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Shotgun sequencing is a DNA analysis method that potentially determines the nucleotide sequence of every DNA fragment in a sample, unlike PCR-based genotyping methods that is widely used in forensic genetics and targets predefined short tandem repeats (STRs) or predefined single nucleotide polymorphisms (SNPs). Shotgun DNA sequencing is particularly useful for highly degraded low-quality DNA samples, such as ancient samples or those from crime scenes. Here, we developed a statistical model for human identification using shotgun sequencing data and developed formulas for calculating the evidential weight as a likelihood ratio (L R). The model uses a dynamic set of binary SNP loci and takes the error rate from shotgun sequencing into consideration in a probabilistic manner. To our knowledge, the method is the first to make this possible. Results from replicated shotgun sequencing of buccal swabs (high-quality samples) and hair samples (low-quality samples) were arranged in a genotype-call confusion matrix to estimate the calling error probability by maximum likelihood and Bayesian inference. Different genotype quality filters may be applied to account for genotyping errors. An error probability of zero resulted in the commonly used L R formula for the weight of evidence. Error probabilities above zero reduced the L R contribution of matching genotypes and increased the L R in the case of a mismatch between the genotypes of the trace and the person of interest. In the latter scenario, the L R increased from zero (occurring when the error probability was zero) to low positive values, which allow for the possibility that the mismatch may be due to genotyping errors. We developed an open-source R package, wgsLR, which implements the method, including estimation of the calling error probability and calculation of L R values. The R package includes all formulas used in this paper and the functionalities to generate the formulas. • Evidential weight as a likelihood ratio (L R) is given for shotgun sequencing data. • The L R accounts for the genotype calling error probability that we can estimate. • The method uses binary SNP loci chosen dynamically based on the trace sample. • We provide an open-source R package, wgsLR, which implements the method. [ABSTRACT FROM AUTHOR]

• Tolerance to humans in Eurasian red squirrels was examined in three European countries and Japan. • Tolerance varied between four different countries. • Squirrels were less tolerant to humans when the trees and underbrush were taller in sites in the UK. • Squirrels were more tolerant when there was artificial feeding in Japan, while site-scale characteristics were not associated with tolerance in Germany and Finland. • Differences in human-squirrel interaction and conservation status among countries may create variations of tolerance in wildlife. As urbanisation increases, wildlife is more likely to be exposed to humans. Although human disturbance is the main cause of biodiversity loss, some wildlife thrives in anthropogenic environments. Such species show increased behavioural tolerance to humans, which plays an important role in human-wildlife coexistence. However, whether wildlife modulates tolerance differently between regions and cities is inadequately understood. Understanding how animals behaviourally modulate their tolerance to humans at the larger geographical scale can provide useful information to predict behavioural adaptations to urbanisation and adequate management actions for conservation. We examined alert distance (AD), flight initiation distance (FID), and vertical escape distance (VED) in Eurasian red squirrels (Sciurus vulgaris) in four countries (Finland, Germany, Japan, and the UK). Linear mixed models with Tukey's multicomparisons showed that all tolerance measures varied between countries. We subsequently analysed the relationship between site-scale environmental variables (vegetation conditions, presence of artificial feeding, and human population density) and tolerance behaviours separately by country. We found that squirrels were more tolerant in sites with taller trees and more underbrush cover in the UK, and more tolerant in sites with artificial feeding in Japan, while environmental variables were not associated with squirrels' tolerance in Finland and Germany. Our results indicated that regional forms of environmental factors and human-squirrel interactions play a key role in tolerance modification at the larger spatial scale. Our study suggests the importance of considering regional-dependent relationships between tolerance behaviours and environmental characteristics for urban wildlife conservation and management. [ABSTRACT FROM AUTHOR]

Biological trace samples consisting of very few cells pose a challenge to conventional forensic genetic DNA analysis. RNA may be an alternative to DNA when handling low template samples. Whereas each cell only contains two copies of an autosomal DNA segment, the transcriptome retains much of the genomic variation replicated in abundant RNA fragments. In this study, we describe the development of a prototype RNA-based SNP selection set for forensic human identification from low template samples (50 pg gDNA). Whole blood from a subset of the Danish population (41 individuals) and blood stains subjected to degradation at room temperature for up to two weeks were analysed by whole transcriptome shotgun sequencing. Concordance was determined by DNA genotyping with the Infinium Omni5–4 SNP chip. In the 100 protein-coding genes with the most reads, 5214 bi-allelic SNPs with gnomAD minor allele frequencies > 0.1 in the African/African American, East Asian, and (non-Finnish) European populations were identified. Of these, 24 SNPs in 21 genes passed screening in whole blood and degraded blood stains, with a resulting mean match probability of 4.5 ∙ 10−9. Additionally, ancestry informative SNPs and SNPs in genes useful for body fluid identification were identified in the transcriptome. Consequently, shotgun sequencing of RNA from low template samples may be used for a vast host of forensic genetics purposes, including simultaneous human and body fluid identification, leading to direct donor identification in the identified body fluid. • A prototype RNA-based SNP selection set for human identification was developed. • The HID SNP loci were evaluated in both whole blood and degraded blood stains. • The SNP selection set yielded a high discriminatory power. • SNPs in body-fluid-specific mRNAs directly link body fluid and donor information. • RNA-based SNP profiling may be used for ancestry inference. [ABSTRACT FROM AUTHOR]

The performance of Verogen's ForenSeq mtDNA Whole Genome Kit and its possible application in a forensic setting was evaluated. We carried out three sequencing runs that included 1) a dilution series of two different samples (200–6.25 pg input nuclear DNA), 2) casework samples, and 3) hair, blood, and buccal samples from the same individual. For samples described in 1), the method was tested using different reagent volume in the PCR and library building (recommended volume or half-volume). Reference samples were analysed in duplicates, and positive and negative controls were included in every run. All mtDNA profiles were known from previous genotyping with the Precision ID mtDNA Whole Genome Panel or using long range PCR and the MiSeq instrument. The average read depth per sample was > 1200x in all samples, although some regions had read depth 50x. Using half volume of reagents did not compromise the quality of the results and can be a cost-effective alternative for reference samples. For an analytical threshold of 10%, complete concordance was observed between the duplicates and previous results. Heteroplasmy was consistent with prior observations although frequencies varied up to 10% compared to previous results. [ABSTRACT FROM AUTHOR]

