse events were fatigue (23.1%), headache (19.8%), and nausea (11.0%). CONCLUSIONS: The combination tablet of elbasvir and grazoprevir, with or without ribavirin, was highly efficacious in inducing an SVR12 in patients with HCV genotype 1, 4, or 6 infection failed by previous treatment with peg-interferon and ribavirin, including patients with cirrhosis and/or a prior null response. The treatment was generally well tolerated. ClinicalTrials.gov Number: NCT02105701.

Discovery of 3(S)-thiomethyl pyrrolidine ERK inhibitors for oncology.

Compound 5 (SCH772984) was identified as a potent inhibitor of ERK1/2 with excellent selectivity against a panel of kinases (0/231 kinases tested @ 100 nM) and good cell proliferation activity, but suffered from poor PK (rat AUC PK @10 mpk = 0 µM h; F% = 0) which precluded further development. In an effort to identify novel ERK inhibitors with improved PK properties with respect to 5, a systematic exploration of sterics and composition at the 3-position of the pyrrolidine led to the discovery of a novel 3(S)-thiomethyl pyrrolidine analog 28 with vastly improved PK (rat AUC PK @10 mpk = 26 µM h; F% = 70).

MBTH: A novel approach to rapid, spectrophotometric quantitation of total algal carbohydrates.

A high-throughput and robust application of the 3-methyl-2-benzothiazolinone hydrazone (MBTH) method was developed for carbohydrate determination in microalgae. The traditional phenol-sulfuric acid method to quantify carbohydrates is strongly affected by algal biochemical components and exhibits a highly variable response to microalgal monosaccharides. We present a novel use of the MBTH method to accurately quantify carbohydrates in hydrolyzate after acid hydrolysis of algal biomass, without a need for neutralization. The MBTH method demonstrated consistent and sensitive quantitation of algae-specific monosaccharides down to 5 µg mL-1 without interference from other algae acidic hydrolyzate components.

Accuracy and Efficiency of Recording Pediatric Early Warning Scores Using an Electronic Physiological Surveillance System Compared With Traditional Paper-Based Documentation.

Pediatric Early Warning Scores are advocated to assist health professionals to identify early signs of serious illness or deterioration in hospitalized children. Scores are derived from the weighting applied to recorded vital signs and clinical observations reflecting deviation from a predetermined "norm." Higher aggregate scores trigger an escalation in care aimed at preventing critical deterioration. Process errors made while recording these data, including plotting or calculation errors, have the potential to impede the reliability of the score. To test this hypothesis, we conducted a controlled study of documentation using five clinical vignettes. We measured the accuracy of vital sign recording, score calculation, and time taken to complete documentation using a handheld electronic physiological surveillance system, VitalPAC Pediatric, compared with traditional paper-based charts. We explored the user acceptability of both methods using a Web-based survey. Twenty-three staff participated in the controlled study. The electronic physiological surveillance system improved the accuracy of vital sign recording, 98.5% versus 85.6%, P < .02, Pediatric Early Warning Score calculation, 94.6% versus 55.7%, P < .02, and saved time, 68 versus 98 seconds, compared with paper-based documentation, P < .002. Twenty-nine staff completed the Web-based survey. They perceived that the electronic physiological surveillance system offered safety benefits by reducing human error while providing instant visibility of recorded data to the entire clinical team.

Susceptibilities of genotype 1a, 1b, and 3 hepatitis C virus variants to the NS5A inhibitor elbasvir.

Elbasvir is an investigational NS5A inhibitor with in vitro activity against multiple HCV genotypes. Antiviral activity of elbasvir was measured in replicons derived from wild-type or resistant variants of genotypes 1a, 1b, and 3. The barrier to resistance was assessed by the number of resistant colonies selected by exposure to various elbasvir concentrations. In a phase 1b dose-escalating study, virologic responses were determined in 48 noncirrhotic adult men with chronic genotype 1 or 3 infections randomized to placebo or elbasvir from 5 to 50 mg (genotype 1) or 10 to 100 mg (genotype 3) once daily for 5 days. The NS5A gene was sequenced from plasma specimens obtained before, during, and after treatment. Elbasvir suppressed the emergence of resistance-associated variants (RAVs) in vitro in a dose-dependent manner. Variants selected by exposure to high elbasvir concentrations typically encoded multiple amino acid substitutions (most commonly involving loci 30, 31, and 93), conferring high-level elbasvir resistance. In the monotherapy study, patients with genotype 1b had greater reductions in HCV RNA levels than patients with genotype 1a at all elbasvir doses; responses in patients with genotype 3 were generally less pronounced than for genotype 1, particularly at lower elbasvir doses. M28T, Q30R, L31V, and Y93H in genotype 1a, L31V and Y93H in genotype 1b, and A30K, L31F, and Y93H in genotype 3 were the predominant RAVs selected by elbasvir monotherapy. Virologic findings in patients were consistent with the preclinical observations. NS5A-RAVs emerged most often at amino acid positions 28, 30, 31, and 93 in both the laboratory and clinical trial. (The MK-8742 P002 trial has been registered at ClinicalTrials.gov under identifier NCT01532973.).

