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).

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.

Broad toxicological effects of per-/poly- fluoroalkyl substances (PFAS) on the unicellular eukaryote, Tetrahymena pyriformis.

Per-/Poly- fluoroalkyl substances represent emerging persistent organic pollutants. Their toxic effects can be broad, yet little attention has been given to organisms at the microscale. To address this knowledge shortfall, the unicellular eukaryote Tetrahymena pyriformis was exposed to increasing concentrations (0-5000 µM) of PFOA/PFOS and monitored for cellular motility, division and function (i.e., phagocytosis), reactive oxygen species generation and total protein levels. Both PFOA/PFOS exposure had negative impacts on T. pyriformis, including reduced motility, delayed cell division and oxidative imbalance, with each chemical having distinct toxicological profiles. T. pyriformis represents a promising candidate for assessing the biological effects these emerging anthropogenically-derived contaminants in a freshwater setting.

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 howY. 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.

Effects of Fluorodeoxyglucose Conjugated and Unconjugated Iron Oxide Magnetic Nanoparticles on Macrophages: a Pilot Study

Abstract The effects of fluorodeoxyglucose conjugated iron oxide magnetic nanoparticles (FDGMNP) on macrophages are presented using a yeast substrate. Iron oxide magnetic nanoparticles (MNP) were synthesized by partially reducing FeCl3, then conjugated with (3-aminopropyl) triethoxysilane (APTES) after silication with tetraethyl orthosilicate. Silanated MMP nanoparticles were combined with fluorodeoxyglucose (FDG). Fluorodeoxyglucose iron oxide magnetic nanoparticles (FDGMNP) and its unconjugated control (MNP) were added (1mL) to the cells from the murine macrophage-like, Abelson murine leukemia virus transformed cell line RAW 264.7 (American Type Culture Collection number TIB-71) cell culture wells at different concentrations from 90-3.6 μg/mL. Cells were placed on the magnet plate for 30 min before incubating at 37°C, 5% CO2 overnight. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium) assay was performed to measure cell viability. Our results demonstrate that iron based nanoparticles can be linked to macrophages (elements of the immune system that attack bacteria) without the function of the macrophages being affected, ie no detrimental effects to the macrophages were evident in these experiments. We conclude that neither FDGMNP nor MNP had a detrimental effect on macrophage function.

The insect, Galleria mellonella, is a compatible model for evaluating the toxicology of okadaic acid.

The polyether toxin, okadaic acid, causes diarrhetic shellfish poisoning in humans. Despite extensive research into its cellular targets using rodent models, we know little about its putative effect(s) on innate immunity. We inoculated larvae of the greater wax moth, Galleria mellonella, with physiologically relevant doses of okadaic acid by direct injection into the haemocoel (body cavity) and/or gavage (force-feeding). We monitored larval survival and employed a range of cellular and biochemical assays to assess the potential harmful effects of okadaic acid. Okadaic acid at concentrations ≥ 75 ng/larva (≥ 242 µg/kg) led to significant reductions in larval survival (> 65%) and circulating haemocyte (blood cell) numbers (> 50%) within 24 h post-inoculation. In the haemolymph, okadaic acid reduced haemocyte viability and increased phenoloxidase activities. In the midgut, okadaic acid induced oxidative damage as determined by increases in superoxide dismutase activity and levels of malondialdehyde (i.e. lipid peroxidation). Our observations of insect larvae correspond broadly to data published using rodent models of shellfish-poisoning toxidrome, including complementary LD50 values: 206-242 µg/kg in mice, ~ 239 µg/kg in G. mellonella. These data support the use of this insect as a surrogate model for the investigation of marine toxins, which offers distinct ethical and financial incentives.

Characterizing the Mechanisms of Nonopsonic Uptake of Cryptococci by Macrophages.

The pathogenic fungus Cryptococcus enters the human host via inhalation into the lung and is able to reside in a niche environment that is serum- (opsonin) limiting. Little is known about the mechanism by which nonopsonic phagocytosis occurs via phagocytes in such situations. Using a combination of soluble inhibitors of phagocytic receptors and macrophages derived from knockout mice and human volunteers, we show that uptake of nonopsonized Cryptococcus neoformans and C. gattii via the mannose receptor is dependent on macrophage activation by cytokines. However, although uptake of C. neoformans is via both dectin-1 and dectin-2, C. gattii uptake occurs largely via dectin-1. Interestingly, dectin inhibitors also blocked phagocytosis of unopsonized Cryptococci in wax moth (Galleria mellonella) larvae and partially protected the larvae from infection by both fungi, supporting a key role for host phagocytes in augmenting early disease establishment. Finally, we demonstrated that internalization of nonopsonized Cryptococci is not accompanied by the nuclear translocation of NF-κB or its concomitant production of proinflammatory cytokines such as TNF-α. Thus, nonopsonized Cryptococci are recognized by mammalian phagocytes in a manner that minimizes proinflammatory cytokine production and potentially facilitates fungal pathogenesis.