In this pilot study, we made dilutions of DNA/RNA co-extractions (1000–12.5 pg gDNA) and whole transcriptome shotgun sequenced RNA on a NovaSeq 6000 platform (Illumina). Whole blood samples from two individuals were sequenced in triplicate for each dilution. More than 450,000 RNA variants were identified to have overlapping positions with the gnomAD 3.0 DNA variant database in the 50 genes with most reads. Between 7000 and 45,000 of these loci were covered with at least 70 reads in the various dilutions. A total of 171 SNPs with global minor allele frequencies > 0.1 were identified in all dilutions with RNA input equivalent to 12.5 pg gDNA. Of these, 56 were genotyped with the Infinium Omni5–4 SNP-chip (Illumina) using DNA from the two individuals and 53 SNP genotypes in 22 autosomal genes were confirmed between RNA and DNA. The mean match probability based on the most polymorphic SNP in the 22 genes was 5 × 10−8. Thus, shotgun RNA sequencing of low template samples may provide both human identification and tissue identification. [ABSTRACT FROM AUTHOR]

Samples collected for forensic case work may be of varying quality and quantity. The sample DNA is often quantified prior to short tandem repeats (STR) profile analysis with methods such as Quantifiler™Trio (QFT). The QFT measures the quantity of DNA as well as an internal PCR control (IPC) and a degradation index (DI). The aim of this study was to use IPC and DI measurements to identify samples, which would benefit from a modified PCR amplification set-up when generating the STR profiles. The sample quality of 6287 single source case work samples were categorized as 'Good', 'Partly degraded', 'Highly degraded', 'Inhibited' and 'Degraded and Inhibited' based on the peak height ratios in the electropherogram data. The DI and IPC were correlated with the assigned quality categories of the samples. Samples categorized into the degraded and/or inhibited categories were found to have statistically significantly different DI and IPC compared to samples categorized as 'Good'. This indicates that the additional information gained from the QFT can be useful to identify degraded and/or inhibited samples prior to the STR-profile analysis. Future work will re-evaluate the criteria of inclusion in the sample quality groups and implement multi source samples. [ABSTRACT FROM AUTHOR]

The placenta sheds extracellular vesicles (EVs), including exosomes, into the maternal circulation. We recently demonstrated that this trafficking of EVs is bi-directional; with uptake of macrophage exosomes by the placenta inducing cytokine release. The specificity of this response is currently unknown. THP-1 cells were cultured as monocytes or differentiated to macrophages, and EVs isolated by ultra-centrifugation. The effect of EVs on human placental explants was measured by cytokine ELISA/luminex. Macrophage, but not monocyte, EVs induce the release of pro-inflammatory cytokines by the placenta. Thus, placental responses to immune cell EVs, including exosomes, reflects the phenotype of the source cell. [ABSTRACT FROM AUTHOR]

In a previous study we presented an assay for targeted mRNA sequencing for the identification of human body fluids, optimised for the Illumina MiSeq/FGx MPS platform. This assay, together with an additional in-house designed assay for the Ion Torrent PGM/S5 platform, was the basis for a collaborative exercise within 17 EUROFORGEN and EDNAP laboratories, in order to test the efficacy of targeted mRNA sequencing to identify body fluids. The task was to analyse the supplied dried body fluid stains and, optionally, participants’ own bona fide or mock casework samples of human origin, according to specified protocols. The provided primer pools for the Illumina MiSeq/FGx and the Ion Torrent PGM/S5 platforms included 33 and 29 body fluid specific targets, respectively, to identify blood, saliva, semen, vaginal secretion, menstrual blood and skin. The results demonstrated moderate to high count values in the body fluid or tissue of interest with little to no counts in non-target body fluids. There was some inter-laboratory variability in read counts, but overall the results of the laboratories were comparable in that highly expressed markers showed high read counts and less expressed markers showed lower counts. We performed a partial least squares (PLS) analysis on the data, where blood, menstrual blood, saliva and semen markers and samples clustered well. The results of this collaborative mRNA massively parallel sequencing (MPS) exercise support targeted mRNA sequencing as a reliable body fluid identification method that could be added to the repertoire of forensic MPS panels. [ABSTRACT FROM AUTHOR]

Background Pectus excavatum is classified using the Haller Index (HI) or the Correction Index. However, no correlation between the HI and CI and cardiopulmonary impairment has been described in detail. Methods This prospective cohort study included 99 otherwise healthy patients with pectus excavatum who underwent cardiopulmonary exercise testing and magnetic resonance imaging at inspiration and expiration to correlate cardiopulmonary function with the grade of thoracic dysmorphia. Results Probands with an HI exceeding 3.25 had first an increase in heart rate at anaerobic threshold (from 148.0 ± 16.0 beats/min to 155.9 ± 15.0 beats/min, p = 0.036), with an HI of more than 3.6 a reduction in oxygen pulse at anaerobic threshold (from 10.7 ± 2.6 mL/beat to 9.3 ± 2.9 mL/beat, p = 0.017), with an HI exceeding 3.8 a reduction of maximum oxygen pulse (from 13.9 ± 3.4 mL/beat to 11.9 ± 3.7 mL/beat, p = 0.010), and with an HI of exceeding 4.0 a decline in maximum oxygen uptake (from 43.7 ± 6.5 mL · kg –1 · min –1 to 40.4 ± 7.4 mL · kg –1 · min –1 , p = 0.025). The CI of more 27% reflects cardiopulmonary changes earlier than the corresponding HI exceeding 3.25 ( p = 0.01 for maximum oxygen pulse; p = 0.017 for oxygen pulse at anaerobic threshold; p = 0.015 for heart rate at anaerobic threshold). Conclusions The inspiratory HI and CI reflect the effect of pectus excavatum on cardiopulmonary function. The cardiopulmonary system reacts first with an increase in heart rate at anaerobic threshold, followed by a decrease in stroke volume at anaerobic threshold and maximum stroke volume. Increased severity of the deformity then leads to a decrease in cardiac output. [ABSTRACT FROM AUTHOR]