Virologic resistance analysis from a phase 2 study of MK-5172 combined with pegylated interferon/ribavirin in treatment-naive patients with hepatitis C virus genotype 1 infection.

BACKGROUND: Virologic failure following treatment of hepatitis C virus (HCV) genotype 1 with direct-acting antiviral agents is often accompanied by the emergence of resistant variants. MK-5172 is an investigational once-daily protease inhibitor. We analyzed variants in treatment-naive noncirrhotic patients with virologic failure on MK-5172 (100-800 mg/day) plus pegylated interferon alfa/ribavirin (peg-IFN/RBV) during a phase 2 trial. METHODS: Population and selective clonal sequencing were performed at baseline and at virologic failure in the 4 MK-5172 dosing arms. MK-5172 activity was determined using a mutant replicon assay. RESULTS: Six of 266 (2.3%) MK-5172 recipients satisfied prespecified criteria for virologic failure, all with genotype 1a infection. Five patients with virologic failure were in the MK-5172 100-mg arm, including 4 patients with low plasma MK-5172 levels documented during triple therapy. Variants associated with >4-fold loss of potency were detected in 3 of the 4 patients with genotype 1a breakthrough while on MK-5172. The fifth patient had undetectable HCV-RNA levels at the end of triple therapy but subsequently broke through during the peg-IFN/RBV tail 16 weeks after completion of MK-5172. Three patients had D168 variants at virologic failure, including 2 with the D168A variant associated with a 95-fold loss of potency. The sole apparent relapse was actually a genotype 3a reinfection in the MK-5172 200-mg group. CONCLUSIONS: Virologic failure occurred uncommonly (6/266 [2.3%]) in MK-5172/peg-IFN/RBV recipients. The most prevalent treatment-emergent variants were detected at the D168 locus. D168A variants conferring approximately 2-log reduction in MK-5172 susceptibility emerged in 2 of the 4 evaluable patients with genotype 1a breakthrough. Clinical Trials Registration. NCT01353911.

Plastic ingestion and trace element contamination of Manx shearwaters Puffinus puffinus on the Faroe Islands.

Procellariiform seabirds can accumulate high levels of plastic in their gastrointestinal tracts, which can cause physical damage and potentially provides a contamination route for trace elements. We examined plastic ingestion and trace element contamination of fledgling Manx shearwaters Puffinus puffinus that were harvested for human consumption in 2003 and 2018 on Skúvoy, Faroe Islands (North Atlantic Ocean). Overall, 88% of fledglings contained plastic in their gastrointestinal tracts, with a mean (± SD) of 7.2 ± 6.6 items weighing 0.007 ± 0.016 g. Though the incidence was similar, fledglings ingested significantly more plastic in 2018 compared to 2003. Hepatic trace element concentrations were unrelated to plastic ingestion. Hepatic carbon (δ13C) and nitrogen (δ15N) stable isotope values were significantly lower in birds sampled in 2018 versus 2003, potentially reflecting further offshore feeding at lower trophic levels. Future research is needed to understand the extent of plastic ingestion by Faroe Islands seabirds.

New method for discovery of starch phenotypes in growing microalgal colonies.

To identify algal strains with altered starch metabolism from a large pool of candidates of growing algal colonies, we have developed a novel, high-throughput screening tool by combining gaseous bleaching of replica transferred colonies and subsequent iodine staining to visualize starch. Screening of healthy growing colonies of microalgae has not been possible previously because high levels of chlorophyll make the detection of starch with an iodine stain impossible. We demonstrated that chlorine dioxide (ClO(2)) removes essentially all chlorophyll from the colonies and enables high-throughput screening of, for example, a population of mutagenized cells or a culture collection isolated in a bioprospecting project.

Centenarian lifespans of three freshwater fish species in Arizona reveal the exceptional longevity of the buffalofishes (Ictiobus).

