Local FK506 dose-dependent study using a novel three-dimensional organotypic assay

Local administration of FK506, an FDA approved immunosuppressant with neuroregenerative properties, is a promising technique to achieve improved peripheral nerve regeneration while preventing the side effects associated with the systemic administration of this drug. Although considerable research has been devoted to the development of clinically suitable systems for local delivery of FK506 to the site of nerve injury and repair, the optimal dose of FK506 for enhancement of axon regeneration in the peripheral nerve has not yet been established. To this end, we devised a three-dimensional (3D) organotypic assay capable of mimicking the peripheral nerve. This assay consisted of a neonatal rat dorsal root ganglion (DRG) extending its neurites into the native peripheral nerve scaffold provided by an acellular nerve allograft (ANA). A novel 3D compartmented cell culture system was adapted from the 3D organotypic assay to achieve local delivery of FK506 just to the growing neurites in vitro and establish the required local dose of FK506 for peripheral nerve regeneration. A bimodal dose response was observed by culturing the entire DRG–ANA construct with media containing different concentrations of FK506. Low drug concentration of 1 pg/ml and high drug concentration of 100 ng/ml lead to the longest neurite extension in vitro. Furthermore, regardless of the FK506 concentration, concentrating the drug to the growing neurites resulted in significant increase in both neurite extension and neurite density, an effect that was not observed with the FK506 delivery to both neurites and neural cell bodies within DRG. The findings in this study provide valuable insight into the optimal local dose of FK506 for peripheral nerve regeneration. Furthermore, for the first time, this study suggests the potential interaction of FK506 with axons at the level of the growth cone.     

Nerve Cross-Bridging to Enhance Nerve Regeneration in a Rat Model of Delayed Nerve Repair

There are currently no available options to promote nerve regeneration through chronically denervated distal nerve stumps. Here we used a rat model of delayed nerve repair asking of prior insertion of side-to-side cross-bridges between a donor tibial (TIB) nerve and a recipient denervated common peroneal (CP) nerve stump ameliorates poor nerve regeneration. First, numbers of retrogradely-labelled TIB neurons that grew axons into the nerve stump within three months, increased with the size of the perineurial windows opened in the TIB and CP nerves. Equal numbers of donor TIB axons regenerated into CP stumps either side of the cross-bridges, not being affected by target neurotrophic effects, or by removing the perineurium to insert 5-9 cross-bridges. Second, CP nerve stumps were coapted three months after inserting 0-9 cross-bridges and the number of 1) CP neurons that regenerated their axons within three months or 2) CP motor nerves that reinnervated the extensor digitorum longus (EDL) muscle within five months was determined by counting and motor unit number estimation (MUNE), respectively. We found that three but not more cross-bridges promoted the regeneration of axons and reinnervation of EDL muscle by all the CP motoneurons as compared to only 33% regenerating their axons when no cross-bridges were inserted. The same 3-fold increase in sensory nerve regeneration was found. In conclusion, side-to-side cross-bridges ameliorate poor regeneration after delayed nerve repair possibly by sustaining the growth-permissive state of denervated nerve stumps. Such autografts may be used in human repair surgery to improve outcomes after unavoidable delays.     

Paleoceanographic Insights on Recent Oxygen Minimum Zone Expansion: Lessons for Modern Oceanography

Climate-driven Oxygen Minimum Zone (OMZ) expansions in the geologic record provide an opportunity to characterize the spatial and temporal scales of OMZ change. Here we investigate OMZ expansion through the global-scale warming event of the most recent deglaciation (18-11 ka), an event with clear relevance to understanding modern anthropogenic climate change. Deglacial marine sediment records were compiled to quantify the vertical extent, intensity, surface area and volume impingements of hypoxic waters upon continental margins. By integrating sediment records (183-2,309 meters below sea level; mbsl) containing one or more geochemical, sedimentary or microfossil oxygenation proxies integrated with analyses of eustatic sea level rise, we reconstruct the timing, depth and intensity of seafloor hypoxia. The maximum vertical OMZ extent during the deglaciation was variable by region: Subarctic Pacific (~600-2,900 mbsl), California Current (~330-1,500 mbsl), Mexico Margin (~330-830 mbsl), and the Humboldt Current and Equatorial Pacific (~110-3,100 mbsl). The timing of OMZ expansion is regionally coherent but not globally synchronous. Subarctic Pacific and California Current continental margins exhibit tight correlation to the oscillations of Northern Hemisphere deglacial events (Termination IA, Bølling-Allerød, Younger Dryas and Termination IB). Southern regions (Mexico Margin and the Equatorial Pacific and Humboldt Current) exhibit hypoxia expansion prior to Termination IA (~14.7 ka), and no regional oxygenation oscillations. Our analyses provide new evidence for the geographically and vertically extensive expansion of OMZs, and the extreme compression of upper-ocean oxygenated ecosystems during the geologically recent deglaciation.     