Sodium hypochlorite is an efficient reagent for removal of unwanted DNA from laboratory surfaces. Here, we tested two different chlorine wipes and compared their performance to a 0.9–1.8% hypochlorite solution. WipeClean Chlorine Disinfection wipes contain > 0.1 g sodium hypochlorite/kg, whereas WetWipe Chlorine Desinfection wipes contain > 1000 ppm active chlorine. Clean surfaces were contaminated with 10 µL 0.5 ng/µL of massively parallel sequencing libraries. The DNA was dried and left for 45 min before any treatment. The surfaces were cleaned using either 1) a 0.9–1.8% hypochlorite solution and clean wipes, 2) a WipeClean wipe, 3) a WetWipe, or 4) the surface was not cleaned. All experiments were repeated three times. Subsequently, the surfaces were swabbed using cotton swabs. DNA was extracted from the swabs and the DNA concentrations were determined in quadruplicates by real-time PCR. This protocol was repeated after the soft plastic wrapping around the wipes were left open or closed for several weeks. The results showed that the WipeClean wipes efficiently removed DNA for up to four weeks after the box with the wipes were opened, whereas the WetWipe wipes dried faster and gradually lost their cleaning effect. [ABSTRACT FROM AUTHOR]

The Precision ID Identity Panel was used to type 109 Somali individuals in order to obtain allele frequencies for the Somali population. These frequencies were used to establish a Somali HID-SNP database, which will be used for the biostatistic calculations in family and immigration cases. Genotypes obtained with the Precision ID Identity Panel were found to be almost in complete concordance with genotypes obtained with the SNP for ID PCR-SBE-CE assay. In seven SNP loci, silent alleles were identified, of which most were previously described in the literature. The project also set out to compare different AmpliSeq™ workflows to investigate the possibility of using automated library building in forensic genetic case work. In order to do so, the SNP typing of the Somalis was performed using three different workflows: 1) manual library building and sequencing on the Ion PGM™, 2) automated library building using the Biomek ® 3000 and sequencing on the Ion PGM™, and 3) automated library building using the Ion Chef™ and sequencing on the Ion S5™. AmpliSeq™ workflows were compared based on coverage, locus balance, noise, and heterozygote balance. Overall, the Ion Chef™/Ion S5™ workflow was found to give the best results and required least hands-on time in the laboratory. However, the Ion Chef™/Ion S5™ workflow was also the most expensive. The number of libraries that may be constructed in one Ion Chef™ library building run was limited to eight, which is too little for high throughput workflows. The Biomek ® 3000/Ion PGM™ workflow was found to perform similarly to the manual/Ion PGM™ workflow. This argues for the use of automated library building in forensic genetic case work. Automated library building decreases the workload of the laboratory staff, decreases the risk of pipetting errors, and simplifies the daily workflow in forensic genetic laboratories. [ABSTRACT FROM AUTHOR]

The urban age in China offers good opportunities for progress and innovation in the country, and also poses serious challenges. The urban expansion to the surrounding rural land creates multiple challenges and conflicts for urban governance. The paper will explore approaches and potential solutions for these challenges, including governance innovations and tools for transformation. It presents the lessons learnt from a number of projects, which were opening new potentials by fostering transformative governance, fruitful inclusive consultations at local, regional and national level, and learning from multi-stakeholder dialogues to achieve sustainable urbanisation. In taking the urban age with inclusive procedures and solutions forward, we conclude that China's valuable experiences in urbanisation efforts should be shared with other actors at urban, national and international levels. [ABSTRACT FROM AUTHOR]

Background: Diagnosis of tuberculosis (TB) remains difficult, particularly in resource-limited settings. The development of nucleic acid-based tests for detection of Mycobacterium tuberculosis complex (MTBC) has significantly increased sensitivity compared to conventional smear microscopy and provides results within a matter of hours compared to weeks for the current gold-standard, liquid culture.Methods: In this study we performed side-by-side comparison of mycobacterial detection assays on sputum samples from 285 subjects presenting with symptoms suggestive of TB in The Gambia and a cross-sectional cohort of 156 confirmed TB patients with a median of 2 months of treatment. A novel assay, Loop-Mediated Amplification test for TB (TB-LAMP), was compared to smear microscopy, MGIT culture and GeneXpert MTB/RIF for all samples.Results: When culture was used as the reference standard, we found an overall sensitivity for TB-LAMP of 99% (95% CI: 94.5-99.8) and specificity of 94% (95% CI: 89.3-96.7). When latent class analysis was performed, TB-LAMP had 98.6% (95% CI: 95.9-100) sensitivity and 99% (95% CI: 98.2-100) specificity compared to 91.1% (95% CI: 86.1-96) sensitivity and 100% (95% CI: 98.2-100) specificity for MGIT culture. GeneXpert had the highest sensitivity 99.1% (95% CI: 97.1-100) but the lowest specificity 96% (95% CI: 92.6-98.3). Both TB-LAMP and GeneXpert showed high sensitivity and specificity regardless of age or strain of infection.Conclusion: Our findings show the diagnostic utility of both GeneXpert and TB-LAMP in The Gambia. Whilst TB-LAMP requires less infrastructure, it is unable to detect drug-resistant patterns and therefore would be most suitable as a screening test for new TB cases in peripheral health clinics. [ABSTRACT FROM AUTHOR]

Objectives: To determine risk factors for GBS colonisation in Gambian mothers and in their infants from birth to day 60-89 of age.Methods: Swabs and breastmilk from mothers/infant pairs were collected and cultured on selective agar. Negative samples were analysed for GBS DNA via real-time PCR. Positive isolates were serotyped using multiplex PCR and gel-agarose electrophoresis.Results: Seven hundred and fifty women/infant pairs were recruited. 253 women (33.7%) were GBS-colonised at delivery. The predominant serotypes were: V (55%), II (16%), III (10%), Ia (8%) and Ib (8%). 186 infants were colonised (24.8%) at birth, 181 (24.1%) at 6 days and 96 at day 60-89 (14%). Infants born before 34 weeks of gestation and to women with rectovaginal and breastmilk colonisation at delivery had increased odds of GBS colonisation at birth. Season of birth was associated with increased odds of persistent infant GBS colonisation (dry season vs. wet season AOR 2.9; 95% CI 1.6-5.2).Conclusion: GBS colonisation is common in Gambian women at delivery and in their infants to day 60-89 and is dominated by serotype V. In addition to maternal colonisation, breastmilk and season of birth are important risk factors for infant GBS colonisation. [ABSTRACT FROM AUTHOR]

