The Identification and Characterization of Copy Number Variants in the Bovine Genome

Separate domestication events and strong selective pressures have created diverse phenotypes among existing cattle populations; however, the genetic determinants underlying most phenotypes are currently unknown. Bos taurus taurus (Bos taurus) and Bos taurus indicus (Bos indicus) cattle are subspecies of domesticated cattle that are characterized by unique morphological and metabolic traits. Because of their divergence, they are ideal model systems to understand the genetic basis of phenotypic variation. Here, we developed DNA and structural variant maps of cattle genomes representing the Bos taurus and Bos indicus breeds. Using this data, we identified genes under selection and biological processes enriched with functional coding variants between the two subspecies. Furthermore, we examined genetic variation at functional non-coding regions, which were identified through epigenetic profiling of indicative histone- and DNA-methylation modifications. Copy number variants, which were frequently not imputed by flanking or tagged SNPs, represented the largest source of genetic divergence between the subspecies, with almost half of the variants present at coding regions. We identified a number of divergent genes and biological processes between Bos taurus and Bos indicus cattle; however, the extent of functional coding variation was relatively small compared to that of functional non-coding variation. Collectively, our findings suggest that copy number and functional non-coding variants may play an important role in regulating phenotypic variation among cattle breeds and subspecies

Gene therapy targeting SARM1 blocks pathological axon degeneration in mice

Axonal degeneration (AxD) following nerve injury, chemotherapy, and in several neurological disorders is an active process driven by SARM1, an injury-activated NADase. Axons of SARM1-null mice exhibit greatly delayed AxD after transection and in models of neurological disease, suggesting that inhibiting SARM1 is a promising strategy to reduce pathological AxD. Unfortunately, no drugs exist to target SARM1. We, therefore, developed SARM1 dominant-negatives that potently block AxD in cellular models of axotomy and neuropathy. To assess efficacy in vivo, we used adeno-associated virus-mediated expression of the most potent SARM1 dominant-negative and nerve transection as a model of severe AxD. While axons of vehicle-treated mice degenerate rapidly, axons of mice expressing SARM1 dominant-negative can remain intact for \u3e10 d after transection, similar to the protection observed in SARM1-null mice. We thus developed a novel in vivo gene therapeutic to block pathological axon degeneration by inhibiting SARM1, an approach that may be applied clinically to treat manifold neurodegenerative diseases characterized by axon loss

Group Vibrational Mode Assignments as a Broadly Applicable Tool for Characterizing Ionomer Membrane Structure as a Function of Degree of Hydration

Infrared spectra of Nafion, Aquivion, and the 3M membrane were acquired during total dehydration of fully hydrated samples. Fully hydrated exchange sites are in a sulfonate form with a C₃V local symmetry. The mechanical coupling of the exchange site to a side chain ether link gives rise to vibrational group modes that are classified as C₃V modes. These mode intensities diminish concertedly with dehydration. When totally dehydrated, the sulfonic acid form of the exchange site is mechanically coupled to an ether link with no local symmetry. This gives rise to C₁ group modes that emerge at the expense of C₃V modes during dehydration. Membrane IR spectra feature a total absence of C₃V modes when totally dehydrated, overlapping C₁ and C₃V modes when partially hydrated, and a total absence of C₁ modes when fully hydrated. DFT calculated normal mode analyses complemented with molecular dynamics simulations of Nafion with overall λ (λ_(Avg)) values of 1, 3, 10, 15 and 20 waters/exchange site, were sectioned into sub-cubes to enable the manual counting of the distribution of λ_(local) values that integrate to λ_(Avg) values. This work suggests that at any state of hydration, IR spectra are a consequence of a distribution of λ_(local) values. Bond distances and the threshold value of λ_(local), for exchange site dissociation, were determined by DFT modelling and used to correlate spectra to manually counted λ_(local) distributions

