The Underlying Effects of Celiac Disease and Subsequent Implications on Deployment in the United States Army.

INTRODUCTION: The purpose of this review is to provide an overview of the etiology, pathology, and treatments for celiac disease (CD), as well as to provide context as to how CD impacts the U.S. military. MATERIALS AND METHODS: To conduct this review, the authors surveyed recent epidemiology and immunology literature in order to provide a detailed summary of the current understanding of CD, its diagnosis, and the real-world impacts within the Department of Defense (DoD). RESULTS: We described the gluten proteins and both the immune response in CD. We further describe the underlying genetic risk factors and diagnosis and pathogenesis of the disease and conclude the review with a discussion of how current DoD regulations impact U.S. military readiness. CONCLUSION: Celiac disease (CD) is an autoimmune disorder that results in damage to the small intestine. Ingestion of gluten in a CD patient is usually followed by villous atrophy in the small intestine, often along with other gastrointestinal symptoms. Around 1% of patients diagnosed with CD can experience complications if gluten-free diet is not followed, including intestinal lymphoma and hyposplenism. Therefore, a patient showing possible symptoms should discuss the diagnostic process with their healthcare providers to ensure adequate understanding of serological and genetic tests along with the histological examination of intestinal biopsy. Patients should seek consults with registered dietitians to structure their diets appropriately. Considering the prevalence and incidence of CD and gluten intolerances are increasing, the military should consider providing gluten-free Meals Ready-to-Eat as an option for all service members. Given the retention of service members with CD, subsequent admission of personnel with mild CD that does not affect the duties will allow the DoD access to a growing population of fully capable service members with critical technical skills who are eager to serve the USA.

Progress and Future Directions of the NCAA-DoD Concussion Assessment, Research, and Education (CARE) Consortium and Mind Matters Challenge at the US Service Academies.

Despite the significant impact that concussion has on military service members, significant gaps remain in our understanding of the optimal diagnostic, management, and return to activity/duty criteria to mitigate the consequences of concussion. In response to these significant knowledge gaps, the US Department of Defense (DoD) and the National Collegiate Athletic Association (NCAA) partnered to form the NCAA-DoD Grand Alliance in 2014. The NCAA-DoD CARE Consortium was established with the aim of creating a national multisite research network to study the clinical and neurobiological natural history of concussion in NCAA athletes and military Service Academy cadets and midshipmen. In addition to the data collected for the larger CARE Consortium effort, the service academies have pursued military-specific lines of research relevant to operational and medical readiness associated with concussion. The purpose of this article is to describe the structure of the NCAA-DoD Grand Alliance efforts at the service academies, as well as discuss military-specific research objectives and provide an overview of progress to date. A secondary objective is to discuss the challenges associated with conducting large-scale studies in the Service Academy environment and highlight future directions for concussion research endeavors across the CARE Service Academy sites.

Persistent motor dysfunction despite homeostatic rescue of cerebellar morphogenesis in the Car8 waddles mutant mouse.

BACKGROUND: Purkinje cells play a central role in establishing the cerebellar circuit. Accordingly, disrupting Purkinje cell development impairs cerebellar morphogenesis and motor function. In the Car8wdl mouse model of hereditary ataxia, severe motor deficits arise despite the cerebellum overcoming initial defects in size and morphology. METHODS: To resolve how this compensation occurs, we asked how the loss of carbonic anhydrase 8 (CAR8), a regulator of IP3R1 Ca2+ signaling in Purkinje cells, alters cerebellar development in Car8wdl mice. Using a combination of histological, physiological, and behavioral analyses, we determined the extent to which the loss of CAR8 affects cerebellar anatomy, neuronal firing, and motor coordination during development. RESULTS: Our results reveal that granule cell proliferation is reduced in early postnatal mutants, although by the third postnatal week there is enhanced and prolonged proliferation, plus an upregulation of Sox2 expression in the inner EGL. Modified circuit patterning of Purkinje cells and Bergmann glia accompany these granule cell adjustments. We also find that although anatomy eventually normalizes, the abnormal activity of neurons and muscles persists. CONCLUSIONS: Our data show that losing CAR8 only transiently restricts cerebellar growth, but permanently damages its function. These data support two current hypotheses about cerebellar development and disease: (1) Sox2 expression may be upregulated at sites of injury and contribute to the rescue of cerebellar structure and (2) transient delays to developmental processes may precede permanent motor dysfunction. Furthermore, we characterize waddles mutant mouse morphology and behavior during development and propose a Sox2-positive, cell-mediated role for rescue in a mouse model of human motor diseases.