Introduction The gastrointestinal hormone GLP-1 is released from enteroendocrine L-cells and augments postprandial insulin secretion. In patients with type 2 diabetes, the incretin effect is markedly diminished. It is unclear, whether this is due to a reduction in the abundance of L-cells in the intestine. Methods Ileal tissue samples from 10 patients with and 10 patients without diabetes that underwent surgery for the removal of colon tumors were included. Tissue sections were stained for GLP-1, Ki67, TUNEL and chromogranin A. Results The number of L-cells was not different between patients with and without diabetes in either crypts (1.81 ± 0.21% vs. 1.49 ± 0.24%, respectively; p = 0.31) or villi (1.07 ± 0.16% vs. 0.83 ± 0.10%, respectively; p = 0.23). L-cell number was higher in crypts than in villi (p < 0.0001). L-cell replication was detected rarely and not different between the groups. L-cell apoptosis was similar in patients with and without diabetes in both crypts (7.84 ± 2.77% vs. 8.65 ± 3.77%, p = 0.85) and villi (4.48 ± 2.89% vs. 8.62 ± 4.64%, p = 0.42). Chromogranin A staining was found in a subset of L-cells only. Conclusions Intestinal L-cell density is higher in crypts than in villi. Chromogranin A is not a prerequisite for GLP-1 production. L-cell density and turnover are not different between patients with and without diabetes. Thus, alterations in the number of GLP-1 producing cells do not explain the reduced incretin effect in patients with type 2 diabetes. [ABSTRACT FROM AUTHOR]

The current Ebola outbreak in West Africa has already caused substantial mortality and dire human and economic consequences. It continues to represent an alarming public health threat in the region and beyond and jeopardizes the provision of health care and other services in the affected countries. The scale of the epidemic has accelerated research efforts for diagnostics, treatment, and prevention galvanized through increased availability of funding. Our knowledge relating to the virus, disease pathogenesis, risk factors, dynamics of transmission, and epidemic control is increasing, and sociocultural factors have emerged as critical determinants for the success and failure of control efforts. However, there is a long way to go. In this review we summarize the current knowledge, examine the sociocultural context in West Africa, and outline priority areas for future research. [ABSTRACT FROM AUTHOR]

The interest in tire wear particles (TWPs), generated from abrasion of tires, have gained traction over the past few years, both in regards to quantifying particulate emissions, leaching of different compounds, toxicity, and analytical methods. The life of a tire, from cradle to end-of-life, crosses over different scenarios during its lifetime and transcends environmental compartments and legislative areas, underlining the need for a collective approach. Sustainability for a tire encompasses the use of raw materials, recycling of raw materials, circular economy and material sourcing. The tire industry is currently making significant efforts towards a greener and more sustainable production considering reduction of CO 2 -emissions, recycling, material sources and implementing the use of biomass from plants rather than oil-derived alternatives. In this paper, we aim to analyze and discuss the need for environmental regulation of tires in order to provide a series of targeted recommendations for future legislation. Our study shows that the numerous regulations related to tires focus on chemicals, manufacturing, raw materials, use of tires on roads, waste handling, safety and polycyclic aromatic hydrocarbons (PAHs) in different life cycle stages of a tire. However, none directly addresses the contribution of TWPs to the environment. Despite the overall good intentions of the existing regulations, there is a lack of focus on the compounds that partition from the tire and disperse in the environment, their mixture effects, and the transformative products from the parent compounds in the environment. Therefore, a renewed focus is needed on risk assessment of complex mixtures like TWPs. Thus, transparency in regard to use of chemicals in TWP, mixtures, minimization of emissions, and capture of particulate pollution should be a priority. [Display omitted] • The complexity of tire wear particles (TWPs) through their life cycle complicates risk assessment. • Abrasion of TWPs and leaching of their chemicals should warrant environmental regulation. • Read-across is not a sufficient tool for assessing the hazard potential of TWPs. • TWP sustainability needs to move focus from recycling and material sourcing to abrasion and chemical constituents. • TWP risk assessment needs to target abrasion of particles, chemical components, and mixture effects. [ABSTRACT FROM AUTHOR]

Objectives Vitamin D insufficiency is common in subjects with type 2 diabetes. Observational studies suggest that vitamin D plays a role in the pathogenesis of type 2 diabetes. However, results of intervention studies have been inconsistent. We investigated the effects of improving vitamin D status on insulin sensitivity, insulin secretion, and inflammatory markers in patients with type 2 diabetes. Materials/methods A double blind, randomized, placebo controlled trial was conducted. Sixteen patients with type 2 diabetes and hypovitaminosis D were recruited. Eight patients received colecalciferol and (280 μg daily for 2 weeks, 140 μg daily for 10 weeks) and 8 patients received identical placebo tablets for 12 weeks. Before and after intervention, patients underwent IVGTT, hyperinsulinemic euglycemic clamp, assessment of baseline high-frequency insulin pulsatility, glucose-entrained insulin pulsatility, DXA scans, 24-hour-ambulatory blood pressure monitorings, and fasting blood samples. Results Serum-25(OH) vitamin D and serum-1,25(OH)2 vitamin D increased significantly after 12 weeks in the intervention group (p = 0.01, p = 0.004). Serum-25(OH) vitamin D was also significantly higher in the vitamin D group compared to the placebo group (p = 0.02) after intervention. Although no significant changes in insulin sensitivity, inflammation, blood pressure, lipid profile, or HbA1c were found, we observed borderline (p between 0.05 and 0.10) improvements of insulin secretion, in terms of c-peptide levels, first phase incremental AUC insulin and insulin secretory burst mass. Conclusions Improvement in vitamin D status does not improve insulin resistance, blood pressure, inflammation or HbA1c, but might increase insulin secretion in patients with established type 2 diabetes. [ABSTRACT FROM AUTHOR]

