Semaphorin 3A Binds to the Perineuronal Nets via Chondroitin Sulfate Type E Motifs in Rodent Brains

Chondroitin sulfate (CS) and the CS-rich extracellular matrix structures called perineuronal nets (PNNs) restrict plasticity and regeneration in the CNS. Plasticity is enhanced by chondroitinase ABC treatment that removes CS from its core protein in the chondroitin sulfate proteoglycans or by preventing the formation of PNNs, suggesting that chondroitin sulfate proteoglycans in the PNNs control plasticity. Recently, we have shown that semaphorin3A (Sema3A), a repulsive axon guidance molecule, localizes to the PNNs and is removed by chondroitinase ABC treatment (Vo, T., Carulli, D., Ehlert, E. M., Kwok, J. C., Dick, G., Mecollari, V., Moloney, E. B., Neufeld, G., de Winter, F., Fawcett, J. W., and Verhaagen, J. (2013) Mol. Cell. Neurosci. 56C, 186–200). Sema3A is therefore a candidate for a PNN effector in controlling plasticity. Here, we characterize the interaction of Sema3A with CS of the PNNs. Recombinant Sema3A interacts with CS type E (CS-E), and this interaction is involved in the binding of Sema3A to rat brain-derived PNN glycosaminoglycans, as demonstrated by the use of CS-E blocking antibody GD3G7. In addition, we investigate the release of endogenous Sema3A from rat brain by biochemical and enzymatic extractions. Our results confirm the interaction of Sema3A with CS-E containing glycosaminoglycans in the dense extracellular matrix of rat brain. We also demonstrate that the combination of Sema3A and PNN GAGs is a potent inhibitor of axon growth, and this inhibition is reduced by the CS-E blocking antibody. In conclusion, Sema3A binding to CS-E in the PNNs may be a mechanism whereby PNNs restrict growth and plasticity and may represent a possible point of intervention to facilitate neuronal plasticity.     

Experimental infections of Orchitophrya stellarum (Scuticociliata) in American blue crabs (Callinectes sapidus) and fiddler crabs (Uca minax)

Outbreaks of an unidentified ciliate have occurred on several occasions in blue crabs from Chesapeake Bay held during winter months in flow-through systems. The parasite was initially thought to be Mesanophrys chesapeakensis, but molecular analysis identified it as Orchitophyra stellarum, a facultative parasite of sea stars (Asteroidea). We investigated the host-parasite association of O. stellarum in the blue crab host. Crabs were inoculated with the ciliate, or they were held in bath exposures after experimentally induced autotomy of limbs in order to determine potential mechanisms for infection. Crabs inoculated with the ciliate, or exposed to it after experimental autotomy, rapidly developed fatal infections. Crabs that were not experimentally injured, but were exposed to the ciliate, rarely developed infections; thus, indicating that the parasite requires a wound or break in the cuticle as a portal of entry. For comparative purposes, fiddler crabs, Uca minax, were inoculated with the ciliate in a dose-titration experiment. Low doses of the ciliate (10 per crab) were sometimes able to establish infections, but high intensity infections developed quickly at doses over 500 ciliates per crab. Chemotaxis studies were initiated to determine if the ciliate preferentially selected blue crab serum (BCS) over other nutrient sources. Cultures grown on medium with BCS or fetal bovine serum showed some conditioning in their selection for different media, but the outcome in choice experiments indicated that the ciliate was attracted to BCS and not seawater. Our findings indicate that O. stellarum is a facultative parasite of blue crabs. It can cause infections in exposed crabs at 10–15 °C, but it requires a portal of entry for successful host invasion, and it may find injured hosts using chemotaxis.     