During the 1910s three buffalofish species (Catostomidae: Ictiobus cyprinellus, I. bubalus, I. niger) were reared in ponds along the Mississippi River. Individuals of these buffalofishes were transported to locations across the United States to support or establish commercial fisheries, including Roosevelt Lake, Arizona in 1918. During the 1930s-1960s a commercial fishery existed on Roosevelt Lake, ending by 1970. Scarce information exists on Arizona buffalofishes since. From 2018 to 2023 we studied buffalofishes from nearby Apache Lake (adjacent and downstream of Roosevelt Lake) in collaboration with anglers. Here we show that > 90% of buffalofishes captured from Apache Lake are more than 80 years old and that some of the original buffalofishes from the Arizona stocking in 1918 are likely still alive. Using unique markings on old-age buffalofishes, we demonstrate how individuals are identified and inform dozens of recaptures. We now know all species of USA Ictiobus can live more than 100 years, making it the only genus of animal besides marine rockfishes (Sebastes) for which three or more species have been shown to live > 100 years. Our citizen-science collaboration has revealed remarkable longevity for freshwater fishes and has fundamentally redefined our understanding of the genus Ictiobus itself.

High-performance liquid chromatography/high-resolution multiple stage tandem mass spectrometry using negative-ion-mode hydroxide-doped electrospray ionization for the characterization of lignin degradation products.

In the search for a replacement for fossil fuel and the valuable chemicals currently obtained from crude oil, lignocellulosic biomass has become a promising candidate as an alternative biorenewable source for crude oil. Hence, many research efforts focus on the extraction, degradation, and catalytic transformation of lignin, hemicellulose, and cellulose. Unfortunately, these processes result in the production of very complex mixtures. Further, while methods have been developed for the analysis of mixtures of oligosaccharides, this is not true for the complex mixtures generated upon degradation of lignin. For example, high-performance liquid chromatography/multiple stage tandem mass spectrometry (HPLC/MS(n)), a tool proven to be invaluable in the analysis of complex mixtures derived from many other biopolymers, such as proteins and DNA, has not been implemented for lignin degradation products. In this study, we have developed an HPLC separation method for lignin degradation products that is amenable to negative-ion-mode electrospray ionization (ESI doped with NaOH), the best method identified thus far for ionization of lignin-related model compounds without fragmentation. The separated and ionized compounds are then analyzed by MS(3) experiments to obtain detailed structural information while simultaneously performing high-resolution measurements to determine their elemental compositions in the two parts of a commercial linear quadrupole ion trap/Fourier-transform ion cyclotron resonance mass spectrometer. A lignin degradation product mixture was analyzed using this method, and molecular structures were proposed for some components. This methodology significantly improves the ability to analyze complex product mixtures that result from degraded lignin.

Corrigendum: A Two-Person Neuroscience Approach for Social Anxiety: A Paradigm With Interbrain Synchrony and Neurofeedback.

[This corrects the article DOI: 10.3389/fpsyg.2021.568921.].

A mechanistic understanding of polyethylene biodegradation by the marine bacterium Alcanivorax.

Polyethylene (PE) is one of the most recalcitrant carbon-based synthetic materials produced and, currently, the most ubiquitous plastic pollutant found in nature. Over time, combined abiotic and biotic processes are thought to eventually breakdown PE. Despite limited evidence of biological PE degradation and speculation that hydrocarbon-degrading bacteria found within the plastisphere is an indication of biodegradation, there is no clear mechanistic understanding of the process. Here, using high-throughput proteomics, we investigated the molecular processes that take place in the hydrocarbon-degrading marine bacterium Alcanivorax sp. 24 when grown in the presence of low density PE (LDPE). As well as efficiently utilising and assimilating the leachate of weathered LDPE, the bacterium was able to reduce the molecular weight distribution (Mw from 122 to 83 kg/mol) and overall mass of pristine LDPE films (0.9 % after 34 days of incubation). Most interestingly, Alcanivorax acquired the isotopic signature of the pristine plastic and induced an extensive array of metabolic pathways for aliphatic compound degradation. Presumably, the primary biodegradation of LDPE by Alcanivorax sp. 24 is possible via the production of extracellular reactive oxygen species as observed both by the material's surface oxidation and the measurement of superoxide in the culture with LDPE. Our findings confirm that hydrocarbon-biodegrading bacteria within the plastisphere may in fact have a role in degrading PE.

A Two-Person Neuroscience Approach for Social Anxiety: A Paradigm With Interbrain Synchrony and Neurofeedback.