Construction and operation of buildings are a major contributor to environmental impacts. Assessments of environmental impacts of buildings often focus on new buildings, while studies on the existing building stock often focus on energy efficiency renovation. However, historical and traditional buildings are of cultural and visual importance, which should be kept. Hence, a key question is if maintaining original aesthetic via restoration is at the expense of the environment or if it is possible maintain the original appearance of the building while keeping environmental impacts comparable to that of energy renovation. To investigate this, we conducted a Life-Cycle Assessment (LCA) of two options: a restoration and a renovation of an old building from 1887 located on Bornholm, Denmark. The restoration scenario focuses on the use of traditional materials and maintaining the original appearance of the house. The renovation scenario focuses on the use of modern materials and complying with energy requirements for renovated buildings. The results show that, while the impacts of the two scenarios are of similar magnitude, the restoration scenario performs slightly better in the majority of the impact categories in the default LCA-model. A Monte Carlo simulation showed that the restoration scenario performs better in, at least, 90% of the simulations for 8 of the 15 impact categories. This study indicates that restoration is a potentially viable alternative to renovation as a means for maintaining the original appearance of historical buildings to keep cultural heritage while also keeping environmental impacts at levels similar to renovation. • Life-Cycle Assessment of restoration and renovation of a building from 1887. • Restoration can keep impacts similar to renovation and maintain cultural heritage. • Need for future studies to corroborate and generalize findings. [ABSTRACT FROM AUTHOR]

• Significant different gene expression levels were observed between adults with CP and typical developing (TD) adults in various genes. • Differences in muscle contraction (MYH1 & MYBPC2), mitochondrial function (ATP5J, CYCS & NDUFB6), and calcium handling (CAMK2B & ATP2A). • Variant frequencies of the mtDNA and the mitochondrial content did not differ when comparing muscle tissue from adult CP and TD individuals. • The ion levels of Ca2+, Na+, K+ and Cl− were significantly different in adults with CP when compared to TD adults. Cerebral palsy (CP) is the most common cause of movement disorders in children. Next generation sequencing (NGS) studies have previously shown that expression levels are fundamentally different in children with CP compared to typically developing (TD). However, given that children are in full development, we might expect gene expression levels to change once maturity is reached. Therefore, the main purpose of this study was to investigate gene expression levels of 93 target genes in adults with CP using NGS on muscle biopsies of the gastrocnemius, taken from 22 participants (n = 12 adults with CP; n = 10 TD adults). Subsequently, we carried out NGS of the mitochondrial genome to identify mtDNA variants, and additionally we studied the mitochondrial content using transmission electron microscopy images of the gastrocnemius muscle. Finally, we compared systemic ion levels between TD adults and adults with CP. Differential gene expression levels were found in genes involved in muscle contraction (MYH1 and MYBPC2), mitochondrial function kATP5J, CYCS and NDUFB6), calcium handling (CAMK2B and ATP2A), metabolism (LPL) , muscle signaling (MYC, CREB1, ACVR2B, LMNA and TRIM54), and ECM (TNC). There was no statistical significant difference between CP and TD for mtDNA variant frequencies and mitochondrial content. The ion levels of Ca2+, Na+ and K+ were statistically significantly reduced while the Cl− levels were significant increased in adults with CP compared to TD adults. These results highlight that most transcriptional differences are related to muscle function in adults with CP and that mitochondrial function might be altered but not mitochondrial content. [ABSTRACT FROM AUTHOR]

Abstract: Methanogenic communities in 200L biogas reactors containing liquid manure were investigated for 33d. The reactors were consecutively fed with casein, starch and cream. Real-time PCR with primers targeting the gene for methyl coenzyme-M reductase (mcrA) resulted in copy numbers of up to 2.1×109 gdrymass−1. Single strand conformation polymorphism (SSCP) analysis revealed a stable community consisting of few hydrogenotrophic methanogens. One of the two most abundant species was closely related to Methanospirillum hungatei, whereas the other one was only distantly related to other methanogens, with Methanopyrus kandleri being the closest cultivated relative. Most probable number (MPN) cultivations were accomplished with a sample from a 600m3 reactor from which all manures used in the experiments originated, and equal cell counts of ca. 109 gdrymass−1 were found for cultivations with acetate, H2 and methanol. SSCP analysis of these samples and sequencing of the DNA bands identified different hydrogenotrophic methanogens in all samples, and acetoclastic methanogens closely related to Methanosarcina mazei in the samples cultivated with acetate and methanol. As the acetoclastic species were not found in any other SSCP sample, it was supposed that the ammonia values in the manure of the laboratory biogas reactor, which ranged from 2.48 to 3.61gNH4-NL−1, inhibited the growth of the acetoclastic methanogens. [Copyright &y& Elsevier]

Summary: The International Conference on Human Immunity to Tuberculosis, held in April 2012 in Atlanta, Georgia, USA provided the opportunity to discuss the current state of knowledge, important knowledge gaps, and approaches to overcoming obstacles to better understand protective and pathological immunity. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: The impact of a capnoperitoneum on the known blood pressure (BP) difference of the upper and lower limb was studied in piglets. Methods: Eleven German Landrace piglets (body weight, 4.3-7.4 kg; mean body weight, 6.2 kg) were studied. Arterial lines were placed in the right carotid and right femoral artery for pressure monitoring. Intraabdominal pressure levels were increased in steps of 6 mm Hg up to 24 mm Hg. Results: We found that elevated intraabdominal pressures up to 24 mm Hg did not change the preexisting systolic BP difference between the carotid and femoral arteries. Systolic femoral artery pressure constantly remained 5% higher than its carotid counterpart. In addition, mean and diastolic values were not affected. Conclusions: Arterial BP measurements recorded at the legs of piglets when abdominal pressure is increased by up to 24 mm Hg can be used for intraoperative assessment of systemic arterial BP. [Copyright &y& Elsevier]

