Improving ancient DNA read mapping against modern reference genomes

ABSTRACT: BACKGROUND: Next-Generation Sequencing has revolutionized our approach to ancient DNA (aDNA) research, by providing complete genomic sequences of ancient individuals and extinct species. However, the recovery of genetic material from long-dead organisms is still complicated by a number of issues, including post-mortem DNA damage and high levels of environmental contamination. Together with error profiles specific to the type of sequencing platforms used, these specificities could limit our ability to map sequencing reads against modern reference genomes and therefore limit our ability to identify endogenous ancient reads, reducing the efficiency of shotgun sequencing aDNA. RESULTS: In this study, we compare different computational methods for improving the accuracy and sensitivity of aDNA sequence identification, based on shotgun sequencing reads recovered from Pleistocene horse extracts using Illumina GAIIx and Helicos Heliscope platforms. We show that the performance of the Burrows Wheeler Aligner (BWA), that has been developed for mapping of undamaged sequencing reads using platforms with low rates of indel-types of sequencing errors, can be employed at acceptable run-times by modifying default parameters in a platform-specific manner. We also examine if trimming likely damaged positions at read ends can increase the recovery of genuine aDNA fragments and if accurate identification of human contamination can be achieved using a strategy previously suggested based on best hit filtering. We show that combining our different mapping and filtering approaches can increase the number of high-quality endogenous hits recovered by up to 33%. CONCLUSIONS: We have shown that Illumina and Helicos sequences recovered from aDNA extracts could not be aligned to modern reference genomes with the same efficiency unless mapping parameters are optimized for the specific types of errors generated by these platforms and by post-mortem DNA damage. Our findings have important implications for future aDNA research, as we define mapping guidelines that improve our ability to identify genuine aDNA sequences, which in turn could improve the genotyping accuracy of ancient specimens. Our framework provides a significant improvement to the standard procedures used for characterizing ancient genomes, which is challenged by contamination and often low amounts of DNA material.     

An Australian Multi‐Regional Waste Supply‐Use Framework

The production of waste creates both direct and indirect environmental impacts. A range of strategies are available to reduce the generation of waste by industry and households, and to select waste treatment approaches that minimize environmental harm. However, evaluating these strategies requires reliable and detailed data on waste production and treatment. Unfortunately, published Australian waste data are typically highly aggregated, published by a variety of entities in different formats, and do not form a complete time‐series. We demonstrate a technique for constructing a multi‐regional waste supply‐use (MRWSU) framework for Australia using information from numerous waste data sources. This is the first MRWSU framework to be constructed (to the authors' knowledge) and the first sub‐national waste input‐output framework to be constructed for Australia. We construct the framework using the Industrial Ecology Virtual Laboratory (IELab), a cloud‐hosted computational platform for building Australian multi‐regional input‐output tables. The structure of the framework complies with the System of Environmental‐Economic Accounting (SEEA). We demonstrate the use of the MRWSU framework by calculating waste footprints that enumerate the full supply chain waste production for Australian consumers.     

Characterization of oxidative stress in blood from diabetic vs. hypercholesterolaemic patients,using a novel synthesized marker

In the present study, we extend our novel concept of designing and using exogenous markers for the characterization of oxidative stress (OS) and OS-associated diseases. The aim was to use such a synthetic compound as a tool for studying OS in blood from diabetic and hypercholesterolaemic (Hc) patients. The marker used N-linoleoyl tyrosine (LT) was constructed from tyrosine and linoleic acid (LA); both components are known to be easily oxidized upon exposure to different types of reactive oxygen/nitrogen species (ROS/RNS), and to generate specific oxidized products, depending on the type of oxidants present in vivo. Using the LT probe, we showed that the ratios of oxidized LT to total LT (Ox-LT/LT) is significantly higher in blood samples obtained from diabetic patients, than in Hc patients or healthy control subjects. LC/MS analysis revealed that blood from diabetic patients oxidizes the marker with predominant formation of Ox-LT hydroperoxide (LT-OOH) and epoxide (epoxy-LT), where the LA moiety is oxidized to hydroperoxide and to epoxide, respectively. Analysis of oxysterol levels in these samples (GC/MS) revealed that the blood of both diabetic and Hc patients contained significantly more oxysterols than blood of control subjects. Consumption of pomegranate juice by diabetic patients for 3 months suppressed their blood capacity to oxidize the LT and similarly also reduced their blood oxysterol/total cholesterol ratio by 93%. The use of an exogenous marker to characterize OS in blood samples yields important information on the extent of OS, and can provide a fingerprint for the early identification of different pathological conditions associated with OS.     

