Xdrop: Targeted sequencing of long DNA molecules from low input samples using droplet sorting

Long‐read sequencing can resolve regions of the genome that are inaccessible to short reads, and therefore are ideal for genome‐gap closure, solving structural rearrangements and sequencing through repetitive elements. Here we introduce the Xdrop technology: a novel microfluidic‐based system that allows for targeted enrichment of long DNA molecules starting from only a few nanograms of DNA. Xdrop is based on the isolation of long DNA fragments in millions of droplets, where the droplets containing a target sequence of interest are fluorescently labeled and sorted using flow cytometry. The final product from the Xdrop procedure is an enriched population of long DNA molecules that can be investigated by sequencing. To demonstrate the capability of Xdrop, we performed enrichment of the human papilloma virus 18 integrated into the genome of human HeLa cells. Analysis of the sequencing reads resolved three HPV18‐chr8 integrations at base‐pair resolution, and the captured fragments extended up to 30 kb into the human genome at the integration sites. Further, we enriched the complete TP53 locus in a leukemia cell line and could successfully phase coexisting mutations using PacBio sequencing. In summary, our results show that Xdrop is an efficient enrichment technology for studying complex genomic regions.     

DiceCT Analysis of the Extreme Gouging Adaptations Within the Masticatory Apparatus of the Aye-Aye (Daubentonia madagascariensis)

Relative to all other primates, the aye-aye (Daubentonia madagascariensis) exists at the extremes of both morphology and behavior. Its specialized anatomy—which includes hypselodont incisors and highly derived manual digits—reflects a dietary niche, unique among primates, which combines tap-foraging with gouging to locate and extract wood-boring larvae. Here, we explore the impact of this extreme dietary ecology upon the masticatory musculature of this taxon with reference to a second, similarly sized but highly generalist lemuriform—the mongoose lemur (Eulemur mongoz). Using non-destructive, high-resolution diffusible iodine-based contrast-enhanced computed tomography techniques, we reconstruct the three-dimensional volumes of eight masticatory muscles, and, for the first time in strepsirrhines, isolate and visualize their constituent muscle fascicles in situ and in three dimensions. Using these data, we report muscle volumes, forces, and fascicle lengths from each muscle portion, as well as their orientation relative to two standardized anatomical planes. Our findings demonstrate the overbuilt nature of the aye-aye's masticatory apparatus, in which each muscle possesses an absolutely and relatively larger muscle volume and PCSA than its counterpart in the mongoose lemur. Likewise, for several adductor muscles, aye-ayes also possess relatively greater fascicle lengths. Finally, we note several unusual features within the lateral pterygoid of the aye-aye—the muscle most responsible for jaw protrusion—that relate to force maximization and reorientation. As this jaw motion is critical to gouging, we interpret these differences to reflect highly specific specializations that facilitate the aye-aye's extreme subsistence strategy. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:282–294, 2020. © 2019 American Association for Anatomy     

The movement of a nerve in a magnetic field: application to MRI Lorentz effect imaging

Direct detection of neural activity with MRI would be a breakthrough innovation in brain imaging. A Lorentz force method has been proposed to image nerve activity using MRI; a force between the action currents and the static MRI magnetic field causes the nerve to move. In the presence of a magnetic field gradient, this will cause the spins to precess at a different frequency, affecting the MRI signal. Previous mathematical modeling suggests that this effect is too small to explain the experimental data, but that model was limited because the action currents were assumed to be independent of position along the nerve and because the magnetic field was assumed to be perpendicular to the nerve. In this paper, we calculate the nerve displacement analytically without these two assumptions. Using realistic parameter values, the nerve motion is <5 nm, which induced a phase shift in the MRI signal of <0.02°. Therefore, our results suggest that Lorentz force imaging is beyond the capabilities of current technology.     

Optimization of PAMAM-gold nanoparticle conjugation for gene therapy

The development of efficient and biocompatible non-viral vectors for gene therapy remains a great challenge, and exploiting the properties of both nanoparticle carriers and cationic polymers is an attractive approach. In this work, we have developed gold nanoparticle (AuNP) polyamidoamine (PAMAM) conjugates for use as non-viral transfection agents. AuPAMAM conjugates were prepared by crosslinking PAMAM dendrimers to carboxylic-terminated AuNPs via EDC and sulfo-NHS chemistry. EDC and sulfo-NHS have been utilized widely and in numerous applications such as amino acid coupling; however, their use in the coupling of PAMAM dendrimers to AuNPs presents new challenges to form effective and stable constructs for delivery that have not yet been examined. Enhanced colloidal stability and DNA condensation ability was established by probing two critical synthetic parameters: the reaction rate of the PAMAM crosslinking step, and the amine to carboxyl ratio. Based on this work, increasing the amine to carboxyl ratio during conjugation of PAMAM onto AuNPs yielded the optimal vector with respect to colloidal stability and transfection efficiency in vitro. AuPAMAM conjugates present attractive candidates for non-viral gene delivery due to their commercial availability, ease of fabrication and scale-up, high yield, high transfection efficiency and low cytotoxicity.     

