Ice-free cryopreservation of heart valve allografts: better extracellular matrix preservation in vivo and preclinical results

The purpose of this study was evaluation of an ice-free cryopreservation method for heart valves in an allogeneic juvenile pulmonary sheep implant model and comparison with traditionally frozen cryopreserved valves. Hearts of 15 crossbred Whiteface sheep were procured in Minnesota. The valves were processed in South Carolina and the pulmonary valves implanted orthotopically in 12 black faced Heidschnucke sheep in Germany. The ice-free cryopreserved valves were cryopreserved in 12.6?mol/l cryoprotectant (4.65, 4.65, and 3.31?mol/l of dimethylsulfoxide, formamide and 1,2-propanediol) and stored at ?80°C. Frozen valves were cryopreserved by controlled slow rate freezing in 1.4?mol/l dimethylsulfoxide and stored in vapor-phase nitrogen. Aortic valve tissues were used to evaluate the impact of preservation without implantation. Multiphoton microscopy revealed reduced but not significantly damaged extracellular matrix before implantation in frozen valves compared with ice-free tissues. Viability assessment revealed significantly less metabolic activity in the ice-free valve leaflets and artery samples compared with frozen tissues (P?<?0.05). After 3 and 6?months in vivo valve function was determined by two-dimensional echo-Doppler and at 7?months the valves were explanted. Severe valvular stenosis with right heart failure was observed in recipients of frozen valves, the echo data revealed increased velocity and pressure gradients compared to ice-free valve recipients (P?=?0.0403, P?=?0.0591). Histo-pathology showed significantly thickened leaflets in the frozen valves (P?<?0.05) and infiltrating CD3+ T-cells (P?<?0.05) compared with ice-free valve leaflets. Multiphoton microscopy at explant revealed reduced inducible autofluorescence and extracellular matrix damage in the frozen explants and well preserved structures in the ice-free explant leaflets. In conclusion, ice-free cryopreservation of heart valve transplants at ?80°C avoids ice formation, tissue-glass cracking and preserves extracellular matrix integrity resulting in minimal inflammation and improved hemodynamics in allogeneic juvenile sheep.     

A death effector domain chain DISC model reveals a crucial role for caspase-8 chain assembly in mediating apoptotic cell death

Formation of the death-inducing signaling complex (DISC) is a critical step in death receptor-mediated apoptosis, yet the mechanisms underlying assembly of this key multiprotein complex remain unclear. Using quantitative mass spectrometry, we have delineated the stoichiometry of the native TRAIL DISC. While current models suggest that core DISC components are present at a ratio of 1:1, our data indicate that FADD is substoichiometric relative to TRAIL-Rs or DED-only proteins; strikingly, there is up to 9-fold more caspase-8 than FADD in the DISC. Using structural modeling, we propose an alternative DISC model in which procaspase-8 molecules interact sequentially, via their DED domains, to form a caspase-activating chain. Mutating key interacting residues in procaspase-8 DED2 abrogates DED chain formation in cells and disrupts TRAIL/CD95 DISC-mediated procaspase-8 activation in?a functional DISC reconstitution model. This provides direct experimental evidence for a DISC model in which DED chain assembly drives caspase-8 dimerization/activation, thereby triggering cell death.     

Widespread state-dependent shifts in cerebellar activity in locomoting mice

Excitatory drive enters the cerebellum via mossy fibers, which activate granule cells, and climbing fibers, which activate Purkinje cell dendrites. Until now, the coordinated regulation of these pathways has gone unmonitored in spatially resolved neuronal ensembles, especially in awake animals. We imaged cerebellar activity using functional two-photon microscopy and extracellular recording in awake mice locomoting on an air-cushioned spherical treadmill. We recorded from putative granule cells, molecular layer interneurons, and Purkinje cell dendrites in zone A of lobule IV/V, representing sensation and movement from trunk and limbs. Locomotion was associated with widespread increased activity in granule cells and interneurons, consistent with an increase in mossy fiber drive. At the same time, dendrites of different Purkinje cells showed increased co-activation, reflecting increased synchrony of climbing fiber activity. In resting animals, aversive stimuli triggered increased activity in granule cells and interneurons, as well as increased Purkinje cell co-activation that was strongest for neighboring dendrites and decreased smoothly as a function of mediolateral distance. In contrast with anesthetized recordings, no 1-10 Hz oscillations in climbing fiber activity were evident. Once locomotion began, responses to external stimuli in all three cell types were strongly suppressed. Thus climbing and mossy fiber representations can shift together within a fraction of a second, reflecting in turn either movement-associated activity or external stimuli.     