Structural understanding of stabilization patterns in engineered bispecific Ig‐like antibody molecules

Bispecific immunoglobulin‐like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin‐beta receptor (LTβR) binding Fv domain of an anti‐LTβR/anti‐TNF‐related apoptosis inducing ligand receptor‐2 (TRAIL‐R2) bispecific immunoglobulin‐like antibody. We further describe a new hierarchical structure‐guided approach toward engineering of antibody‐like molecules to enhance their thermal and chemical stability. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Mechanical stiffness as an improved single-cell indicator of osteoblastic human mesenchymal stem cell differentiation

Although it has been established that cellular stiffness can change as a stem cell differentiates, the precise relationship between cell mechanics and other phenotypic properties remains unclear. Inherent cell heterogeneity and asynchronous differentiation complicate population analysis; therefore, single-cell analysis was employed to determine how changes in cell stiffness correlate with changes in molecular biomarkers during differentiation. Design of a custom gridded tissue culture dish facilitated single-cell comparisons between cell mechanics and other differentiation biomarkers by enabling sequential measurement of cell mechanics and protein biomarker expression at the single cell level. The Young’s modulus of mesenchymal stem cells was shown not only to decrease during chemically-induced osteoblast differentiation, but also to correlate more closely with the day of differentiation than did the relative expression of the traditional osteoblast differentiation markers, bone sialoprotein and osteocalcin. Therefore, cell stiffness, a measurable property of individual cells, may serve as an improved indicator of single-cell osteoblast differentiation compared to traditional biological markers. Revelation of additional osteoblast differentiation indicators, such as cell stiffness, can improve identification and collection of starting cell populations, with applications to mesenchymal stem cell therapies and stem cell-based tissue engineering.     

Pre-treatment of recombinant mouse MFG-E8 downregulates LPS-induced TNF-α production in macrophages via STAT3-mediated SOCS3 activation

Milk fat globule-epidermal growth factor factor 8 (MFG-E8) regulates innate immune function by modulating cellular signaling, which is less understood. Herein, we aimed to investigate the direct anti-inflammatory role of MFG-E8 in macrophages by pre-treatment with recombinant murine MFG-E8 (rmMFG-E8) followed by stimulation with LPS in RAW264.7 cells and in peritoneal macrophages, isolated from wild-type (WT) or MFG-E8(-/-) mice. RAW264.7 cells and mouse peritoneal macrophages treated with rmMFG-E8 significantly downregulated LPS-induced TNF-α mRNA by 25% and 24%, and protein levels by 29% and 23%, respectively (P<0.05). Conversely, peritoneal macrophages isolated from MFG-E8(-/-) mice produced 28% higher levels of TNF-α, as compared to WT mice when treated with LPS. In in vivo, endotoxemia induced by intraperitoneal injection of LPS (5 mg/kg BW), at 4 h after induction, serum level of TNF-α was significantly higher in MFG-E8(-/-) mice (837 pg/mL) than that of WT (570 pg/mL, P<0.05). To elucidate the direct anti-inflammatory effect of MFG-E8, we examined STAT3 and its target gene, SOCS3. Treatment with rmMGF-E8 significantly induced pSTAT3 and SOCS3 in macrophages. Similar results were observed in in vivo treatment of rmMFG-E8 in peritoneal cells and splenic tissues. Pre-treatment with rmMFG-E8 significantly reduced LPS-induced NF-κB p65 contents. These data clearly indicated that rmMFG-E8 upregulated SOCS3 which in turn interacted with NF-κB p65, facilitating negative regulation of TLR4 signaling for LPS-induced TNF-α production. Our findings strongly suggest that MFG-E8 is a direct anti-inflammatory molecule, and that it could be developed as a therapy in attenuating inflammation and tissue injury.     

Living at the edge of the front; reduced predation risk to invasive round goby in a Great Lakes tributary

The objective of this research was to determine if the highly invasive round goby (Neogobius melanostomus) experiences lower predation risk during early stages of invasion. We compared round goby predation rates between a recently invaded area (occupied for ≈1 year) and a longer established area (≈7 years) of the Trent River, Ontario, Canada. Tethering trials were conducted in three habitat types, and comparable habitats in the two areas were similar in water temperature, velocity and depth. Predation rates of tethered round gobies were on average 27% lower in the recently invaded area. Reduced predation in the recently invaded area may be due to the short duration of round goby occupancy and/or differences in predator communities between the two study areas. Data before the round goby invasion suggest that predator communities were similar between the two range areas, but differences in predator abundance cannot be ruled out as a potential mechanism. Other possible mechanisms include a numerical or learned response by predators over time to a novel prey item. Reduced predation rate during the initial stages of invasion may contribute to the fitness of individuals that migrate into areas not previously occupied, and thus facilitate successful range expansion.     

