Gross morphology, histology, and ultrastructure of the olfactory rosette of a critically endangered indicator species, the Delta Smelt, Hypomesus transpacificus.

The Delta Smelt (Hypomesus transpacificus) is a small, semi-anadromous fish native to the San Francisco Bay-Delta Estuary and has been declared as critically endangered. Their olfactory biology, in particular, is poorly understood and a basic description of their sensory anatomy is needed to advance our understanding of the sensory ecology of species to inform conservation efforts to manage and protect them. We provide a description of the gross morphology, histological, immunohistochemical, and ultrastructural features of the olfactory rosette in this fish and discuss some of the functional implications in relation to olfactory ability. We show that Delta Smelt have a multilamellar olfactory rosette with allometric growth. Calretinin immunohistochemistry revealed a diffuse distribution of olfactory receptor neurons within the epithelium. Ciliated, microvillous and crypt neurons were clearly identified using morphological and immunohistochemical features. The olfactory neurons were supported by robust ciliated and secretory sustentacular cells. Although the sense of smell has been overlooked in Delta Smelt, we conclude that the olfactory epithelium has many characteristics of macrosmatic fish. With this study, we provide a foundation for future research into the sensory ecology of this imperiled fish.

Amdoparvovirus Infection in Red Pandas ( Ailurus fulgens).

Aleutian mink disease virus is the type species in the genus Amdoparvovirus, and in mink and other Mustelidae can cause either subclinical disease or fatal chronic immune stimulation and immune complex disease. The authors describe a novel amdoparvovirus in the endangered red panda ( Ailurus fulgens), discovered using viral metagenomics. The authors analyzed the prevalence, tissue distribution, and disease association by PCR, in situ hybridization, electron microscopy, and histology in a group of 6 red pandas from a single zoological collection. The study incorporates a fecal shedding survey and analysis of tissues from 4 necropsied animals over a 12-year span. The tentatively named red panda amdoparvovirus (RpAPV) was detected in the feces and/or tissues of all animals tested. At necropsy of 1 geriatric animal, infection was associated with pyogranulomatous peritonitis, pancreatitis, and myocarditis. Other animals had detectable low-level viral nucleic acid in lymph nodes and both oral and intestinal epithelium at the time of necropsy. Full-length genome sequences of RpAPV strains from 2 animals had 12% sequence divergence, demonstrating genetic diversity even among in-contact animals. RpAPV is a persistent infection in this cohort of red pandas, and has variable clinical expression.

Cell-free expression for nanolipoprotein particles: building a high-throughput membrane protein solubility platform.

Membrane-associated proteins and protein complexes account for approximately a third or more of the proteins in the cell (1, 2). These complexes mediate essential cellular processes; including signal transduc-tion, transport, recognition, bioenergetics and cell-cell communication. In general, membrane proteins are challenging to study because of their insolubility and tendency to aggregate when removed from their protein lipid bilayer environment. This chapter is focused on describing a novel method for producing and solubilizing membrane proteins that can be easily adapted to high-throughput expression screening. This process is based on cell-free transcription and translation technology coupled with nanolipoprotein par ticles (NLPs), which are lipid bilayers confined within a ring of amphipathic protein of defined diameter. The NLPs act as a platform for inserting, solubilizing and characterizing functional membrane proteins. NLP component proteins (apolipoproteins), as well as membrane proteins can be produced by either traditional cell-based or as discussed here, cell-free expression methodologies.

Avian pulmonary proteinosis: six cases and a review of the literature.

Pulmonary alveolar proteinosis (PAP) is a disease of surfactant clearance in which functional abnormalities in alveolar macrophages lead to accumulation of surfactant within alveoli in mammals. Histologic examination of 6 avian autopsies, including 4 chickens, a turkey, and a cockatiel, revealed accumulation of hypereosinophilic densely arrayed lamellar material in the lungs that was magenta by periodic acid-Schiff stain and diastase resistant. Transmission electron microscopy of the proteinaceous material in 2 cases demonstrated alternating electron-dense and electron-lucent lamellae that formed whorls and had a regular periodicity of 6-14 nm, consistent with pulmonary surfactant. Given the anatomic differences between avian and mammalian lungs, we designated the presented condition "pulmonary proteinosis," which can be observed as both an incidental finding or, when severe, may be a contributing factor to death through respiratory failure.