Clustering of body composition,blood pressure and physical activity in Portuguese families

Aim: The purposes of this study were: (i) to identify familial resemblances in body fat, blood pressure (BP) and total physical activity (TPA); (ii) to estimate the magnitude of their genetic and environmental influences; and (iii) to investigate shared familial aggregation among these phenotypes.

Subjects and methods: The sample comprised 260 nuclear families from Portugal. Body fat was assessed by bioelectrical impedance. BP was measured by an oscillometric device. TPA was estimated by the Baecke questionnaire. Familial correlation analyses were performed using Generalized Estimating Equations. Quantitative genetic modelling was used to estimate maximal heritability, genetic and environmental correlations.

Results: Familial intra-trait correlations ranged from 0.15–0.38. Genetic and common environmental factors explained from 30%--44% of fat mass depots and BP and 24% of TPA. Genetic correlations were significant between BP and the fat mass traits (p?p?Conclusions: The results suggest familial resemblance in the variation of body fat, BP and TPA, showing partial pleiotropic effects in the variation in body fat phenotypes and BP. TPA only shares common environmental influences with BP and body fat traits.     

Exercise volume and intensity: a dose–response relationship with health benefits

Introduction

The health benefits of exercise are well established. However, the relationship between exercise volume and intensity and health benefits remains unclear, particularly the benefits of low-volume and intensity exercise.

Purpose

The primary purpose of this investigation was, therefore, to examine the dose–response relationship between exercise volume and intensity with derived health benefits including volumes and intensity of activity well below international recommendations.

Methods

Generally healthy, active participants (n = 72; age = 44 ± 13 years) were assigned randomly to control (n = 10) or one of five 13-week exercise programs: (1) 10-min brisk walking 1×/week (n = 10), (2) 10-min brisk walking 3×/week (n = 10), (3) 30-min brisk walking 3×/week (n = 18), (4) 60-min brisk walking 3×/week (n = 10), and (5) 30-min running 3×/week (n = 14), in addition to their regular physical activity. Health measures evaluated pre- and post-training including blood pressure, body composition, fasting lipids and glucose, and maximal aerobic power (VO₂max).

Results

Health improvements were observed among programs at least 30 min in duration, including body composition and VO₂max: 30-min walking 28.8–34.5 mL kg^?1 min^?1, 60-min walking 25.1–28.9 mL kg^?1 min^?1, and 30-min running 32.4–36.4 mL kg^?1 min^?1. The greater intensity running program also demonstrated improvements in triglycerides.

Conclusion

In healthy active individuals, a physical activity program of at least 30 min in duration for three sessions/per week is associated with consistent improvements in health status.     

Use of Antigen-Specific Interleukin-2 To Differentiate between Cattle Vaccinated with Mycobacterium bovis BCG and Cattle Infected with M. bovis

We describe here the application of a novel bovine interleukin-2 (IL-2) enzyme-linked immunosorbent assay (ELISA) for the measurement of antigen-specific IL-2 in cattle naturally infected with Mycobacterium bovis and in cattle vaccinated with Mycobacterium bovis BCG and then experimentally challenged with pathogenic M. bovis. Supernatants from whole-blood cultures stimulated with mycobacterial antigen (bovine purified protein derivative [PPDB] or the peptide cocktail ESAT6-CFP10) were assessed using a sandwich ELISA consisting of a new recombinant monoclonal fragment capture antibody and a commercially available polyclonal anti-bovine-IL-2. The production of IL-2 was compared to the production of gamma interferon (IFN-γ) in the same antigen-stimulated whole-blood supernatants. The data show that cattle infected with M. bovis produced quantifiable levels of antigen-specific IL-2, while IL-2 levels in cattle vaccinated with M. bovis BCG did not. Furthermore, cattle vaccinated with M. bovis BCG and then challenged with pathogenic M. bovis displayed a more rapid induction of IL-2 but ultimately had lower levels of infection-induced IL-2 than did unvaccinated challenge control cattle. These data suggest that IL-2 responses are not detectable post-BCG vaccination and that these responses may require infection with virulent M. bovis to develop. This may be useful to differentiate infected cattle from uninfected or BCG-vaccinated cattle, although the overall sensitivity is relatively low, particularly in single intradermal comparative cervical tuberculin (SICCT)-negative infected animals. Furthermore, the strength of the IL-2 response may correlate with pathology, which poses interesting questions on the immunobiology of bovine tuberculosis in contrast to human tuberculosis, which is discussed.     

Delayed presentation of a caecal haematoma initially managed as appendicitis

A 15-year-old boy with caecal haematoma required a right hemicolectomy due to development of small bowel obstruction and near caecal perforation having presented several days after an episode of minor trauma. The position of the caecum between intraperitoneal and extraperitoneal bowel requires special treatment considerations. This is a unique case in an adolescent because caecal haematoma usually presents acutely with abdominal pain.