Drug-resistant genotypes and multi-clonality in Plasmodium falciparum analysed by direct genome sequencing from peripheral blood of malaria patients.

Naturally acquired blood-stage infections of the malaria parasite Plasmodium falciparum typically harbour multiple haploid clones. The apparent number of clones observed in any single infection depends on the diversity of the polymorphic markers used for the analysis, and the relative abundance of rare clones, which frequently fail to be detected among PCR products derived from numerically dominant clones. However, minority clones are of clinical interest as they may harbour genes conferring drug resistance, leading to enhanced survival after treatment and the possibility of subsequent therapeutic failure. We deployed new generation sequencing to derive genome data for five non-propagated parasite isolates taken directly from 4 different patients treated for clinical malaria in a UK hospital. Analysis of depth of coverage and length of sequence intervals between paired reads identified both previously described and novel gene deletions and amplifications. Full-length sequence data was extracted for 6 loci considered to be under selection by antimalarial drugs, and both known and previously unknown amino acid substitutions were identified. Full mitochondrial genomes were extracted from the sequencing data for each isolate, and these are compared against a panel of polymorphic sites derived from published or unpublished but publicly available data. Finally, genome-wide analysis of clone multiplicity was performed, and the number of infecting parasite clones estimated for each isolate. Each patient harboured at least 3 clones of P. falciparum by this analysis, consistent with results obtained with conventional PCR analysis of polymorphic merozoite antigen loci. We conclude that genome sequencing of peripheral blood P. falciparum taken directly from malaria patients provides high quality data useful for drug resistance studies, genomic structural analyses and population genetics, and also robustly represents clonal multiplicity

ORMIR_XCT: A Python package for high resolution peripheral quantitative computed tomography image processing

High resolution peripheral quantitative computed tomography (HR-pQCT) is an imaging technique capable of imaging trabecular bone in-vivo. HR-pQCT has a wide range of applications, primarily focused on bone to improve our understanding of musculoskeletal diseases, assess epidemiological associations, and evaluate the effects of pharmaceutical interventions. Processing HR-pQCT images has largely been supported using the scanner manufacturer scripting language (Image Processing Language, IPL, Scanco Medical). However, by expanding image processing workflows outside of the scanner manufacturer software environment, users have the flexibility to apply more advanced mathematical techniques and leverage modern software packages to improve image processing. The ORMIR_XCT Python package was developed to reimplement some existing IPL workflows and provide an open and reproducible package allowing for the development of advanced HR-pQCT data processing workflows

High-Frequency, Low-Magnitude Vibration Does Not Prevent Bone Loss Resulting from Muscle Disuse in Mice following Botulinum Toxin Injection

High-frequency, low-magnitude vibration enhances bone formation ostensibly by mimicking normal postural muscle activity. We tested this hypothesis by examining whether daily exposure to low-magnitude vibration (VIB) would maintain bone in a muscle disuse model with botulinum toxin type A (BTX). Female 16–18 wk old BALB/c mice (N = 36) were assigned to BTX-VIB, BTX-SHAM, VIB, or SHAM. BTX mice were injected with BTX (20 µL; 1 U/100 g body mass) into the left hindlimb posterior musculature. All mice were anaesthetized for 20 min/d, 5 d/wk, for 3 wk, and the left leg mounted to a holder. Through the holder, VIB mice received 45 Hz, ±0.6 g sinusoidal acceleration without weight bearing. SHAM mice received no vibration. At baseline and 3 wk, muscle cross-sectional area (MCSA) and tibial bone properties (epiphysis, metaphysis and diaphysis) were assessed by in vivo micro-CT. Bone volume fraction in the metaphysis decreased 12±9% and 7±6% in BTX-VIB and BTX-SHAM, but increased in the VIB and SHAM. There were no differences in dynamic histomorphometry outcomes between BTX-VIB and BTX nor between VIB and SHAM. Thus, vibration did not prevent bone loss induced by a rapid decline in muscle activity nor produce an anabolic effect in normal mice. The daily loading duration was shorter than would be expected from postural muscle activity, and may have been insufficient to prevent bone loss. Based on the approach used in this study, vibration does not prevent bone loss in the absence of muscle activity induced by BTX

Cryptic Eimeria genotypes are common across the southern but not northern hemisphere

The phylum Apicomplexa includes parasites of medical, zoonotic and veterinary significance. Understanding the global distribution and genetic diversity of these protozoa is of fundamental importance for efficient, robust and long-lasting methods of control. Eimeria spp. cause intestinal coccidiosis in all major livestock animals and are the most important parasites of domestic chickens in terms of both economic impact and animal welfare. Despite having significant negative impacts on the efficiency of food production, many fundamental questions relating to the global distribution and genetic variation of Eimeria spp. remain largely unanswered. Here, we provide the broadest map yet of Eimeria occurrence for domestic chickens, confirming that all the known species (Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox, Eimeria tenella) are present in all six continents where chickens are found (including 21 countries). Analysis of 248 internal transcribed spacer sequences derived from 17 countries provided evidence of possible allopatric diversity for species such as E. tenella (FST values ⩽0.34) but not E. acervulina and E. mitis, and highlighted a trend towards widespread genetic variance. We found that three genetic variants described previously only in Australia and southern Africa (operational taxonomic units x, y and z) have a wide distribution across the southern, but not the northern hemisphere. While the drivers for such a polarised distribution of these operational taxonomic unit genotypes remains unclear, the occurrence of genetically variant Eimeria may pose a risk to food security and animal welfare in Europe and North America should these parasites spread to the northern hemisphere

