Hemocyanin-derived Phenoloxidase; Biochemical and Cellular Investigations of Innate Immunity

Hemocyanins (Hcs) and phenoloxidases (POs) are both members of the type-3 copper protein family, possessing di-cupric active sites which facilitate the binding of dioxygen. While Hcs and POs share a high degree of sequence homology, Hcs have been associated traditionally with oxygen transport whereas POs are catalytic proteins with a role in innate immunity. Evidence gathered in recent years details numerous immune functions for Hc, including an inducible PO activity. Unlike the pro-phenoloxidase activation cascade in arthropods, the endogenous mechanism(s) involved in the conversion of Hc into an immune enzyme is lacking in detail. The overall aim of this research was to characterise the physiological circumstances in which Hc is converted into a PO-like enzyme during immune challenge. A series of biochemical, biophysical and cellular techniques were used to assess the ability of phospholipid liposomes to mimic the well-characterised induction of PO activity in Hc by SDS micelles. Incubation of Hc purified from Limulus polyphemus, in the presence of phosphatidylserine (PS) liposomes, yielded ~ 90% of the PO activity observed upon incubation of Hc with the non-physiological activator, SDS. Phospholipid–induced PO activity in Hc was accompanied by secondary and tertiary structural changes similar to those observed in the presence of SDS. Subsequent analysis revealed that electrostatic interactions appear to be important in the PS-Hc activation complex. In vivo, PS-Hc interactions are assumed to be limited in quiescent cells. However, amebocytes undergoing apoptosis redistribute PS onto the outer leaflet of the plasma membrane, resulting in the potential for increased Hc-PS interactions. In the absence of a reliable culturing technique for L. polyphemus amebocytes, in vitro conditions were optimised for the short term maintenance of this labile cell type. Amebocytes retained viability and functionality in a medium that mimicked most-closely, the biochemical properties of L. polyphemus hemolymph. When presented with a fungal, bacterial or synthetic challenge, ~9% of amebocytes in vitro were found to be phagocytically active. Target internalisation was confirmed via the use of fluorescent quenchers and membrane probes. Within 4 hours of target internalisation, amebocytes underwent apoptosis, characterised by the loss of plasma and mitochondrial membrane potential, increased caspase-3 activity and extracellularisation of PS. Phagocytosis-induced cell death led to a proportional increase in the level of Hc-derived PO activity, suggesting that Hc may be interacting with PS present on terminal amebocyte membranes. The PO activity of Hc was investigated further in order to address an economically important issue; hyperpigmentation in commercial shellfish. While PO enzymes are thought to be the cause of hyperpigmentation in Nephrops norvegicus, evidence presented here suggests that cellular PO is inactivated after freeze-thawing, while extracellular Hc retains stability and displays a heightened level of inducible PO activity under similar treatments. Known PO inhibitors were used successfully to reduce Hc-derived PO activity, with inhibitors assumed to bind Hc in a manner similar to PO-inhibitor complexes. Structural and functional studies of hemocyanins and immune cells presented here provide new insights into the interactions of hemocyanin-activator complexes in invertebrates

Low dose γ-radiation induced effects on wax moth (Galleria mellonella) larvae

Larvae of the greater wax moth Galleria mellonella are common pests of beehives and commercial apiaries, and in more applied settings, these insects act as alternative in vivo bioassays to rodents for studying microbial virulence, antibiotic development, and toxicology. In the current study, our aim was to assess the putative adverse effects of background gamma radiation levels on G. mellonella. To achieve this, we exposed larvae to low (0.014 mGy/h), medium (0.056 mGy/h), and high (1.33 mGy/h) doses of caesium-137 and measured larval pupation events, weight, faecal discharge, susceptibility to bacterial and fungal challenges, immune cell counts, activity, and viability (i.e., haemocyte encapsulation) and melanisation levels. The effects of low and medium levels of radiation were distinguishable from the highest dose rates used – the latter insects weighed the least and pupated earlier. In general, radiation exposure modulated cellular and humoral immunity over time, with larvae showing heightened encapsulation/melanisation levels at the higher dose rates but were more susceptible to bacterial (Photorhabdus luminescens) infection. There were few signs of radiation impacts after 7 days exposure, whereas marked changes were recorded between 14 and 28 days. Our data suggest that G. mellonella demonstrates plasticity at the whole organism and cellular levels when irradiated and offers insight into how such animals may cope in radiologically contaminated environments (e.g. Chornobyl Exclusion Zone)