Oscillating Magnet Array-Based Nanomagnetic Gene Transfection: A Valuable Tool for Molecular Neurobiology Studies.

To develop treatments for neurodegenerative disorders, it is critical to understand the biology and function of neurons in both normal and diseased states. Molecular studies of neurons involve the delivery of small biomolecules into cultured neurons via transfection to study genetic variants. However, as cultured primary neurons are sensitive to temperature change, stress, and shifts in pH, these factors make biomolecule delivery difficult, particularly non-viral delivery. Herein we used oscillating nanomagnetic gene transfection to successfully transfect SH-SY5Y cells as well as primary hippocampal and cortical neurons on different days in vitro. This novel technique has been used to effectively deliver genetic material into various cell types, resulting in high transfection efficiency and viability. From these observations and other related studies, we suggest that oscillating nanomagnetic gene transfection is an effective method for gene delivery into hard-to-transfect neuronal cell types.

Multivalent adhesion molecule 7 clusters act as signaling platform for host cellular GTPase activation and facilitate epithelial barrier dysfunction.

Vibrio parahaemolyticus is an emerging bacterial pathogen which colonizes the gastrointestinal tract and can cause severe enteritis and bacteraemia. During infection, V. parahaemolyticus primarily attaches to the small intestine, where it causes extensive tissue damage and compromises epithelial barrier integrity. We have previously described that Multivalent Adhesion Molecule (MAM) 7 contributes to initial attachment of V. parahaemolyticus to epithelial cells. Here we show that the bacterial adhesin, through multivalent interactions between surface-induced adhesin clusters and phosphatidic acid lipids in the host cell membrane, induces activation of the small GTPase RhoA and actin rearrangements in host cells. In infection studies with V. parahaemolyticus we further demonstrate that adhesin-triggered activation of the ROCK/LIMK signaling axis is sufficient to redistribute tight junction proteins, leading to a loss of epithelial barrier function. Taken together, these findings show an unprecedented mechanism by which an adhesin acts as assembly platform for a host cellular signaling pathway, which ultimately facilitates breaching of the epithelial barrier by a bacterial pathogen.

Enhanced nanomagnetic gene transfection of human prenatal cardiac progenitor cells and adult cardiomyocytes.

Magnetic nanoparticle-based gene transfection has been shown to be an effective, non-viral technique for delivery of both plasmid DNA and siRNA into cells in culture. It has several advantages over other non-viral delivery techniques, such as short transfection times and high cell viability. These advantages have been demonstrated in a number of primary cells and cell lines. Here we report that oscillating magnet array-based nanomagnetic transfection significantly improves transfection efficiency in both human prenatal cardiac progenitor cells and adult cardiomyocytes when compared to static magnetofection, cationic lipid reagents and electroporation, while maintaining high cell viability. In addition, transfection of adult cardiomyocytes was improved further by seeding the cells onto Collagen I-coated plates, with transfection efficiencies of up to 49% compared to 24% with lipid reagents and 19% with electroporation. These results demonstrate that oscillating nanomagnetic transfection far outperforms other non-viral transfection techniques in these important cells.

Regulator of G-Protein Signalling-14 (RGS14) Regulates the Activation of αMß2 Integrin during Phagocytosis.

Integrin-mediated phagocytosis, an important physiological activity undertaken by professional phagocytes, requires bidirectional signalling to/from αMß2 integrin and involves Rap1 and Rho GTPases. The action of Rap1 and the cytoskeletal protein talin in activating αMß2 integrins, in a RIAM-independent manner, has been previously shown to be critical during phagocytosis in mammalian phagocytes. However, the events downstream of Rap1 are not clearly understood. Our data demonstrate that one potential Rap1 effector, Regulator of G-Protein Signalling-14 (RGS14), is involved in activating αMß2. Exogenous expression of RGS14 in COS-7 cells expressing αMß2 results in increased binding of C3bi-opsonised sheep red blood cells. Consistent with this, knock-down of RGS14 in J774.A1 macrophages results in decreased association with C3bi-opsonised sheep red blood cells. Regulation of αMß2 function occurs through the R333 residue of the RGS14 Ras/Rap binding domain (RBD) and the F754 residue of ß2, residues previously shown to be involved in binding of H-Ras and talin1 head binding prior to αMß2 activation, respectively. Surprisingly, overexpression of talin2 or RAPL had no effect on αMß2 regulation. Our results establish for the first time a role for RGS14 in the mechanism of Rap1/talin1 activation of αMß2 during phagocytosis.