Tracking fatty acid kinetics in distinct lipoprotein fractions in vivo: a novel high-throughput approach for studying dyslipidemia in rodent models

Isotopic tracers have been used to examine lipid trafficking for many years, and data from those studies have typically yielded novel insight regarding the pathophysiology of dyslipidemia. Previous experimental designs were suitable for studies in humans because relatively large volumes of plasma could be regularly sampled. We have expanded on the earlier logic by applying high-throughput analytical methods that require reduced sample volumes. Specifically, we have examined the possibility of coupling gel-based separations of lipoproteins (e.g., lipoprint) with LC-MS/MS analyses of complex lipid mixtures as a way to routinely measure the labeling profiles of distinct lipids in discrete lipoprotein subfractions. We demonstrate the ability to measure the incorporation of [U-¹³C]oleate into triglycerides (TG), PLs (PL), and cholesterol esters (CE) in VLDL, LDL, and HDL particles in mice. Although rodent models of dyslipidemia are inherently different from humans because of alterations in enzyme activities and underlying metabolism, rodent models can be used to screen novel compounds for efficacy in altering a given biochemical pathway and therein enable studies of target engagement in vivo. We expect that it is possible to translate our approach for application in other systems, including studies in humans.     

AMP-dependent Kinase Inhibits Oxidative Stress-induced Caveolin-1 Phosphorylation and Endocytosis by Suppressing the Dissociation between c-Abl and Prdx1 Proteins in Endothelial Cells

Caveolin-1 is the primary structural component of endothelial caveolae that is essential for transcellular trafficking of albumin and is also a critical scaffolding protein that regulates the activity of signaling molecules in caveolae. Phosphorylation of caveolin-1 plays a fundamental role in the mechanism of oxidant-induced vascular hyper permeability. However, the regulatory mechanism of caveolin-1 phosphorylation remains unclear. Here we identify a previously unexpected role for AMPK in inhibition of caveolin-1 phosphorylation under oxidative stress. A pharmacological activator of AMPK, 5-amino-4-imidazole carboxamide riboside (AICAR), inhibited oxidative stress-induced phosphorylation of both caveolin-1 and c-Abl, which is the major kinase of caveolin-1, and endocytosis of albumin in human umbilical vein endothelial cell. These effects were abolished by treatment with two specific inhibitors of AICAR, dipyridamole, and 5-iodotubericidin. Consistently, knockdown of the catalytic AMPKα subunit by siRNA abolished the inhibitory effect of AICAR on oxidant-induced phosphorylation of both caveolin-1 and c-Abl. Pretreatment with specific c-Abl inhibitor, imatinib mesylate, and knock down of c-Abl significantly decreased the caveolin-1 phosphorylation after H₂O₂ exposure and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Interestingly, knockdown of Prdx-1, an antioxidant enzyme associated with c-Abl, increased phosphorylation of both caveolin-1 and c-Abl and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Furthermore, co-immunoprecipitation experiment showed that AICAR suppressed the oxidant-induced dissociation between c-Abl and Prdx1. Overall, our results suggest that activation of AMPK inhibits oxidative stress-induced caveolin-1 phosphorylation and endocytosis, and this effect is mediated in part by stabilizing the interaction between c-Abl and Prdx-1.     

Research Commentary: Association of Zoonotic Pathogens with Fresh, Estuarine, and Marine Macroaggregates

Aquatic macroaggregates (flocs ≥0.5 mm) provide an important mechanism for vertical flux of nutrients and organic matter in aquatic ecosystems, yet their role in the transport and fate of zoonotic pathogens is largely unknown. Terrestrial pathogens that enter coastal waters through contaminated freshwater runoff may be especially prone to flocculation due to fluid dynamics and electrochemical changes that occur where fresh and marine waters mix. In this study, laboratory experiments were conducted to evaluate whether zoonotic pathogens (Cryptosporidium, Giardia, Salmonella) and a virus surrogate (PP7) are associated with aquatic macroaggregates and whether pathogen aggregation is enhanced in saline waters. Targeted microorganisms showed increased association with macroaggregates in estuarine and marine waters, as compared with an ultrapure water control and natural freshwater. Enrichment factor estimations demonstrated that pathogens are 2–4 orders of magnitude more concentrated in aggregates than in the estuarine and marine water surrounding the aggregates. Pathogen incorporation into aquatic macroaggregates may influence their transmission to susceptible hosts through settling and subsequent accumulation in zones where aggregation is greatest, as well as via enhanced uptake by invertebrates that serve as prey for marine animals or as seafood for humans.     