Sacrificial templates for manufacturing multidimensional vasculature

Biological systems employ complex, composite architectures that are intimately related to homeostatic functionality. A common necessity underlying many of these systems is the transport of fluids that distribute nutrients, remove waste, and provide thermal regulation. Parallels exist in engineered materials; however, the architectures are comparatively less complex. No single fabrication technique has emerged with the flexibility to create architectures of various size-scale and dimensionality. Esser-Kahn et al. introduced a technique referred to as vaporization of sacrificial components (VaSC) [1]. Poly(lactic acid) PLA fibers are first treated with a catalyst, tin oxalate (SnOx), to lower their depolymerization temperature. The fibers are embedded in a thermoset matrix and then vaporized to leave behind straight channels (1D dimensionality). In this study, we extend the application of sacrificial PLA and VaSC to all levels of spatial dimensionality (0D–3D). Sacrificial PLA templates of each level of dimensionality: 0D-spheres, 1D-fibers, 2D-sheets, and 3D-printed structures are fabricated. Two different tin catalysts (tin oxalate, SnOx, and tin octoate, SnOc) are incorporated into PLA to promote depolymerization at modest temperatures (~200°C). Spheres with diameters averaging 23 μm are fabricated using an emulsion/solvent evaporation technique. Fibers spanning two orders of magnitude in diameter are fabricated using electrospinning (~5 μm) and melt-spinning (~300 μm) techniques. Sheets (~550 μm thick) are hot-pressed and laser cut to form branched planar networks. Fused deposition modeling is used to create a 3D branching tree-like structure. Each template is embedded in epoxy and removed using VaSC (200°C in a vacuum oven, 24–48 h) to reveal the inverse of the template architecture. The effectiveness of VaSC is evaluated using isothermal thermogravimetric analysis (iTGA) at 200°C (ex situ), and by tracking weight of the embedded °C in a vacuum oven (in situ). The templates in epoxy subjected to 200°C choice of catalyst influences the vaporization time with SnOc promoting more rapid removal. Comparison of in situ and ex situ tests reveals a delay in VaSC completion in the embedded state. The structures created using template materials from each level of dimensionality (0D–3D) are evaluated by flow rate testing. Experiments were performed under laminar flow conditions and compared to appropriate predictive models. Structures tested include porous sheets, 1D channels, a 2D-bifurcating network, and a 3D-branched tree-like structure. Flow in porous sheets is compared to Darcy’s law using a porosity-permeability correlation, whereas flow in one-dimensional channels is compared to the Hagen–Poiseuille equation. Computational fluid dynamics simulations of flow in both the 2D and 3D structure are performed in ANSYS FLUENT. Experimental data agreed well with modeling/simulation for every level of dimensionality. Sacrificial templates provide a technique to form multiscale, multidimensional, and interconnected vascular and porous networks in thermosetting polymers. Further work in this area will focus on extending the concept to more types of polymers and improving precision and resolution in complex 2D and 3D structures. REFERENCE [1] Esser-Kahn et al.. Adv. Mater. 2011, 23, 3654