Abstract: Several models explaining species composition of aquatic bryophytes are available for specific regions. However, a more general, conceptual model applicable to a broader range of regions is lacking. We present a conceptual model ranking environmental factors determining submerged bryophyte communities in small mountain streams. It was tested on a dataset of 54 stream sections after removing the effect of stream size and altitude. Species responses were modeled with pH as predictor variable based on 97 stream sites covering six mountain regions all over Germany. Multiple regressions revealed the importance of primary growth factors (light, Ep(CO2)) and substrate for the total submerged bryophyte coverage. The known distinction of hard- and softwater bryoflora was clearly supported. The floristic composition of headwaters was predominantly determined by the bicarbonate/ionic strength complex. Species response to pH values supported this result and thus our conceptual model. The primary growth resources light, Ep(CO2) and availability of coarse streambed material explained one third (R adjusted 2 =0.34) of total submerged bryophyte cover. Disturbances, predominantly spates, reduce biomass but do not affect the basic floristic structure. In conclusion, conceptual models and monitoring methods focusing on aquatic bryophytes need to clearly distinguish “aquatic” from “submersed by chance”. All “aquatic bryophytes” found in Germany can also occur at least temporarily at non-submerged sites. Therefore, a distinction between primary growth factors and additional resources is recommended to disentangle factors determining aquatic bryophyte communities. [Copyright &y& Elsevier]

Abstract: Most evaluations of agri-environment policies conducted in Europe have focused on biodiversity improvement effects. Their effectiveness with respect to protecting existing biodiversity has been less investigated. Biodiversity and landscape protection effects can be expected in marginal agricultural areas, where farming has been less intensive. We investigated (i) the effectiveness of the Ecological Compensation Area (ECA) scheme for biodiversity protection according to different evaluation criteria and (ii) how the effectiveness of the scheme was influenced by climate, socio-economic conditions and farming structures. Vegetation surveys were carried out in 18 municipalities across the Swiss Alps on 162 randomly selected meadows managed as ECAs and on 162 conventionally managed meadows. Average species richness of ECA-meadows per municipality ranged between 27 and 46 vascular plant species per 25m2 (conventionally managed meadows: 16–35 species per 25m2). In lower altitude municipalities (mainly fulltime farming) where ECA meadows constituted 20% of farmland, the difference in species richness between the two land-use types was highly significant and the contribution of ECA-meadows to the grassland species pool was highest. In higher altitude municipalities (mainly part-time farming) where ECA meadows constituted at least 40% of farmland, higher species richness was found. This was associated with a strong botanical similarity of ECA and conventionally managed meadows. We conclude that at lower altitudes, where farming is relatively intensive, the ECA scheme protects a few, economically marginal, species rich and botanically complementary sites, important for maintaining the species pool. At higher altitudes the ECA scheme contributes to protecting existing biodiversity at the landscape scale. As it is more efficient to protect than to restore biodiversity, the protection effect of agri-environmental schemes needs to be adequately recognized. [Copyright &y& Elsevier]

Abstract: Different harvesting methods have been developed for bony augmentation before implantation. The aim of the present study was to assess the viability of endochondral (femoral) and membranous (mandibular) bone cells harvested by different methods under standard conditions in an animal model, and to investigate the surface of the bone in the harvested area. Samples of mandibular and femoral bone were harvested using a drilling burr, a piezoelectrical device, or a Safescraper®. Blocks of bone that had been harvested with cutting forceps were used as controls. The size of the samples was measured and they were examined by conventional microscopy and immunohistochemical analysis; osteoblast-like cells were also cultured. The surface of the harvested area was analysed with scanning and conventional microscopy. There was no significant difference between mandibular and femoral bone in the size of particles harvested, but bone chips were significantly smaller when a drilling device had been used in both harvesting areas. Viability of cells in these smaller particles was significantly less than among cells harvested with a piezoelectrical device or Safescraper®. Scanning microscopy showed a smooth bony surface where a drilling burr or piezoelectrical device had been used, whereas small disruptions were observed after the Safescraper® had been used. Harvesting of particulate bone is feasible using a drilling burr, piezoelectrical device, or Safescraper® from mandibular and femoral bone. The piezoelectrical device and the Safescraper® gave comparable results concerning the viability of osteoblast-like cells, and so are preferred to a drilling burr. [ABSTRACT FROM AUTHOR]

Abstract: The objective was to generate evidence for clinical efficacy and acceptability of a second generation coxib, firocoxib, administered orally for 14 days to lame horses under field conditions compared with a classic nonsteroidal anti-inflammatory drug, vedaprofen, in a prospective, randomized, controlled, double-blinded, multicenter field trial. Ninety-six client-owned horses with American Association of Equine Practitioners score of at least grade 3 lameness or grade 2 lameness plus at least a score of 2 for either pain on palpation, range of motion, or joint swelling were analyzed. Horses were administered 0.1 mg/kg firocoxib orally at 24 hour intervals (n = 48) or 1.0 mg/kg vedaprofen paste at 12 hour intervals for 14 days (single loading dose of 2.0 mg/kg vedaprofen) (n = 48). Physical examinations and lameness evaluations were conducted on Day 1 (V1, before treatment) and on Days 7 (V2) and 14 (V3). Blood chemistry and hematology profiles were also evaluated. With regard to the primary variable, clinical improvement, 83% of the firocoxib-treated horses improved at V3 compared with 65% of vedaprofen-treated horses improved meeting the criteria defined to demonstrate noninferiority of firocoxib to vedaprofen. Health and behavioral abnormalities for side effect detection occurred at the rate of 2% (1 horse) and 8% (4 horses) for firocoxib- and vedaprofen-treated horses, respectively. Changes in hematology and blood chemistry values from V1 to V3 were not significantly different between treatment groups. Firocoxib, formulated as an oral paste was highly effective, well tolerated, and acceptable for the control of pain and inflammation associated with lameness in horses under field conditions. [Copyright &y& Elsevier]