Determination of the size distribution of non-spherical nanoparticles by electric birefringence-based methods.

The in situ determination of the size distribution of dispersed non-spherical nanoparticles is an essential characterization tool for the investigation and use of colloidal suspensions. In this work, we test a size characterization method based on the measurement of the transient behaviour of the birefringence induced in the dispersions by pulsed electric fields. The specific shape of such relaxations depends on the distribution of the rotational diffusion coefficient of the suspended particles. We analyse the measured transient birefringence with three approaches: the stretched-exponential, Watson-Jennings, and multi-exponential methods. These are applied to six different types of rod-like and planar particles: PTFE rods, goethite needles, single- and double-walled carbon nanotubes, sodium montmorillonite particles and gibbsite platelets. The results are compared to electron microscopy and dynamic light scattering measurements. The methods here considered provide good or excellent results in all cases, proving that the analysis of the transient birefringence is a powerful tool to obtain complete size distributions of non-spherical particles in suspension.

T cells presenting viral antigens or autoantigens induce cytotoxic T cell anergy.

In the course of modeling the naturally occurring tumor immunity seen in patients with paraneoplastic cerebellar degeneration (PCD), we discovered an unexpectedly high threshold for breaking CD8+ cytotoxic T cell (CTL) tolerance to the PCD autoantigen, CDR2. While CDR2 expression was previously found to be strictly restricted to immune-privileged cells (cerebellum, testes, and tumors), unexpectedly we have found that T cells also express CDR2. This expression underlies inhibition of CTL activation; CTLs that respond to epithelial cells expressing CDR2 fail to respond to T cells expressing CDR2. This was a general phenomenon, as T cells presenting influenza (flu) antigen also fail to activate otherwise potent flu-specific CTLs either in vitro or in vivo. Moreover, transfer of flu peptide-pulsed T cells into flu-infected mice inhibits endogenous flu-specific CTLs. Our finding that T cells serve as a site of immune privilege, inhibiting effector CTL function, uncovers an autorepressive loop with general biologic and clinical relevance.

Proton-Assisted Recoupling (PAR) in Peptides and Proteins.

Proton-assisted recoupling (PAR) is examined by exploring optimal experimental conditions and magnetization transfer rates in a variety of biologically relevant nuclear spin-systems, including simple amino acids, model peptides, and two proteins-nanocrystalline protein G (GB1), and importantly amyloid beta 1-42 (M0Aß1-42) fibrils. A selective PAR protocol, SUBPAR (setting up better proton assisted recoupling), is described to observe magnetization transfer in one-dimensional spectra, which minimizes experiment time (in comparison to two-dimensional experiments) and thereby enables an efficient assessment of optimal PAR conditions for a desired magnetization transfer. In the case of the peptide spin systems, experimental and simulated PAR data sets are compared on a semiquantitative level, thereby elucidating the interactions influencing PAR magnetization transfer and their manifestations in different spin transfer networks. Using the optimum Rabi frequencies determined by SUBPAR, PAR magnetization transfer trajectories (or buildup curves) were recorded and compared to simulated results for short peptides. PAR buildup curves were also recorded for M0Aß1-42 and examined conjointly with a recent structural model. The majority of salient cross-peak intensities observed in the M0Aß1-42 PAR spectra are well-modeled with a simple biexponential equation, although the fitting parameters do not show any strong correlation to internuclear distances. Nevertheless, these parameters provide a wealth of invaluable semiquantitative structural constraints for the M0Aß1-42. The results presented here offer a complete protocol for recording PAR 13C-13C correlation spectra with high-efficiency and using the resulting information in protein structural studies.