TMEM184b promotes axon degeneration and neuromuscular junction maintenance

UNLABELLED: Complex nervous systems achieve proper connectivity during development and must maintain these connections throughout life. The processes of axon and synaptic maintenance and axon degeneration after injury are jointly controlled by a number of proteins within neurons, including ubiquitin ligases and mitogen activated protein kinases. However, our understanding of these molecular cascades is incomplete. Here we describe the phenotype resulting from mutation of TMEM184b, a protein identified in a screen for axon degeneration mediators. TMEM184b is highly expressed in the mouse nervous system and is found in recycling endosomes in neuronal cell bodies and axons. Disruption of TMEM184b expression results in prolonged maintenance of peripheral axons following nerve injury, demonstrating a role for TMEM184b in axon degeneration. In contrast to this protective phenotype in axons, uninjured mutant mice have anatomical and functional impairments in the peripheral nervous system. Loss of TMEM184b causes swellings at neuromuscular junctions that become more numerous with age, demonstrating that TMEM184b is critical for the maintenance of synaptic architecture. These swellings contain abnormal multivesicular structures similar to those seen in patients with neurodegenerative disorders. Mutant animals also show abnormal sensory terminal morphology. TMEM184b mutant animals are deficient on the inverted screen test, illustrating a role for TMEM184b in sensory-motor function. Overall, we have identified an important function for TMEM184b in peripheral nerve terminal structure, function, and the axon degeneration pathway. SIGNIFICANCE STATEMENT: Our work has identified both neuroprotective and neurodegenerative roles for a previously undescribed protein, TMEM184b. TMEM184b mutation causes delayed axon degeneration following peripheral nerve injury, indicating that it participates in the degeneration process. Simultaneously, TMEM184b mutation causes progressive structural abnormalities at neuromuscular synapses and swellings within sensory terminals, and animals with this mutation display profound weakness. Thus, TMEM184b is necessary for normal peripheral nerve terminal morphology and maintenance. Loss of TMEM184b results in accumulation of autophagosomal structures in vivo, fitting with emerging studies that have linked autophagy disruption and neurological disease. Our work recognizes TMEM184b as a new player in the maintenance of the nervous system

Discovery of Fur binding site clusters in Escherichia coli by information theory models

Fur is a DNA binding protein that represses bacterial iron uptake systems. Eleven footprinted Escherichia coli Fur binding sites were used to create an initial information theory model of Fur binding, which was then refined by adding 13 experimentally confirmed sites. When the refined model was scanned across all available footprinted sequences, sequence walkers, which are visual depictions of predicted binding sites, frequently appeared in clusters that fit the footprints (∼83% coverage). This indicated that the model can accurately predict Fur binding. Within the clusters, individual walkers were separated from their neighbors by exactly 3 or 6 bases, consistent with models in which Fur dimers bind on different faces of the DNA helix. When the E. coli genome was scanned, we found 363 unique clusters, which includes all known Fur-repressed genes that are involved in iron metabolism. In contrast, only a few of the known Fur-activated genes have predicted Fur binding sites at their promoters. These observations suggest that Fur is either a direct repressor or an indirect activator. The Pseudomonas aeruginosa and Bacillus subtilis Fur models are highly similar to the E. coli Fur model, suggesting that the Fur–DNA recognition mechanism may be conserved for even distantly related bacteria

THE EFFECT OF PLASMODIUM FLORIDENSE ON RELATIVE LEUKOCYTE COUNTS OF ANOLIS SAGREI AND A. CAROLINENSIS IN FLORIDA, USA

Native Green Anoles, Anolis carolinensis, and invasive Brown Anoles, Anolis sagrei, are commonly found in Florida and may be infected with the malarial parasite, Plasmodium floridense. Because no studies have directly addressed health effects of the parasite on Florida anoles, we collected blood smears of infected and uninfected anoles from Central and Southwest Florida and compared the overall leukocyte (WBC) counts, eosinophil counts, and heterophil/lymphocyte ratios. Eosinophils are generally elevated in response to protozoal infection and heterophil/lymphocyte ratios are often altered due to stress. A generalized linear model that tested contributions to erythrocyte/leukocyte ratios included infection status and locality as significant factors. We found significant differences in WBC counts between infected and uninfected lizards in Central Florida but not in Southwest Florida. Central Florida anoles also had higher mean WBC counts than Southwest Florida anoles. We did not detect significant differences in eosinophil counts or H/L ratios related to infection status. Our project is the first to examine leukocyte effects of Plasmodium infection in anoles and to provide leukocyte profiles of Anolis lizards. It appears that infected anoles sustain some negative immunological effects, at least in Central Florida. The differences in regions may be caused by the fact that Central Florida anoles still are under continuous interspecific competition whereas the Southwest Florida Brown Anoles are not because of low populations of Green Anoles. Additional studies that address leukocyte levels related to Plasmodium infection are needed to tease out the health and fitness effects on the lizards of Florida