Summary: Growth hormone (GH) and intracellular STAT 5 signalling represents a very old regulatory system and, whereas insulin dominates periprandially, GH may be viewed as the primary anabolic hormone during stress and fasting. GH exerts metabolic effects both directly and through stimulation of IGF-I, insulin, and free fatty acids (FFA). When well nourished the GH-induced stimulation of IGF-I and insulin is important for tissue anabolism whereas during fasting and other catabolic states GH predominantly stimulates the release and oxidation of FFA which leads to decreased glucose and protein oxidation and preservation of LBM. The most prominent metabolic effect of GH is a marked increase in lipolysis and FFA levels. In the periprandial and postabsorptive states the effects of GH on protein metabolism are modest and include increased protein synthesis and decreased breakdown at the whole body level and in muscle together with decreased amino acid degradation/oxidation and decreased hepatic urea formation. During fasting and stress the effects of GH on protein metabolism become more pronounced; lack of GH during fasting increases protein loss and urea production rates by ≈ 50% with a similar increase in muscle protein breakdown. The importance of GH is further substantiated by the observations that adult patients with GH-deficiency are obese and have reduced LBM, and impaired physical performance and acromegaly is characterised by increased LBM and decreased fat mass. [Copyright &y& Elsevier]

Summary: Grasslands of the Swiss Alps provide agricultural goods, ecological services, disaster protection, and scenic beauty. We identified influences of site conditions and management type on grassland biodiversity, and specifically evaluated the effectiveness of the agri-environmental programme in the Alps. Vascular plants and grasshoppers were mapped in conventionally managed cut meadows, cut meadows of the ecological compensation programme (ECA meadows), and conventionally managed pastures (n=324). We determined climatic and topographic site conditions, quantified the degree of remoteness of all plots in GIS, and estimated the habitat diversity in the vicinity of the sampling sites. The data were analysed by discriminant analysis, rank-correlation analysis, non-parametric ANOVA, general linear models, and ANCOVA. Plant biodiversity hot spots were in economically unattractive, remote sites. These were the typical site conditions of ECA meadows. Hence we concluded that the ecological compensation programme was effectively ensuring management at these sites, and thus protecting mountain grassland plant species richness. Grasshoppers seemed not to benefit from mountain ECA meadows, which were probably often situated at the climatic limit of several species. Pastures were the most species rich management type (plants and grasshoppers), but conversion from cut grassland to pastures should be limited, as negative economic and ecological effects have been reported. We propose that the co-existence of well-managed pastures and long-term ECA meadows might best conserve mountain grassland biodiversity. [Copyright &y& Elsevier]

Background: The arterial switch operation has become the surgical approach of choice for d-transposition of the great arteries, but there is an increased awareness of adverse sequelae in some survivors. Long-term patency and normal function of the translocated coronary arteries must be achieved. It is reported that dependent of the prior coronary status, 3% to 11% of all survivors have proximal coronary stenosis or complete occlusion develop after arterial switch operations. However, treatment of these stenoses is still a matter of debate. Late results after percutaneous transluminal coronary angioplasty (PTCA) for coronary stenosis after the arterial switch operation for d-transposition of the great arteries are reported. Methods: Seven children after arterial switch operation for d-transposition of the great arteries who had subsequently undergone PTCA for coronary stenosis were angiographically re-evaluated 3 to 15 months after the initial PTCA and again after 3 to 5 years. Results: All children survived the initial PTCA procedure. There were no late deaths. The degree of stenosis before PTCA ranged from 74% to 97%; immediately after PTCA from 5% to 10%; at 3 to 15 months after PTCA from zero to 6%; and at 3 to 5 years after PTCA from zero to 3%. Three to 5 years after PTCA all children showed normal development of the treated coronary artery. Conclusions: Primary PTCA of stenotic proximal coronary arteries after the arterial switch procedure for d-transposition of the great arteries seems to be an effective treatment with excellent long-term results. [Copyright &y& Elsevier]

Type 2 diabetes mellitus (T2DM) is associated with impaired skeletal muscle function and degeneration of the skeletal muscles. However, the mechanisms underlying the degeneration are not well described in human skeletal muscle. Here we show that skeletal muscle of T2DM patients exhibit degenerative remodeling of the extracellular matrix that is associated with a selective increase of a subpopulation of fibro-adipogenic progenitors (FAPs) marked by expression of THY1 (CD90)—the FAPCD90+. We identify platelet-derived growth factor (PDGF) as a key FAP regulator, as it promotes proliferation and collagen production at the expense of adipogenesis. FAPsCD90+ display a PDGF-mimetic phenotype, with high proliferative activity, clonogenicity, and production of extracellular matrix. FAPCD90+ proliferation was reduced by in vitro treatment with metformin. Furthermore, metformin treatment reduced FAP content in T2DM patients. These data identify a PDGF-driven conversion of a subpopulation of FAPs as a key event in the fibrosis development in T2DM muscle. [Display omitted] • Type 2 diabetes is associated with fibro-fatty degeneration of skeletal muscle • CD34+ FAPs represent a unique cell population responsible for fibro-fatty infiltration • CD90+ FAPs display enhanced cell division and collagen production mediated by PDGF • The FAPsCD90+ accumulate in skeletal muscle of type 2 diabetic patients Pathological tissue transformation, including fibro-fatty organ degeneration, is a hallmark of type 2 diabetes. Farup et al. demonstrate that degeneration of skeletal muscle in individuals with type 2 diabetes is associated with the accumulation of a subset of fibro-adipogenic progenitors, expressing CD90, endowed with enhanced progenitor potential and collagen production. [ABSTRACT FROM AUTHOR]

The aim was to investigate whether travelling to less-resourced destinations influences the composition of faecal microbiota in generally healthy adults. In this prospective observational study, 47 adults (median age, 24 years; 73% females) travelled from Sweden to distant destinations for 1–12 weeks. Five faecal samples, two before and three after travel, were analysed by 16S amplicon massive parallel sequencing. Subjects had taken no antibiotics within three months of each sampling. The overall composition of faecal microbiota was not affected by travel. However, when looking at the relative abundance of individual bacterial taxa, Enterobacteriaceae demonstrated a 10-fold increase immediately after the trip as compared to the samples taken before travelling. Conversely, the relative abundance of Christensenellaceae had decreased equally much. Both these changes were reversible within nine weeks. International travel, even to less-resourced countries, did not appear to alter the overall diversity of human faecal microbiota as studied here after travelling. However, Enterobacteriaceae bacteria, often associated with infection, inflammation, and antibiotic resistance, showed dramatically elevated levels, and Christensenellaceae , frequently associated with healthy conditions, demonstrated remarkably declined levels in relative abundance as detected immediately after travel. Both these changes returned to original pre-travel levels within nine weeks. [ABSTRACT FROM AUTHOR]