Identification of a thermo-regulated glutamine-binding protein from Yersinia pestis.

Here we present modeling and NMR spectroscopic evidence that the function of a Yersinia pestis pMT1 plasmid protein, designated as orf38, is most likely a glutamine binding protein. The modeling was homology-based at a very low level of sequence identity ( approximately 16%) and involved structural comparison of multiple templates, as well as template-substrate interaction analyses. Transferred nuclear Overhauser and saturation transfer difference experiments were used to characterize the binding of sugars and amino acids to orf38. The identification and characterization of an unknown protein function using the strategy presented here has applicability to a variety of research areas, including functional genomics and proteomics efforts.

Peptide stabilized amphotericin B nanodisks.

Nanometer scale apolipoprotein A-I stabilized phospholipid disk complexes (nanodisks; ND) have been formulated with the polyene antibiotic amphotericin B (AMB). The present studies were designed to evaluate if a peptide can substitute for the function of the apolipoprotein component of ND with respect to particle formation and stability. An 18-residue synthetic amphipathic alpha-helical peptide, termed 4F (Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH(2)), solubilized vesicles comprised of egg phosphatidylcholine (egg PC), dipentadecanoyl PC or dimyristoylphosphatidylcholine (DMPC) at rates greater than or equal to solubilization rates observed with human apolipoprotein A-I (apoA-I; 243 amino acids). Characterization studies revealed that interaction with DMPC induced a near doubling of 4F tryptophan fluorescence emission quantum yield (excitation 280 nm) and a approximately 7 nm blue shift in emission wavelength maximum. Inclusion of AMB in the vesicle substrate resulted in formation of 4F AMB-ND. Spectra of AMB containing particles revealed the antibiotic is a highly effective quencher of 4F tryptophan fluorescence emission, giving rise to a Ksv=7.7 x 10(4). Negative stain electron microscopy revealed that AMB-ND prepared with 4F possessed a disk shaped morphology similar to ND prepared without AMB or prepared with apoA-I. In yeast and pathogenic fungi growth inhibition assays, 4F AMB-ND was as effective as apoA-I AMB-ND. The data indicate that AMB-ND generated using an amphipathic peptide in lieu of apoA-I form a discrete population of particles that possess potent biological activity. Given their intrinsic versatility, peptides may be preferred for scale up and clinical application of AMB-ND.

Phase transitions of hexadecane in poly(alkyl methacrylate) core-shell microcapsules.

Microcapsules containing subfemtoliter volumes of n-hexadecane (HD) within a 4-40 nm thick shell of poly(alkyl methacrylates) were prepared. The size of the HD drop was varied between 50 and 140 nm. The alkyl substituents on the methacrylate monomer were varied to alter the surface tension between the HD and the polymer shell in order to investigate the effects of surface tension on the freezing point of the HD. The size dependence of the supercooling as predicted by the G-T equation was not observed in our systems. An effect on the magnitude of supercooling with variation in the side chains was observed, where freezing the HD in capsules with bulkier side chains requires a greater magnitude of supercooling. This is in agreement with the increased hydrophobic character of the polymers and also correlates with the decrease in glass transition temperature of the polymer. We also observed aging of the capsules, which could be accelerated by heating.

Quantifying size distributions of nanolipoprotein particles with single-particle analysis and molecular dynamic simulations.