Social anxiety disorder has been widely recognised as one of the most commonly diagnosed mental disorders. Individuals with social anxiety disorder experience difficulties during social interactions that are essential in the regular functioning of daily routines; perpetually motivating research into the aetiology, maintenance and treatment methods. Traditionally, social and clinical neuroscience studies incorporated protocols testing one participant at a time. However, it has been recently suggested that such protocols are unable to directly assess social interaction performance, which can be revealed by testing multiple individuals simultaneously. The principle of two-person neuroscience highlights the interpersonal aspect of social interactions that observes behaviour and brain activity from both (or all) constituents of the interaction, rather than analysing on an individual level or an individual observation of a social situation. Therefore, two-person neuroscience could be a promising direction for assessment and intervention of the social anxiety disorder. In this paper, we propose a novel paradigm which integrates two-person neuroscience in a neurofeedback protocol. Neurofeedback and interbrain synchrony, a branch of two-person neuroscience, are discussed in their own capacities for their relationship with social anxiety disorder and relevance to the paradigm. The newly proposed paradigm sets out to assess the social interaction performance using interbrain synchrony between interacting individuals, and to employ a multi-user neurofeedback protocol for intervention of the social anxiety.

Accelerated aging of fast pyrolysis bio-oil: a new method based on carbonyl titration.

Fast pyrolysis bio-oils are known to age upon storage at room temperature, resulting in changes to both physical properties (increase in viscosity) and chemical composition (decrease in carbonyl content). A widely used accelerated aging test consists of holding samples at 80 °C for 24 hours, with viscosity measurement before and after heat treatment. Unfortunately, the viscosity measurement has high variability, and cannot be applied to samples that have phase separated. Here, we show that carbonyl content is a much better metric for tracking bio-oil aging. Furthermore, results from different accelerated aging protocols (for varying times at both 40 °C and 80 °C) are compared to actual room temperature storage for over 3 years. Based on this, we show that the accepted accelerated aging test (80 °C for 24 hours) is too severe a treatment, and results in more extensive aging than would occur with over 3 years of storage at room temperature. A new aging protocol is proposed: heat treatment at 80 °C for 2 hours, with carbonyl quantification before and after. This protocol correlates to room temperature storage for 1-3 months. Finally, samples were also kept in cold storage (at both 9 °C and -17 °C) for over 3 years. Unexpectedly, these samples also showed a substantial reduction in carbonyl content (by up to 25%), indicating that bio-oil aging still progresses at low temperatures. Both physical and chemical changes will occur in samples in cold storage, which has implications for the archiving of bio-oil samples.

Evidence for the onset of mining activities during the 13th century in Poland using lead isotopes from lake sediment cores.

Efforts to study how human activities have influenced the environment since the end of the Roman period to present day are lacking for North Central Europe. Here, we present new lead (Pb) isotope data determined from two sediment cores collected from ancient lakes spanning the last 1500 years, located in the Kuyavian-Pomeranian Voivodeship, Poland. Study sites at Radzyn Chelminski and Rywald were used to differentiate Pb sources. Radzyn Chelminski is located in the vicinity of a late Medieval Teutonic Order castle and town, while Rywald is situated within a relatively pristine area until the 19th century when it became used for agricultural purpose. Core samples were analyzed for Pb concentration and isotopes (206Pb, 207Pb, 208Pb). Bayesian modeling was used to isolate the anthropogenic signal at each site over time. For both sites, Pb enrichment factors relative to titanium (Ti) and upper continental crust values range from 13 to 159. Lead isotopic ratios range from background, pre-anthropogenic local values (206Pb/207Pb = 1.31 ±â€¯0.03‰, 208Pb/206Pb = 1.97 ±â€¯0.04‰) to anthropogenic values (SW Poland coal, ore, slag 206Pb/207Pb = 1.17 ±â€¯0.01‰, 208Pb/206Pb = 2.09 ±â€¯0.01‰). Modeled anthropogenic contribution varies greatly over time, ranging from 14 to 100%. At Radzyn Chelminski, modeled anthropogenic Pb contribution and measured Pb concentration follow similar trends. However, at Rywald, from around A.D.1000 to 1400 CE these profiles diverge significantly. Our new insights highlight different sources of Pb from the 12th century to present day: (1) short range agricultural activities from the town, and (2) long range mining activities. Additionally, prior to the 12th century, our data suggest continental anthropogenic activity possibly favored by a warmer climate.