Lipolytic Activity and the Utilization of Fatty Acids Associated with Bat Sebum by Pseudogymnoascus destructans

Mycopathologia - Pseudogymnoascus destructans is the causative agent of a fungal infection of bats known as white-nose syndrome (WNS). Since its discovery in 2006, it has been responsible for...     

MyoD(-/-) satellite cells in single-fiber culture are differentiation defective and MRF4 deficient

MyoD-deficient mice are without obvious deleterious muscle phenotype during embryogenesis and fetal development, and adults in the laboratory have grossly normal skeletal muscle and life span. However, a previous study showed that in the context of muscle degeneration on a mdx (dystrophin null) genetic background, animals lacking MyoD have a greatly intensified disease phenotype leading to lethality not otherwise seen in mdx mice. Here we have examined MyoD(-/-) adult muscle fibers and their associated satellite cells in single myofiber cultures and describe major phenotypic differences found at the tissue, cellular, and molecular levels. The steady-state number of satellite cells on freshly isolated MyoD(-/-) fibers was elevated and abnormal branched fiber morphologies were observed, the latter suggesting chronic muscle regeneration in vivo. Single-cell RNA coexpression analyses were performed for c-met, m-cadherin, and the four myogenic regulatory factors (MRFs.) Most mutant satellite cells entered the cell cycle and upregulated expression of myf5, both characteristic early steps in satellite cell maturation. However, they later failed to normally upregulate MRF4, displayed a major deficit in m-cadherin expression, and showed a significant diminution in myogenin-positive status compared with wildtype. MyoD(-/-) satellite cells formed unusual aggregate structures, failed to fuse efficiently, and showed greater than 90% reduction in differentiation efficiency relative to wildtype. A further survey of RNAs encoding regulators of growth and differentiation, cell cycle progression, and cell signaling revealed similar or identical expression profiles for most genes as well as several noteworthy differences. Among these, GDF8 and Msx1 were identified as potentially important regulators of the quiescent state whose expression profile differs between mutant and wildtype. Considered together, these data suggest that activated MyoD(-/-) satellite cells assume a phenotype that resembles in some ways a developmentally "stalled" cell compared to wildtype. However, the MyoD(-/-) cells are not merely developmentally immature, as they also display novel molecular and cellular characteristics that differ from any observed in wild-type muscle precursor counterparts of any stage.     

Eph/ephrin interactions modulate muscle satellite cell motility and patterning

During development and regeneration, directed migration of cells, including neural crest cells, endothelial cells, axonal growth cones and many types of adult stem cells, to specific areas distant from their origin is necessary for their function. We have recently shown that adult skeletal muscle stem cells (satellite cells), once activated by isolation or injury, are a highly motile population with the potential to respond to multiple guidance cues, based on their expression of classical guidance receptors. We show here that, in vivo, differentiated and regenerating myofibers dynamically express a subset of ephrin guidance ligands, as well as Eph receptors. This expression has previously only been examined in the context of muscle-nerve interactions; however, we propose that it might also play a role in satellite cell-mediated muscle repair. Therefore, we investigated whether Eph-ephrin signaling would produce changes in satellite cell directional motility. Using a classical ephrin 'stripe' assay, we found that satellite cells respond to a subset of ephrins with repulsive behavior in vitro; patterning of differentiating myotubes is also parallel to ephrin stripes. This behavior can be replicated in a heterologous in vivo system, the hindbrain of the developing quail, in which neural crest cells are directed in streams to the branchial arches and to the forelimb of the developing quail, where presumptive limb myoblasts emigrate from the somite. We hypothesize that guidance signaling might impact multiple steps in muscle regeneration, including escape from the niche, directed migration to sites of injury, cell-cell interactions among satellite cell progeny, and differentiation and patterning of regenerated muscle.     

Neural cell adhesion molecule (NCAM) marks adult myogenic cells committed to differentiation