Defective phagocyte association during infection of Galleria mellonella with Yersinia pseudotuberculosis is detrimental to both insect host and microbe

Adhesins facilitate bacterial colonization and invasion of host tissues and are considered virulence factors, but their impact on immune-mediated damage as a driver of pathogenesis remains unclear. Yersinia pseudotuberculosis encodes for a multivalent adhesion molecule (MAM), a mammalian cell entry (MCE) family protein and adhesin. MAMs are widespread in Gram-negative bacteria and enable enteric bacteria to colonize epithelial tissues. Their role in bacterial interactions with the host innate immune system and contribution to pathogenicity remains unclear. Here, we investigated how Y. pseudotuberculosis MAM contributes to pathogenesis during infection of the Galleria mellonella insect model. We show that Y. pseudotuberculosis MAM is required for efficient bacterial binding and uptake by hemocytes, the host phagocytes. Y. pseudotuberculosis interactions with insect and mammalian phagocytes are determined by bacterial and host factors. Loss of MAM, and deficient microbe–phagocyte interaction, increased pathogenesis in G. mellonella. Diminished phagocyte association also led to increased bacterial clearance. Furthermore, Y. pseudotuberculosis that failed to engage phagocytes hyperactivated humoral immune responses, most notably melanin production. Despite clearing the pathogen, excessive melanization also increased phagocyte death and host mortality. Our findings provide a basis for further studies investigating how microbe- and host-factors integrate to drive pathogenesis in a tractable experimental system

Effect of acute ultraviolet radiation on Galleria mellonella health and immunity

For humans, acute and chronic overexposure to ultraviolet (UV) radiation can cause tissue damage in the form of sunburn and promote cancer(s). The immune-modulating properties of UV radiation and health-related consequences are not well known. Herein, we used the larvae of the wax moth Galleria mellonella, to determine UV-driven changes in cellular components of innate immunity. From immune cell (haemocyte) reactivity and the production of antimicrobial factors, these insects share many functional similarities with mammalian cellular innate immunity. After exposing insects to UVA or UVB for up to two hours, we monitored larval viability, susceptibility to infection, haemolymph (blood) physiology and faecal discharge. Prolonged exposure of larvae to UVB coincided with decreased survival, enhanced susceptibility to bacterial challenge, melanin synthesis in the haemolymph, compromised haemocyte functionality and changes in faecal (bacterial) content. We contend G. mellonella is a reliable in vivo model for assessing the impact of UV exposure at the whole organism and cellular levels

Source region and growth analysis of narrowband Z-mode emission at Saturn

Intense Z-mode emission is observed in the lower density region near the inner edge of the Enceladus torus at Saturn, where these waves may resonate with MeV electrons. The source mechanism of this emission, which is narrow-banded and most intense near 5 kHz, is not well understood. We survey the Cassini Radio and Plasma Wave Science data to isolate several probable source regions near the inner edge of the Enceladus density torus. Electron phase space distributions are obtained from the Cassini Electron Spectrometer, part of the Cassini Plasma Spectrometer investigation. We perform a plasma wave growth analysis to conclude that an electron temperature anisotropy and possibly a weak loss cone can drive the Z mode as observed. Electrostatic electron acoustic waves and perhaps weak beam modes are also found to be unstable coincident with the Z mode. Quasi-steady conditions near the Enceladus density torus may result in the observations of narrowband Z-mode emission at Saturn

Parvovirus B19 infection in sickle cell disease: An analysis from the Centers for Disease Control haemoglobinopathy blood surveillance project

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155921/1/tme12671_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155921/2/tme12671.pd

Diamagnetic depression observations at Saturn’s magnetospheric cusp by the Cassini spacecraft