Self-assembly of purified apolipoproteins and phospholipids results in the formation of nanometer-sized lipoprotein complexes, referred to as nanolipoprotein particles (NLPs). These bilayer constructs are fully soluble in aqueous environments and hold great promise as a model system to aid in solubilizing membrane proteins. Size variability in the self-assembly process has been recognized for some time, yet limited studies have been conducted to examine this phenomenon. Understanding the source of this heterogeneity may lead to methods to mitigate heterogeneity or to control NLP size, which may be important for tailoring NLPs for specific membrane proteins. Here, we have used atomic force microscopy, ion mobility spectrometry, and transmission electron microscopy to quantify NLP size distributions on the single-particle scale, specifically focusing on assemblies with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a recombinant apolipoprotein E variant containing the N-terminal 22 kDa fragment (E422k). Four discrete sizes of E422k/DMPC NLPs were identified by all three techniques, with diameters centered at approximately 14.5, 19, 23.5, and 28 nm. Computer simulations suggest that these sizes are related to the structure and number of E422k lipoproteins surrounding the NLPs and particles with an odd number of lipoproteins are consistent with the double-belt model, in which at least one lipoprotein adopts a hairpin structure.

Different apolipoproteins impact nanolipoprotein particle formation.

Spontaneous interaction of purified apolipoproteins and phospholipids results in formation of lipoprotein particles with nanometer-sized dimensions; we refer to these assemblies as nanolipoprotein particles or NLPs. These bilayer constructs can serve as suitable mimetics of biological membranes and are fully soluble in aqueous environments. We made NLPs from dimyristoylphospatidylcholine (DMPC) in combination with each of four different apolipoproteins: apoA-I, Delta-apoA-I fragment, apoE4 fragment, and apolipophorin III (apoLp-III) from the silk moth B. mori. Predominately discoidal in shape, these particles have diameters between 10 and 20 nm, share uniform heights between 4.5 and 5 nm, and can be produced in yields ranging between 40 and 60%. The particular lipoprotein, the lipid to lipoprotein ratio, and the assembly parameters determine the size and homogeneity of nanolipoprotein particles and indicate that apoA-I NLP preparations are smaller than the larger apoE422K and apoLp-III NLP preparations.

pH-induced conformational change of the rotavirus VP4 spike: implications for cell entry and antibody neutralization.

The rotavirus spike protein, VP4, is a major determinant of infectivity and neutralization. Previously, we have shown that trypsin-enhanced infectivity of rotavirus involves a transformation of the VP4 spike from a flexible to a rigid bilobed structure. Here we show that at elevated pH the spike undergoes a drastic, irreversible conformational change and becomes stunted, with a pronounced trilobed appearance. These particles with altered spikes, at a normal pH of 7.5, despite the loss of infectivity and the ability to hemagglutinate, surprisingly exhibit sialic acid (SA)-independent cell binding in contrast to the SA-dependent cell binding exhibited by native virions. Remarkably, a neutralizing monoclonal antibody that remains bound to spikes throughout the pH changes (pH 7 to 11 and back to pH 7) completely prevents this conformational change, preserving the SA-dependent cell binding and hemagglutinating functions of the virion. A hypothesis that emerges from the present study is that high-pH treatment triggers a conformational change that mimics a post-SA-attachment step to expose an epitope recognized by a downstream receptor in the rotavirus cell entry process. This process involves sequential interactions with multiple receptors, and the mechanism by which the antibody neutralizes is by preventing this conformational change.

Structures of rotavirus reassortants demonstrate correlation of altered conformation of the VP4 spike and expression of unexpected VP4-associated phenotypes.

Numerous prior studies have indicated that viable rotavirus reassortants containing structural proteins of heterologous parental origin may express unexpected phenotypes, such as changes in infectivity and immunogenicity. To provide a structural basis for alterations in phenotypic expression, a three-dimensional structural analysis of these reassortants was conducted. The structures of the reassortants show that while VP4 generally maintains the parental structure when moved to a heterologous protein background, in certain reassortants, there are subtle alterations in the conformation of VP4. The alterations in VP4 conformation correlated with expression of unexpected VP4-associated phenotypes. Interactions between heterologous VP4 and VP7 in reassortants expressing unexpected phenotypes appeared to induce the conformational alterations seen in VP4.