• In a collaborative exercise we tested dried body fluid stains with an Illumina 33plex mRNA panel for body fluid/tissue identification and a 35plex cSNP panel for assignment of body fluids/tissues to donors. • We predicted the origin of the stains using a PLS-DA model, where most of the single source samples were correctly predicted. • We were able to associate donors with body fluids in mixtures of different body fluids as well as in stains where both donors have contributed the same body fluid. • cSNP genotype calling can be difficult in RNA due to pseudo-homozygous phenomena. In a previous EUROFORGEN/EDNAP collaborative exercise, we tested two assays for targeted mRNA massively parallel sequencing for the identification of body fluids/tissues, optimized for the Illumina MiSeq/FGx and the Ion Torrent PGM/S5 platforms, respectively. The task of the second EUROFORGEN/EDNAP collaborative exercise was to analyze dried body fluid stains with two different multiplexes, the former Illumina 33plex mRNA panel for body fluid/tissue identification and a 35plex cSNP panel for assignment of body fluids/tissues to donors that was introduced in a proof-of-concept study recently. The coding region SNPs (cSNPs) are located within the body fluid specific mRNA transcripts and represent a direct link between the body fluid and the donor. We predicted the origin of the stains using a partial least squares discriminant analysis (PLS-DA) model, where most of the single source samples were correctly predicted. The mixed body fluid stains showed poorer results, however, at least one component was predicted correctly in most stains. The cSNP data demonstrated that coding region SNPs can give valuable information on linking body fluids/tissues with donors in mixed body fluid stains. However, due to the unfavorable performance of some cSNPs, the interpretation remains challenging. As a consequence, additional markers are needed to increase the discrimination power in each body fluid/tissue category. [ABSTRACT FROM AUTHOR]

In this study, we tested various approaches to remove DNA from hard laboratory surfaces. We contaminated clean surfaces with four different concentrations of massively parallel sequencing libraries. The DNA was dried and left for 45 min and for 24 h, respectively, before any treatment. The surfaces were cleaned with six different methods water, 96% ethanol, water followed by 96% ethanol, 3–6% hypochlorite solution, 0.9–1.8% hypochlorite solution, and no treatment. Subsequently, the surfaces were swabbed using cotton. DNA was extracted from the swabs and the DNA concentrations were determined by real-time PCR. The results showed that leaving the DNA traces for 24 h decreased the amount of amplifiable DNA approximately four times. The concentrations of amplifiable DNA were approximately five times lower after cleaning with 96% ethanol. Cleaning with water and water followed by 96% ethanol reduced the amount of amplifiable DNA 100–200 times, whereas cleaning with hypochlorite removed all traces of amplifiable DNA. This indicated that the ‘mechanical’ cleaning was efficient but cleaning with hypochlorite was superior. In conclusion, it is recommended to clean laboratory surfaces with 0.9–1.8% hypochlorite in order to eliminate laboratory contamination and simultaneously minimise any poisonous gases. [ABSTRACT FROM AUTHOR]

DNA methylation patterns may be used for identification of body fluids and for age estimation of human individuals. We evaluated some of the challenges and pitfalls of studying methylated CpG sites. We compared the methylated CpG analysis of two different methods 1) massively parallel sequencing (MPS) using the Illumina MiSeq and 2) the iPLEX assay on the MassARRAY ® System. On the Illumina MiSeq, the standard deviation of the fraction of methylation was under 3% between replicates, whereas the reproducibility of the MassARRAY ® was very difficult to achieve. We tested the denaturation and conversion times of the Premium Bisulfite kit (Diagenode) in order to optimise the conversion rate and minimise DNA degradation. Finally, we tested the reproducibility of the methylation patterns using the Illumina MiSeq. [ABSTRACT FROM AUTHOR]

To ensure efficient sequencing, the DNA of next-generation sequencing (NGS) libraries must be quantified correctly. Therefore, an accurate, sensitive and stable method for DNA quantification is crucial. In this study, seven different methods for DNA quantification were compared to each other by quantifying NGS libraries for the Ion Torrent™ and Illumina ® platforms as well as dsDNA oligos with known DNA concentrations. Rather large variations in library concentration estimates were observed. The differences between the highest and lowest concentration estimates varied with a factor of 5–100 depending on the library concentration. The Bioanalyzer, TapeStation and Qubit ® instruments gave concentrations closest to the expected when quantifying dsDNA oligos. At very low concentrations (2–4 pg/ul) only the Bioanalyzer could reliably quantify the dsDNA oligos. [ABSTRACT FROM AUTHOR]

Objectives: To investigate a 4-year prospective clinical trial of agalsidase alfa in children with Fabry disease, an X-linked metabolic disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A. Study design: Seventeen (16 boys, 1 girl; age range, 7.3 to 18.4 years) of the 24 children who completed a 6-month, open-label agalsidase alfa study enrolled in a 3.5-year extension study that investigated the safety and potential efficacy of long-term treatment. All 17 patients completed the initial 6-month study, and 10 patients (9 boys) completed the extension study. Results: Agalsidase alfa was well tolerated. In treated boys, there were sustained, statistically-significant improvements in the clinical features of Fabry disease, including reduced plasma globotriaosylceramide levels, reduced pain severity assessed by the Brief Pain Index, and improved heart rate variability. Mean urine globotriaosylceramide levels were reduced to normal range (P < .05 compared with baseline during 1.5 to 4 years). Kidney function and left ventricular mass indexed to height remained stable throughout. Conclusions: This clinical trial demonstrates that treatment with agalsidase alfa was well tolerated and associated with improvement of Fabry disease–related features. [Copyright &y& Elsevier]