Although recent advances in broad-scale gene expression analysis have dramatically increased our knowledge of the repertoire of mRNAs present in multiple cell types, it has become increasingly clear that examination of the expression, localization, and associations of the encoded proteins will be critical for determining their functional significance. In particular, many signaling receptors, transducers, and effectors have been proposed to act in higher-order complexes associated with physically distinct areas of the plasma membrane. Adult muscle stem cells (satellite cells) must, upon injury, respond appropriately to a wide range of extracellular stimuli: the role of such signaling scaffolds is therefore a potentially important area of inquiry. To address this question, we first isolated detergent-resistant membrane fractions from primary satellite cells, then analyzed their component proteins using liquid chromatography-tandem mass spectrometry. Transmembrane and juxtamembrane components of adhesion-mediated signaling pathways made up the largest group of identified proteins; in particular, neural cell adhesion molecule (NCAM), a multifunctional cell-surface protein that has previously been associated with muscle regeneration, was significant. Immunohistochemical analysis revealed that not only is NCAM localized to discrete areas of the plasma membrane, it is also a very early marker of commitment to terminal differentiation. Using flow cytometry, we have sorted physically homogeneous myogenic cultures into proliferating and differentiating fractions based solely upon NCAM expression.     

Muscle spindle alterations precede onset of sensorimotor deficits in Charcot–Marie–Tooth type 2E

Charcot–Marie–Tooth (CMT) is the most common inherited peripheral neuropathy, affecting approximately 2.8 million people. The CMT leads to distal neuropathy that is characterized by reduced motor nerve conduction velocity, ataxia, muscle atrophy and sensory loss. We generated a mouse model of CMT type 2E (CMT2E) expressing human neurofilament light E396K (hNF‐L^E396K), which develops decreased motor nerve conduction velocity, ataxia and muscle atrophy by 4 months of age. Symptomatic hNF‐L^E396K mice developed phenotypes that were consistent with proprioceptive sensory defects as well as reduced sensitivity to mechanical stimulation, while thermal sensitivity and auditory brainstem responses were unaltered. Progression from presymptomatic to symptomatic included a 50% loss of large diameter sensory axons within the fifth lumbar dorsal root of hNF‐L^E396K mice. Owing to proprioceptive deficits and loss of large diameter sensory axons, we analyzed muscle spindle morphology in presymptomatic and symptomatic hNF‐L^E396K and hNF‐L control mice. Muscle spindle cross‐sectional area and volume were reduced in all hNF‐L^E396K mice analyzed, suggesting that alterations in muscle spindle morphology occurred prior to the onset of typical CMT pathology. These data suggested that CMT2E pathology initiated in the muscle spindles altering the proprioceptive sensory system. Early sensory pathology in CMT2E could provide a unifying hypothesis for the convergence of pathology observed in CMT.     

Inhibitors of foot and mouth disease virus targeting a novel pocket of the RNA-dependent RNA polymerase

Background

Foot-and-Mouth Disease Virus (FMDV) is a picornavirus that infects cloven-hoofed animals and leads to severe losses in livestock production. In the case of an FMD outbreak, emergency vaccination requires at least 7 days to trigger an effective immune response. There are currently no approved inhibitors for the treatment or prevention of FMDV infections.

Methodology/Principal Findings

Using a luciferase-based assay we screened a library of compounds and identified seven novel inhibitors of 3Dpol, the RNA-dependent RNA polymerase of FMDV. The compounds inhibited specifically 3Dpol (IC₅₀s from 2-17 µM) and not other viral or bacterial polymerases. Enzyme kinetic studies on the inhibition mechanism by compounds 5D9 and 7F8 showed that they are non-competitive inhibitors with respect to NTP and nucleic acid substrates. Molecular modeling and docking studies into the 3Dpol structure revealed an inhibitor binding pocket proximal to, but distinct from the 3Dpol catalytic site. Residues surrounding this pocket are conserved among all 60 FMDV subtypes. Site directed mutagenesis of two residues located at either side of the pocket caused distinct resistance to the compounds, demonstrating that they indeed bind at this site. Several compounds inhibited viral replication with 5D9 suppressing virus production in FMDV-infected cells with EC₅₀ = 12 µM and EC₉₀ = 20 µM).

Significance

We identified several non-competitive inhibitors of FMDV 3Dpol that target a novel binding pocket, which can be used for future structure-based drug design studies. Such studies can lead to the discovery of even more potent antivirals that could provide alternative or supplementary options to contain future outbreaks of FMD.     

Cultural barriers to compassionate care--patients' and health professionals' perspectives

Cultural barriers to compassionate care are presented from two perspectives in healthcare: the patient and the health professional. Content areas include autonomy clashes between the patient and health professional, end-of-life values and preferences, cultural responses to pain and pain management, and the problem of stereotypical and uncaring behaviors toward people different from ourselves. Suggestions to address content areas caused by conflicting value systems include (1) a review of the literature on diversity, and (2) discovering one's sensitivity through exposure to various ethnic and cultural groups.