The retrograde frequency response of passive dendritic trees constrains the nonlinear firing behaviour of a reduced neuron model

Our goal was to investigate how the propagation of alternating signals (i.e. AC), like action potentials, into the dendrites influenced nonlinear firing behaviour of motor neurons using a systematically reduced neuron model. A recently developed reduced modeling approach using only steady-current (i.e. DC) signaling was analytically expanded to retain features of the frequency-response analysis carried out in multicompartment anatomically reconstructed models. Bifurcation analysis of the extended model showed that the typically overlooked parameter of AC amplitude attenuation was positively correlated with the current threshold for the activation of a plateau potential in the dendrite. Within the multiparameter space map of the reduced model the region demonstrating "fully-bistable" firing was bounded by directional DC attenuation values that were negatively correlated to AC attenuation. Based on these results we conclude that analytically derived reduced models of dendritic trees should be fit on DC and AC signaling, as both are important biophysical parameters governing the nonlinear firing behaviour of motor neurons.     

The role of dorsal root ganglia activation and brain-derived neurotrophic factor in multiple sclerosis

Multiple sclerosis (MS) is characterized by focal destruction of the white matter of the brain and spinal cord. The exact mechanisms underlying the pathophysiology of the disease are unknown. Many studies have shown that MS is predominantly an autoimmune disease with an inflammatory phase followed by a demyelinating phase. Recent studies alongside current treatment strategies, including glatiramer acetate, have revealed a potential role for brain-derived neurotrophic factor (BDNF) in MS. However, the exact role of BDNF is not fully understood. We used the experimental autoimmune encephalomyelitis (EAE) model of MS in adolescent female Lewis rats to identify the role of BDNF in disease progression. Dorsal root ganglia (DRG) and spinal cords were harvested for protein and gene expression analysis every 3 days post-disease induction (pdi) up to 15 days. We show significant increases in BDNF protein and gene expression in the DRG of EAE animals at 12 dpi, which correlates with peak neurological disability. BDNF protein expression in the spinal cord was significantly increased at 12 dpi, and maintained at 15 dpi. However, there was no significant change in mRNA levels. We show evidence for the anterograde transport of BDNF protein from the DRG to the dorsal horn of the spinal cord via the dorsal roots. Increased levels of BDNF within the DRG and spinal cord in EAE may facilitate myelin repair and neuroprotection in the CNS. The anterograde transport of DRG-derived BDNF to the spinal cord may have potential implications in facilitating central myelin repair and neuroprotection.     

Lack of Innate Interferon Responses during SARS Coronavirus Infection in a Vaccination and Reinfection Ferret Model

In terms of its highly pathogenic nature, there remains a significant need to further define the immune pathology of SARS-coronavirus (SARS-CoV) infection, as well as identify correlates of immunity to help develop vaccines for severe coronaviral infections. Here we use a SARS-CoV infection-reinfection ferret model and a functional genomics approach to gain insight into SARS immunopathogenesis and to identify correlates of immune protection during SARS-CoV-challenge in ferrets previously infected with SARS-CoV or immunized with a SARS virus vaccine. We identified gene expression signatures in the lungs of ferrets associated with primary immune responses to SARS-CoV infection and in ferrets that received an identical second inoculum. Acute SARS-CoV infection prompted coordinated innate immune responses that were dominated by antiviral IFN response gene (IRG) expression. Reinfected ferrets, however, lacked the integrated expression of IRGs that was prevalent during acute infection. The expression of specific IRGs was also absent upon challenge in ferrets immunized with an inactivated, Al(OH)₃-adjuvanted whole virus SARS vaccine candidate that protected them against SARS-CoV infection in the lungs. Lack of IFN-mediated immune enhancement in infected ferrets that were previously inoculated with, or vaccinated against, SARS-CoV revealed 9 IRG correlates of protective immunity. This data provides insight into the molecular pathogenesis of SARS-CoV and SARS-like-CoV infections and is an important resource for the development of CoV antiviral therapeutics and vaccines.     