The magnetospheric cusp is a region where shocked solar wind plasma can enter a planetary magnetosphere, after magnetic reconnection has occurred at the dayside magnetopause or in the lobes. The dense plasma that enters the high‐latitude magnetosphere creates diamagnetic effects whereby a depression is observed in the magnetic field. We present observations of the cusp events at Saturn’s magnetosphere where these diamagnetic depressions are found. The data are subtracted from a magnetic field model, and the calculated magnetic pressure deficits are compared to the particle pressures. A high plasma pressure layer in the magnetosphere adjacent to the cusp is discovered to also depress the magnetic field, outside of the cusp. This layer is observed to contain energetic He++ (up to ∼100 keV) from the solar wind as well as heavy water group ions (W+) originating from the moon Enceladus. We also find a modest correlation of diamagnetic depression strength to solar wind dynamic pressure and velocity; however, unlike at Earth, there is no correlation found with He++ counts.Key PointsDiamagnetic depressions are found in the cusp and are observed to continue into the adjacent magnetosphereA heated plasma layer of mixed composition is found to depress the adjacent magnetospheric fieldDiamagnetic depression strength is correlated to solar wind dynamic pressure and velocity but not to the observed He++ counts, like at EarthPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137687/1/jgra53517_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137687/2/jgra53517-sup-0001-supinfo.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137687/3/jgra53517.pd

Cassini plasma observations of Saturn's magnetospheric cusp

The magnetospheric cusp is a funnel-shaped region where shocked solar wind plasma is able to enter the high latitude magnetosphere via the process of magnetic reconnection. The plasma observations include various cusp signatures such as ion energy dispersions as well as diamagnetic effects. We present an overview analysis of the cusp plasma observations at the Saturnian magnetosphere from the Cassini spacecraft era. A comparison of the observations is made as well as classification into groups due to varying characteristics. The locations of the reconnection site are calculated and shown to vary along the subsolar magnetopause. We show the first in situ evidence for lobe reconnection that occurred at nearly the same time as dayside reconnection for one of the cusp crossings. Evidence for 'bursty' and more 'continous' reconnection signatures are observed in different cusp events. The events are compared to solar wind propagation models and it is shown that magnetic reconnection and plasma injection into the cusp can occur for a variety of upstream conditions. These are important results because they show that Saturn's magnetospheric interaction with the solar wind and the resulting cusp signatures are dynamic, and that plasma injection in the cusp occurs due to a variety of solar wind conditions. Furthermore, reconnection can proceed at a variety of locations along the magnetopause

Estimating Uranium Partition Coefficients from Laboratory Adsorption Isotherms

An estimated 330 metric tons of uranium have been buried in the radioactive waste Subsurface Disposal Area (SDA) at the Idaho National Engineering and Environmental Laboratory (INEEL). An assessment of uranium transport parameters is being performed to decrease the uncertainty in risk and dose predictions derived from computer simulations of uranium fate and transport to the underlying Snake River Plain Aquifer. Uranium adsorption isotherms have been measured in the laboratory and fit with a Freundlich isotherm. The Freundlich n parameter was statistically identical for 14 sediment samples. The Freundlich Kf for seven samples, where material properties have been measured, is correlated to sediment surface area. Based on these empirical observations, a model has been derived for adsorption of uranium on INEEL sedimentary materials using surface complexation theory. The model was then used to predict the range of adsorption conditions to be expected at the SDA. Adsorption in the deep vadose zone is predicted to be stronger than in near-surface sediments because the total dissolved carbonate decreases with depth

Welfare in farmed decapod crustaceans, with particular reference to Penaeus vannamei

The farming of decapod crustaceans is a key economic driver in many countries, with production reaching around 9.4 million tonnes (USD 69.3 billion) in 2018. These efforts are currently dominated by the farming of Pacific whiteleg shrimp, Penaeus vannamei, which translates into approximately 167 billion farmed P. vannamei being harvested annually. Further production growth is expected in the future and hence the need for more research into its health and welfare is required. Herein, from an extensive survey of the available literature, we scrutinise farming practices and the challenges associated with the production of P. vannamei from an animal-centric welfare perspective (1), we propose potential welfare indicators (2) and we critically review current scientific evidence of sentience in penaeid shrimp among other commercially important decapods (3), since it is plausible that in the near future not only the largest, but in fact all decapod crustaceans will receive welfare protection. This review highlights that despite the wide knowledge on crustacean stress physiology and immunology as well as disease control, still little is known about some key parameters related to the five welfare dimensions. We recommend that further research should focus on developing a systematic integrated welfare assessment encompassing all the different aspects of the crustaceans farming and life cycle up to slaughter. Furthermore, direct and indirect species-specific operational welfare indicators should be developed for all decapod crustaceans currently farmed, similar to the ones suggested in this review for P. vannamei