3D MAS NMR Experiment Utilizing Through-Space 15N-15N Correlations.

We demonstrate a novel 3D NNC magic angle spinning NMR experiment that generates 15N-15N internuclear contacts in protein systems using an optimized 15N-15N proton assisted recoupling (PAR) mixing period and a 13C dimension for improved resolution. The optimized PAR condition permits the acquisition of high signal-to-noise 3D data that enables backbone chemical shift assignments using a strategy that is complementary to current schemes. The spectra can also provide distance constraints. The utility of the experiment is demonstrated on an M0Aß1-42 fibril sample that yields high-quality data that is readily assigned and interpreted. The 3D NNC experiment therefore provides a powerful platform for solid-state protein studies and is broadly applicable to a variety of systems and experimental conditions.

Intrinsic Axonal Growth and the Drive for Regeneration.

Following damage to the adult nervous system in conditions like stroke, spinal cord injury, or traumatic brain injury, many neurons die and most of the remaining spared neurons fail to regenerate. Injured neurons fail to regrow both because of the inhibitory milieu in which they reside as well as a loss of the intrinsic growth capacity of the neurons. If we are to develop effective therapeutic interventions that promote functional recovery for the devastating injuries described above, we must not only better understand the molecular mechanisms of developmental axonal growth in hopes of re-activating these pathways in the adult, but at the same time be aware that re-activation of adult axonal growth may proceed via distinct mechanisms. With this knowledge in hand, promoting adult regeneration of central nervous system neurons can become a more tractable and realistic therapeutic endeavor.

Atomic Resolution Structure of Monomorphic Aß42 Amyloid Fibrils.

Amyloid-ß (Aß) is a 39-42 residue protein produced by the cleavage of the amyloid precursor protein (APP), which subsequently aggregates to form cross-ß amyloid fibrils that are a hallmark of Alzheimer's disease (AD). The most prominent forms of Aß are Aß1-40 and Aß1-42, which differ by two amino acids (I and A) at the C-terminus. However, Aß42 is more neurotoxic and essential to the etiology of AD. Here, we present an atomic resolution structure of a monomorphic form of AßM01-42 amyloid fibrils derived from over 500 (13)C-(13)C, (13)C-(15)N distance and backbone angle structural constraints obtained from high field magic angle spinning NMR spectra. The structure (PDB ID: 5KK3 ) shows that the fibril core consists of a dimer of Aß42 molecules, each containing four ß-strands in a S-shaped amyloid fold, and arranged in a manner that generates two hydrophobic cores that are capped at the end of the chain by a salt bridge. The outer surface of the monomers presents hydrophilic side chains to the solvent. The interface between the monomers of the dimer shows clear contacts between M35 of one molecule and L17 and Q15 of the second. Intermolecular (13)C-(15)N constraints demonstrate that the amyloid fibrils are parallel in register. The RMSD of the backbone structure (Q15-A42) is 0.71 ± 0.12 Å and of all heavy atoms is 1.07 ± 0.08 Å. The structure provides a point of departure for the design of drugs that bind to the fibril surface and therefore interfere with secondary nucleation and for other therapeutic approaches to mitigate Aß42 aggregation.

Extrinsic and intrinsic signals converge on the Runx1/CBFß transcription factor for nonpeptidergic nociceptor maturation.