Involvement of regulatory volume decrease in the migration of nasopharyngeal carcinoma cells

The transwell chamber migration assay and CCD digital camera imaging techniques were used to investigate the relationship between regulatory volume decrease (RVD) and cell migration in nasopharyngeal carcinoma cells (CNE-2Z cells). Both migrated and non-migrated CNE-2Z cells, when swollen by 47% hypotonic solution, exhibited RVD which was inhibited by extracellular application of chloride channel blockers adenosine 5‘-triphosphate (ATP), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen. However, RVD rate in migrated CNE-2Z cells was bigger than that of non-migrated cells and the sensitivity of migrated cells to NPPB and tamoxifen was higher than that of nonmigrated cells. ATP, NPPB and tamoxifen also inhibited migration of CNE-2Z cells. The inhibition of migration was positively correlated to the blockage of RVD, with a correlation coefficient (r) = 0.99, suggesting a functional relationship between RVD and cell migration. We conclude that RVD is involved in cell migration and RVD may play an important role in migratory process in CNE-2Z cells.     

A new bacteria-free strategy induced by MaGal2 facilitates pinewood nematode escape immune response from its vector beetle

Symbiotic microbes play a crucial role in regulating parasite–host interactions; however, the role of bacterial associates in parasite–host interactions requires elucidation. In this study, we showed that, instead of introducing numerous symbiotic bacteria, dispersal of 4th-stage juvenile (J_IV) pinewood nematodes (PWNs), Bursaphelenchus xylophilus, only introduced few bacteria to its vector beetle, Monochamus alternatus (Ma). J_IV showed weak binding ability to five dominant bacteria species isolated from the beetles’ pupal chamber. This was especially the case for binding to the opportunistic pathogenic species Serratia marcescens; the nematodes’ bacteria binding ability at this critical stage when it infiltrates Ma for dispersal was much weaker compared with Caenorhabditis elegans, Diplogasteroides asiaticus, and propagative-stage PWN. The associated bacterium S. marcescens, which was isolated from the beetles’ pupal chambers, was unfavorable to Ma, because it caused a higher mortality rate upon injection into tracheae. In addition, S. marcescens in the tracheae caused more immune effector disorders compared with PWN alone. Ma_Galectin2 (MaGal2), a pattern-recognition receptor, was up-regulated following PWN loading. Recombinant MaGal2 protein formed aggregates with five dominant associated bacteria in vitro. Moreover, MaGal2 knockdown beetles had up-regulated prophenoloxidase gene expression, increased phenoloxidase activity, and decreased PWN loading. Our study revealed a previously unknown strategy for immune evasion of this plant pathogen inside its vector, and provides novel insights into the role of bacteria in parasite–host interactions.     

Does Isolated Unilateral Hip or Knee Osteoarthritis Lead to Adverse Changes in Extremity Composition?

BackgroundWhile muscle atrophy is a function of normal aging, loss of muscle in the setting of hip and knee osteoarthritis (OA) has been observed using radiographic studies. There is limited data available regarding changes in extremity composition using bioimpedance (BIA). The purpose of this study was to assess the changes in extremity composition in patients with isolated, unilateral hip or knee OA using BIA.MethodsPatients presenting to our institution’s adult reconstruction clinic from February 2020 to April 2021 were retrospectively reviewed to identify those with isolated, unilateral hip and knee OA. The InBody 770 Body Composition Analyzer (InBody USA, Cerritos, California) was used to perform a complete body composition assessment, per protocol. Lean extremity mass (LEM), fat mass (FM), intracellular water (ICW), extremity body water (EBW = ICW + extracellular water (ECW)) and phase angle (PA) were determined. Differences between the affected (OA) and unaffected (no OA) extremities were compared using t-tests.Results38 patients had isolated hip OA. The mean age was 60.8 (±11.7) years, mean BMI was 31.7 (±6.8) kg/m2, and 39.5% were female. LEM, FM, EBW, ICW, and PA were significantly decreased in the hip OA extremity (LEM: 20.0 vs. 20.4 kg, p=0.0008, FM: 8.8 vs. 8.9 kg, p=0.0049, EBW: 15.7 vs 16.0, p=0.0011, ICW: 9.5 vs. 9.7 L, p=0.0004, PA: 4.5 vs 4.9º, p<0.0001). There were 25 patients with isolated knee OA. Mean age was 62.8 (±11.3) years, mean BMI was 33.6 (±6.9) kg/m2, and 52.0% were female. FM and PA were significantly lower in the knee OA extremity (11.3 vs 11.4 kg, p=0.0291, 4.5 vs 4.9º, p<0.0001). There were no significant differences in LEM, EBW, and ICW between the knee OA extremity and the unaffected extremity.ConclusionPatients with isolated, unilateral hip OA had decreased LEM, FM, EBW, and ICW in the affected extremity. Both unilateral hip and knee OA was associated with decreased PA, suggestive of greater underlying dysfunction in muscle or cellular performance. Further study is needed to better define when these abnormalities develop, how they progress over time, and the impact of targeted interventions in reversing these changes. Level of Evidence: III