An open dataset of Plasmodium falciparum genome variation in 7,000 worldwide samples.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed.  Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination

Cartilage imaging of a rabbit knee using dual-energy X-ray microscopy and 1.0 T and 9.4 T magnetic resonance imaging

Background/Objective: Osteoarthritis is a common chronic disease of the joints characterised by the degeneration of articular cartilages and subchondral bone. The most common diagnostic imaging used clinically is X-ray; however, it cannot directly image cartilage. Magnetic resonance imaging (MRI) is well suited for cartilage imaging, but it requires costly and lengthy scans. For preclinical work, microcomputed tomography provides high spatial resolution and contrast for bone, however, its standard application is not well suited for cartilage imaging. Methods: We performed a preliminary investigation into the use of dual-energy X-ray microscopy (XRM) for cartilage imaging and analysis of a rabbit knee, and compared it to the MRI results from 9.4 T and 1.0 T small-animal scanners. Results: The XRM images offer a higher image resolution (∼25 μm nominal isotropic resolution) compared with the MRI (50–86 μm in plane, and 250 μm slice thickness). The cartilage-thickness measurements using the dual-energy XRM are on average 3.8% (femur) and 5.1% (tibia) thicker estimates than the 9.4 T MRI results. The cartilage-thickness measurements using the 1.0 T MRI are on average 10.9% (femur) and 2.3% (tibia) thinner estimates than the 9.4 T MRI results. Conclusion: Our results suggest that the dual-energy XRM for articular-cartilage analysis is feasible and comparable to the MRI. This technology will provide good support for high-resolution animal-osteoarthritis studies, and in the future, it may be possible to apply dual energy in a clinical setting

Locomotory behaviour of early tetrapods from Blue Beach, Nova Scotia, revealed by novel microanatomical analysis

Evidence for terrestriality in early tetrapods is fundamentally contradictory. Fossil trackways attributed to early terrestrial tetrapods long predate the first body fossils from the Late Devonian. However, the Devonian body fossils demonstrate an obligatorily aquatic lifestyle. Complicating our understanding of the transition from water to land is a pronounced gap in the fossil record between the aquatic Devonian taxa and presumably terrestrial tetrapods from the later Early Carboniferous. Recent work suggests that an obligatorily aquatic habit persists much higher in the tetrapod tree than previously recognized. Here, we present independent microanatomical data of locomotor capability from the earliest Carboniferous of Blue Beach, Nova Scotia. The site preserves limb bones from taxa representative of Late Devonian to mid-Carboniferous faunas as well as a rich trackway record. Given that bone remodels in response to functional stresses including gravity and ground reaction forces, we analysed both the midshaft compactness profiles and trabecular anisotropy, the latter using a new whole bone approach. Our findings suggest that early tetrapods retained an aquatic lifestyle despite varied limb morphologies, prior to their emergence onto land. These results suggest that trackways attributed to early tetrapods be closely scrutinized for additional information regarding their creation conditions, and demand an expansion of sampling to better identify the first terrestrial tetrapods

Reproducibility and repeatability of a semi-automated pipeline to quantify trapeziometacarpal joint angles using dynamic computed tomography

Abstract Background The trapeziometacarpal (TMC) joint is a mechanically complex joint and is commonly affected by musculoskeletal diseases such as osteoarthritis. Quantifying in vivo TMC joint biomechanics, such as joint angles, with traditional reflective marker-based methods can be difficult due to the joint’s location in the hand. Dynamic computed tomography (CT) can facilitate the quantification of TMC joint motion by continuously capturing three-dimensional volumes over time. However, post-processing of dynamic CT datasets can be time intensive and automated methods are needed to reduce processing times to allow for application to larger clinical studies. The purpose of this work is to introduce a fast, semi-automated pipeline to quantify joint angles from dynamic CT scans of the TMC joint and evaluate the associated error in joint angle and translation computation by means of a reproducibility and repeatability study. Methods Ten cadaveric hands were scanned with dynamic CT using a passive motion device to move thumbs in a radial abduction–adduction motion. Static CT scans and high-resolution peripheral quantitative CT scans were also acquired to generate high-resolution bone meshes. Abduction–adduction, flexion–extension, and axial rotation angles were computed using a joint coordinate system. Reproducibility and repeatability were assessed using intraclass correlation coefficients, Bland–Altman analysis, and root mean square errors. Target registration errors were computed to evaluate errors associated with image registration. Results We found good repeatability for flexion–extension, abduction–adduction, and axial rotation angles. Reproducibility was moderate for all three angles. Joint translations exhibited greater repeatability than reproducibility. Specimens with greater joint degeneration had lower repeatability and reproducibility. We found that the difference in resulting joint angles and translations were likely due to differences in segment coordinate system definition between multiple raters, rather than due to registration errors. Conclusions The proposed semi-automatic processing pipeline was fast, repeatable, and moderately reproducible when quantifying TMC joint angles and translations. This work provides a range of errors for TMC joint angles from dynamic CT scans using manually selected anatomical landmarks

Strain

<p>(<b>A</b>) <b>and acceleration</b> (<b>B</b>) <b>vs. time for a 0.2 </b><b>s portion of one trial.</b> The strain gauge was fixed to the anterior surface of the proximal tibia. The accelerometer was fixed to the leg holder.</p