Evidence of Subclinical mtDNA Alterations in HIV-Infected Pregnant Women Receiving Combination Antiretroviral Therapy Compared to HIV-Negative Pregnant Women

Introduction

Combination antiretroviral therapy (cART) can effectively prevent vertical transmission of HIV but there is potential risk of adverse maternal, foetal or infant effects. Specifically, the effect of cART use during pregnancy on mitochondrial DNA (mtDNA) content in HIV-positive (HIV+) women is unclear. We sought to characterize subclinical alterations in peripheral blood mtDNA levels in cART-treated HIV+ women during pregnancy and the postpartum period.

Methods

This prospective longitudinal observational cohort study enrolled both HIV+ and HIV-negative (HIV-) pregnant women. Clinical data and blood samples were collected at three time points in pregnancy (13-<23 weeks, 23-<30 weeks, 30–40 weeks), and at delivery and six weeks post-partum in HIV+ women. Peripheral blood mtDNA to nuclear DNA (nDNA) ratio was measured by qPCR.

Results

Over a four year period, 63 HIV+ and 42 HIV- women were enrolled. HIV+ women showed significantly lower mtDNA/nDNA ratios compared to HIV- women during pregnancy (p = 0.003), after controlling for platelet count and repeated measurements using a multivariable mixed-effects model. Ethnicity, gestational age (GA) and substance use were also significantly associated with mtDNA/nDNA ratio (p≤0.02). Among HIV+ women, higher CD4 nadir was associated with higher mtDNA/nDNA ratios (p<0.0001), and these ratio were significantly lower during pregnancy compared to the postpartum period (p<0.0001).

Conclusions

In the context of this study, it was not possible to distinguish between mtDNA effects related to HIV infection versus cART therapy. Nevertheless, while mtDNA levels were relatively stable over time in both groups during pregnancy, they were significantly lower in HIV+ women compared to HIV- women. Although no immediate clinical impact was observed on maternal or infant health, lower maternal mtDNA levels may exert long-term effects on women and children and remain a concern. Improved knowledge of such subclinical alterations is another step toward optimizing the safety and efficacy of cART regimens during pregnancy.     

Acoustic localization of terrestrial wildlife: Current practices and future opportunities

Autonomous acoustic recorders are an increasingly popular method for low‐disturbance, large‐scale monitoring of sound‐producing animals, such as birds, anurans, bats, and other mammals. A specialized use of autonomous recording units (ARUs) is acoustic localization, in which a vocalizing animal is located spatially, usually by quantifying the time delay of arrival of its sound at an array of time‐synchronized microphones. To describe trends in the literature, identify considerations for field biologists who wish to use these systems, and suggest advancements that will improve the field of acoustic localization, we comprehensively review published applications of wildlife localization in terrestrial environments. We describe the wide variety of methods used to complete the five steps of acoustic localization: (1) define the research question, (2) obtain or build a time‐synchronizing microphone array, (3) deploy the array to record sounds in the field, (4) process recordings captured in the field, and (5) determine animal location using position estimation algorithms. We find eight general purposes in ecology and animal behavior for localization systems: assessing individual animals' positions or movements, localizing multiple individuals simultaneously to study their interactions, determining animals' individual identities, quantifying sound amplitude or directionality, selecting subsets of sounds for further acoustic analysis, calculating species abundance, inferring territory boundaries or habitat use, and separating animal sounds from background noise to improve species classification. We find that the labor‐intensive steps of processing recordings and estimating animal positions have not yet been automated. In the near future, we expect that increased availability of recording hardware, development of automated and open‐source localization software, and improvement of automated sound classification algorithms will broaden the use of acoustic localization. With these three advances, ecologists will be better able to embrace acoustic localization, enabling low‐disturbance, large‐scale collection of animal position data.     