The generation of diverse neuronal subtypes involves specification of neural progenitors and, subsequently, postmitotic neuronal differentiation, a relatively poorly understood process. Here, we describe a mechanism whereby the neurotrophic factor NGF and the transcription factor Runx1 coordinate postmitotic differentiation of nonpeptidergic nociceptors, a major nociceptor subtype. We show that the integrity of a Runx1/CBFß holocomplex is crucial for NGF-dependent nonpeptidergic nociceptor maturation. NGF signals through the ERK/MAPK pathway to promote expression of Cbfb but not Runx1 prior to maturation of nonpeptidergic nociceptors. In contrast, transcriptional initiation of Runx1 in nonpeptidergic nociceptor precursors is dependent on the homeodomain transcription factor Islet1, which is largely dispensable for Cbfb expression. Thus, an NGF/TrkA-MAPK-CBFß pathway converges with Islet1-Runx1 signaling to promote Runx1/CBFß holocomplex formation and nonpeptidergic nociceptor maturation. Convergence of extrinsic and intrinsic signals to control heterodimeric transcription factor complex formation provides a robust mechanism for postmitotic neuronal subtype specification.

HyperBIRD: a sensitivity-enhanced approach to collecting homonuclear-decoupled proton NMR spectra.

Samples prepared following dissolution dynamic nuclear polarization (DNP) enable the detection of NMR spectra from low-γ nuclei with outstanding sensitivity, yet have limited use for the enhancement of abundant species like (1)H nuclei. Small- and intermediate-sized molecules, however, show strong heteronuclear cross-relaxation effects: spontaneous processes with an inherent isotopic selectivity, whereby only the (13)C-bonded protons receive a polarization enhancement. These effects are here combined with a recently developed method that delivers homonuclear-decoupled (1)H spectra in natural abundance samples based on heteronuclear couplings to these same, (13)C-bonded nuclei. This results in the HyperBIRD methodology; a single-shot combination of these two effects that can simultaneously simplify and resolve complex, congested (1)H NMR spectra with many overlapping spin multiplets, while achieving 50-100 times sensitivity enhancements over conventional thermal counterparts.

Whole-mount imaging of mouse embryo sensory axon projections.

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkA(taulacZ) mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkA(taulacZ) line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkA(taulacZ)/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

Synchronized and concurrent experiments in Moving Tube NMR: using separate sample volumes for different pulse sequences.

This study presents a new application of sample shuttling with a long NMR tube (Moving Tube NMR, MT-NMR) as a method for collecting different experiments synchronously or even concurrently using separate sample regions. Synchronized experiments were performed using an automated shuttling apparatus to move different sample regions into the coil between transients such that each experiment was collected using a separate, specific sample segment. Additionally, a 2D NOESY spectrum and a double quantum filtered COSY (DQCOSY) spectrum were collected concurrently by shuttling between two different sample regions during the NOESY mixing time. These applications of the Moving Tube technique show that it is a useful platform for compounded data acquisition to optimize spectrometer time by minimizing measurement times and avoiding problems arising from instrument and sample instabilities. Furthermore, collecting a DQCOSY during a 2D NOESY mixing time opens a wide array of possibilities, as this principle can be applied to collect any experiment during a NOESY mixing time provided that the mixing period is longer than the sum of the sample shuttling time plus a complete scan of the intermittent experiment. While this methodology relies on the use of a long sample tube, it does not require excessive sample volumes, as two milliliters is enough to constitute multiple sample regions.

B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS.

Activation of intrinsic growth programs that promote developmental axon growth may also facilitate axon regeneration in injured adult neurons. Here, we demonstrate that conditional activation of B-RAF kinase alone in mouse embryonic neurons is sufficient to drive the growth of long-range peripheral sensory axon projections in vivo in the absence of upstream neurotrophin signaling. We further show that activated B-RAF signaling enables robust regenerative growth of sensory axons into the spinal cord after a dorsal root crush as well as substantial axon regrowth in the crush-lesioned optic nerve. Finally, the combination of B-RAF gain-of-function and PTEN loss-of-function promotes optic nerve axon extension beyond what would be predicted for a simple additive effect. We conclude that cell-intrinsic RAF signaling is a crucial pathway promoting developmental and regenerative axon growth in the peripheral and central nervous systems.

Heteronuclear cross-relaxation effects in the NMR spectroscopy of hyperpolarized targets.