Dramatic phenotypic plasticity within species of Siphomutabilus n. g. (Digenea: Cryptogonimidae) from Indo-Pacific caesionines (Perciformes: Lutjanidae)

A survey of Indo-Pacific lutjanids of the subfamily Caesioninae revealed the presence of Siphodera gurukun Machida, 1910 and two new cryptogonimid taxa from off Heron and Lizard Islands on the Great Barrier Reef, Australia, Ningaloo Reef, Western Australia and Rasdhoo Atoll, Maldives. A combined morphological and genetic characterisation of these species shows that they form a clade distinct from the type-species of Siphodera Linton, 1910, S. vinaledwardsii (Linton, 1901), and warrants the proposal of a new genus. Here we propose Siphomutabilus n. g. and transfer Siphodera gurukun Machida, 1986 as the type-species, Siphomutabilus gurukun (Machida, 1986) n. comb. Siphodera aegyptensis Hassanine & Gibson, 2005 is transferred to Siphomutabilus as S. aegyptensis (Hassanine & Gibson, 2005) n. comb. based on morphological and ecological similarities. Siphomutabilus raritas n. sp. is described from Caesio cuning (Bloch) off Lizard Island and S. bitesticulatus n. sp. is described from Pterocaesio marri Schultz off Heron Island. The two new species are unique in that they have two testes, making their morphology broadly consistent with that of Metadena Linton, 1910, yet the molecular analyses conducted here indicates that they are unequivocally united with Siphomutabilus gurukun (which has multiple testes) to the exclusion of Metadena lutiani (Yamaguti, 1942), which was sequenced here. The dramatic phenotypic plasticity observed among such closely related species of Siphomutabilus suggests a secondary modification of what is generally considered a robust generic diagnostic character within this and other digenean families, highlighting the need for a combined morphological and molecular diagnostic approach when characterising these taxa. Siphodera Linton, 1910 is amended to include just two species, the type-species S. vinaledwardsii (Linton, 1901) Linton, 1910 and S. cirrhiti Yamaguti, 1970, which are distinguished by their lack of oral spines and multiple testes that are primarily extracaecal. Siphodera ghanensis Fischthal & Thomas, 1968 is considered a species incertae sedis here based on significant morphological and ecological differences compared with species of Siphodera and Siphomutabilus n. g.     

Identification of Pauses during Formation of HIV-1 Virus Like Particles

HIV Gag polymerizes on the plasma membrane to form virus like particles (VLPs) that have similar diameters to wild-type viruses. We use multicolor, dual-penetration depth, total internal reflection fluorescence microscopy, which corrects for azimuthal movement, to image the assembly of individual VLPs from the time of nucleation to the recruitment of VPS4 (a component of the endosomal sorting complexes required for transport, and which marks the final stage of VLP assembly). Using a mathematical model for assembly and maximum-likelihood comparison of fits both with and without pauses, we detect pauses during Gag polymerization in 60% of VLPs. Pauses range from 2 to 20 min, with an exponentially distributed duration that is independent of cytosolic Gag concentration. VLPs assembled with late domain mutants of Gag (which do not recruit the key endosomal sorting complexes required for transport proteins Alix or TSG101) exhibit similar pause distributions. These pauses indicate that a single rate-limiting event is required for continuation of assembly. We suggest that pauses are either related to incorporation of defects in the hexagonal Gag lattice during VLP assembly or are caused by shortcomings in interactions of Gag with essential and still undefined cellular components during formation of curvature on the plasma membrane.