Group I mGluR5 metabotropic glutamate receptors regulate proliferation of neuronal progenitors in specific forebrain developmental domains

Major classical neurotransmitters including GABA and glutamate play novel morphogenic roles during development of the mammalian CNS. During forebrain neurogenesis, glutamate regulates neuroblast proliferation in different germinal domains using receptor subtype-specific mechanisms. For example, ionotropic N -methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptors mediate distinct proliferative effects in ventral or dorsal forebrain germinal domains, and regulate the correct number of neurons that populate the striatum or cerebral cortex. Recent work suggests metabotropic receptors may also mediate glutamate's proliferative effects. Group I mGluR5 receptor subtypes are highly expressed in forebrain germinal zones. Using in vitro and in vivo methods, we demonstrate mGluR5 receptor activation plays an important role in neuroblast proliferation in the ventral telencephalon, and helps determine the complement of striatum projection neurons. mGluR5 receptor-mediated effects on striatal neuronal progenitors are restricted mainly to early cycling populations in the ventricular zone, with little effect on secondary proliferative populations in the subventricular zone. In contrast to proliferative effects in the ventral telencephalon, mGluR5 receptors do not modulate proliferation of dorsal telencephalon-derived cortical neuroblasts. Heterogeneous domain-specific proliferative effects of glutamate-mediated by specific receptor subtypes provide an important developmental mechanism allowing generation of the correct complement of neuronal subtypes that populate the mammalian forebrain.     

Lubiprostone activates non-CFTR-dependent respiratory epithelial chloride secretion in cystic fibrosis mice

Periciliary fluid balance is maintained by the coordination of sodium and chloride channels in the apical membranes of the airways. In the absence of the cystic fibrosis transmembrane regulator (CFTR), chloride secretion is diminished and sodium reabsorption exaggerated. ClC-2, a pH- and voltage-dependent chloride channel, is present on the apical membranes of airway epithelial cells. We hypothesized that ClC-2 agonists would provide a parallel pathway for chloride secretion. Using nasal potential difference (NPD) measurements, we quantified lubiprostone-mediated Cl(-) transport in sedated cystic fibrosis null (gut-corrected), C57Bl/6, and A/J mice during nasal perfusion of lubiprostone (a putative ClC-2 agonist). Baseline, amiloride-inhibited, chloride-free gluconate-substituted Ringer with amiloride and low-chloride Ringer plus lubiprostone (at increasing concentrations of lubiprostone) were perfused, and the NPD was continuously recorded. A clear dose-response relationship was detected in all murine strains. The magnitude of the NPD response to 20 muM lubiprostone was -5.8 +/- 2.1 mV (CF, n = 12), -8.1 +/- 2.6 mV (C57Bl/6 wild-type, n = 12), and -5.3 +/- 1.2 mV (AJ wild-type, n = 8). A cohort of ClC-2 knockout mice did not respond to 20 muM lubiprostone (n = 6, P = 0.27). In C57Bl/6 mice, inhibition of CFTR with topical application of CFTR inhibitor-172 did not abolish the lubiprostone response, thus confirming the response seen is independent of CFTR regulation. RT-PCR confirmed expression of ClC-2 mRNA in murine lung homogenate. The direct application of lubiprostone in the CF murine nasal airway restores nearly normal levels of chloride secretion in nasal epithelia.     

Exploring Nitrilase Sequence Space for Enantioselective Catalysis

Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10⁶ to 10¹⁰ members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.