Dissolution dynamic nuclear polarization (DNP) enables high-sensitivity solution-phase NMR experiments on long-lived nuclear spin species such as (15)N and (13)C. This report explores certain features arising in solution-state (1)H NMR upon polarizing low-γ nuclear species. Following solid-state hyperpolarization of both (13)C and (1)H, solution-phase (1)H NMR experiments on dissolved samples revealed transient effects, whereby peaks arising from protons bonded to the naturally occurring (13)C nuclei appeared larger than the typically dominant (12)C-bonded (1)H resonances. This enhancement of the satellite peaks was examined in detail with respect to a variety of mechanisms that could potentially explain this observation. Both two- and three-spin phenomena active in the solid state could lead to this kind of effect; still, experimental observations revealed that the enhancement originates from (13)C→(1)H polarization-transfer processes active in the liquid state. Kinetic equations based on modified heteronuclear cross-relaxation models were examined, and found to well describe the distinct patterns of growth and decay shown by the (13)C-bound (1)H NMR satellite resonances. The dynamics of these novel cross-relaxation phenomena were determined, and their potential usefulness as tools for investigating hyperpolarized ensembles and for obtaining enhanced-sensitivity (1)H NMR traces was explored.

Ultrafast NMR T1 relaxation measurements: probing molecular properties in real time.

The longitudinal relaxation properties of NMR active nuclei carry useful information about the site-specific chemical environments and about the mobility of molecular fragments. Molecular mobility is in turn a key parameter reporting both on stable properties, such as size, as well as on dynamic ones, such as transient interactions and irreversible aggregation. In order to fully investigate the latter, a fast sampling of the relaxation parameters of transiently formed molecular species may be needed. Nevertheless, the acquisition of longitudinal relaxation data is typically slow, being limited by the requirement that the time for which the nucleus relaxes be varied incrementally until a complete build-up curve is generated. Recently, a number of single-shot-inversion-recovery methods have been developed capable of alleviating this need; still, these may be challenged by either spectral resolution restrictions or when coping with very fast relaxing nuclei. Here, we present a new experiment to measure the T1s of multiple nuclear spins that experience fast longitudinal relaxation, while retaining full high-resolution chemical shift information. Good agreement is observed between T1s measured with conventional means and T1s measured using the new technique. The method is applied to the real-time investigation of the reaction between D-xylose and sodium borate, which is in turn elucidated with the aid of ancillary ultrafast and conventional 2D TOCSY measurements.

HyperSPASM NMR: a new approach to single-shot 2D correlations on DNP-enhanced samples.

Dissolution DNP experiments are limited to a single or at most a few scans, before the non-Boltzmann magnetization has been consumed. This makes it impractical to record 2D NMR data by conventional, t(1)-incremented schemes. Here a new approach termed HyperSPASM to establish 2D heteronuclear correlations in a single scan is reported, aimed at dealing with this kind of challenge. The HyperSPASM experiment relies on imposing an amplitude-modulation of the data by a single Δt(1) indirect-domain evolution time, and subsequently monitoring the imparted encoding on separate echo and anti-echo pathway signals within a single continuous acquisition. This is implemented via the use of alternating, switching, coherence selection gradients. As a result of these manipulations the phase imparted by a heteronucleus over its indirect domain evolution can be accurately extracted, and 2D data unambiguously reconstructed with a single-shot excitation. The nature of this sequence makes the resulting experiment particularly well suited for collecting indirectly-detected HSQC data on hyperpolarized samples. The potential of the ensuing HyperSPASM method is exemplified with natural-abundance hyperpolarized correlations on model systems.

Tracking cell lineage and fate into cerebellar circuits.

Understanding how cells from different neuronal and glial lineages contribute to functional circuits has been complicated by the difficulty in tracking cells as they integrate into brain circuits. Sudarov et al. (J Neurosci 31(30):11055-11069, 2011) used a powerful genetics-based lineage marking approach to birth date ventricular zone-derived cells in the mouse cerebellum. The authors use their novel tools to elucidate the spatial and temporal dynamics of how distinct ventricular zone lineages are generated and assemble into the cerebellar microcircuitry. In this journal club, we discuss and evaluate the author's major findings.