Insights in to the pathogenesis of axial spondyloarthropathy based on gene expression profiles

Introduction  

Axial spondyloarthropathy (SpA) is a group of inflammatory diseases, with ankylosing spondylitis as the prototype. SpA affects the axial skeleton, entheses, joints and, at times, the eyes. This study tested the hypothesis that SpA is characterized by a distinct pattern of gene expression in peripheral blood of affected individuals compared with healthy controls.     

<Emphasis Type="Italic">Myzostoma fuscomaculatum</Emphasis> (Myzostomida), a new myzostome species from False Bay,South Africa

A new myzostome species, described here as Myzostoma fuscomaculatum n. sp. was collected on Tropiometra carinata in False Bay (South Africa), during a survey of symbionts associated with comatulid crinoid species. M. fuscomaculatum n. sp. occurred only on T. carinata and not on the more common crinoid, Comanthus wahlbergi. It infested 61.7% of the 120 host specimens collected, of which 64.9% (48 specimens) hosted more than one individual (maximum of 32). M. fuscomaculatum n. sp. was always located on the host’s arms and pinnules and was cryptically coloured, closely matching the colour pattern of the host. This is the first record of myzostomes from the cool temperate waters of South Africa’s Atlantic coast. The new species is morphologically close to M. gopalai Subramaniam, 1938, collected on T. encrinus in Madras Harbour. M. fuscomaculatum n. sp. differs from M. gopalai in lacking marginal cirri at the adult stage, the presence of three pairs of digestive diverticula, by the position of its lateral organs and by the shape of the manubrium. Molecular phylogenetic analyses based on 18S and 16S rDNA placed M. fuscomaculatum n. sp. into a clade including Hypomyzostoma, Myzostoma and Mesomyzostoma species. Handling editor: K. Martens Déborah Lanterbecq and Tessa Hempson contributed equally to this work.     

Fatal Chytridiomycosis in the Tyrrhenian Painted Frog

Batrachochytrium dendrobatidis (Bd), the causative agent of the amphibian disease chytridiomycosis, is an important factor in the global decline of amphibians. Within Europe, animals that exhibit clinical signs of the disease have only been reported in Spain despite the pathogen’s wide, but patchy, distribution on the continent. Recently, another occurrence of chytridiomycosis was reported in Euproctus platycephalus, the Sardinian brook newt, on the Mediterranean island of Sardinia, but without any evidence of fatal disease. We report further evidence of the emergence of Bd on Sardinia and the first evidence of lethal chytridiomycosis outside of Spain. Unusual mortalities of the Tyrrhenian painted frog (Discoglossus sardus) were found at three sites in the Limbara mountains of northern Sardinia. Molecular and histological screens of corpses, frogs, and tadpoles from these sites revealed infection with Bd. Infection and mortality occurred at locations that are unusual in terms of the published habitat requirements of the pathogen. Given the endemicity, the IUCN Red List status of the amphibian species on Sardinia, and the occurrence of infection and mortality caused by chytridiomycosis, there is serious reason for concern for the impact that disease emergence may have on the conservation of the amphibians of the island.     

NADPH analog binding to constitutive nitric oxide activates electron transfer and NO synthesis.

We report here that NADPH analogs such as 2'5'ADP, ATP, and 2'AMP paradoxically activate constitutive calcium/calmodulin regulated nitric oxide synthases (cNOS), including the endothelial isoform (eNOS) and the neuronal isoform (nNOS). These activators compete with NADPH by filling the binding site of the adenine moiety of NADPH, but do not occupy the entire NADPH binding domain. Effects of these analogs on cNOS's include increasing the electron transfer rate to external acceptors, as assessed by cytochrome c reductase activity in the absence of calmodulin. In addition, NO synthase activity in the presence of calmodulin (with or without added calcium) was increased by the addition of NADPH analogs. In contrast, the same NADPH analogs inhibit iNOS, the calcium insensitive inducible isoform, which lacks control elements found in constitutive isoforms. Because ATP and ADP are among the effective activators of cNOS isoforms, these effects may be physiologically relevant.