Delivery of short interfering ribonucleic acid-complexed magnetic nanoparticles in an oscillating field occurs via caveolae-mediated endocytosis.

Gene delivery technologies to introduce foreign genes into highly differentiated mammalian cells have improved significantly over the last few decades. Relatively new techniques such as magnetic nanoparticle-based gene transfection technology are showing great promise in terms of its high transfection efficiency and wide-ranging research applications. We have developed a novel gene delivery technique, which uses magnetic nanoparticles moving under the influence of an oscillating magnetic array. Herein we successfully introduced short interfering RNA (siRNA) against green fluorescent protein (GFP) or actin into stably-transfected GFP-HeLa cells or wild-type HeLa and rat aortic smooth muscle cells, respectively. This gene silencing technique occurred in a dose- and cell density- dependent manner, as reflected using fluorescence intensity and adhesion assays. Furthermore, using endocytosis inhibitors, we established that these magnetic nanoparticle-nucleic acid complexes, moving across the cell surface under the influence of an oscillating magnet array, enters into the cells via the caveolae-mediated endocytic pathway.

Signalling mechanisms of the leukocyte integrin αMß2: current and future perspectives.

Integrins are a family of heterodimeric cell adhesion receptors expressed on most cells and are involved in many cellular functions including phagocytosis, a process by which professional phagocytes recognise, bind and internalise foreign materials larger than 0.5 µm in diameter. An example of a phagocytic integrin receptor is αMß2, and this review seeks to provide fresh insights into the current knowledge of this subject. Key areas that this review will emphasise include, the classical understanding of bi-directional signalling to and from αMß2 (aka inside-out and outside-in signalling, respectively). For inside-out signalling, we will review the involvement of the small GTPase, Rap1, FERM-containing proteins such as talin and kindlin-3, some of the kinases, and the GEF, cytohesin-1 and vasodilator-stimulated phosphoprotein (VASP). We also summarise studies into outside-in signalling, focussing on the roles of RhoA and RhoG, and activation of Rac1 through the complex comprising TIAM, 14-3-3 and ß2. We will also consider non-classical signalling processes, which include integrin clustering and membrane ruffling. Through this review, we hope to highlight the importance of αMß2 signalling mechanisms and their relevance to other integrin-mediated events.

Ser756 of ß2 integrin controls Rap1 activity during inside-out activation of αMß2.

During αMß2-mediated phagocytosis, the small GTPase Rap1 activates the ß2 integrin by binding to a region between residues 732 and 761. Using COS-7 cells transfected with αMß2, we show that αMß2 activation by the phorbol ester PMA involves Ser(756) of ß2. This residue is critical for the local positioning of talin and biochemically interacts with Rap1. Using the CaM (calmodulin) antagonist W7, we found Rap1 recruitment and the inside-out activation of αMß2 to be affected. We also report a role for CaMKII (calcium/CaM-dependent kinase II) in the activation of Rap1 during integrin activation. These results demonstrate a distinct physiological role for Ser(756) of ß2 integrin, in conjunction with the actions of talin and Rap1, during αMß2 activation in macrophages.

Rap1 controls activation of the α(M)ß(2) integrin in a talin-dependent manner.

The small GTPase Rap1 and the cytoskeletal protein talin regulate binding of C3bi-opsonised red blood cells (RBC) to integrin α(M)ß(2) in phagocytic cells, although the mechanism has not been investigated. Using COS-7 cells transfected with α(M)ß(2), we show that Rap1 acts on the ß(2) and not the α(M) chain, and that residues 732-761 of the ß(2) subunit are essential for Rap1-induced RBC binding. Activation of α(M)ß(2) by Rap1 was dependent on W747 and F754 in the ß(2) tails, which are required for talin head binding, suggesting a link between Rap1 and talin in this process. Using talin1 knock-out cells or siRNA-mediated talin1 knockdown in the THP-1 monocytic cell line, we show that Rap1 acts upstream of talin but surprisingly, RIAM knockdown had little effect on integrin-mediated RBC binding or cell spreading. Interestingly, Rap1 and talin influence each other's localisation at phagocytic cups, and co-immunoprecipitation experiments suggest that they interact together. These results show that Rap1-mediated activation of α(M)ß(2) in macrophages shares both common and distinct features from Rap1 activation of α(IIb)ß(3) expressed in CHO cells.

Four distinct structural domains in Clostridium difficile toxin B visualized using SAXS.

Clostridium difficile is a nosocomial bacterial pathogen causing antibiotic-associated diarrhea and fatal pseudomembranous colitis. Key virulence factors are toxin A and toxin B (TcdB), two highly related toxins that are members of the large clostridial toxin family. These large multifunctional proteins disrupt cell function using a glucosyltransferase domain that is translocated into the cytosol after vesicular internalization of intact holotoxin. Although substantial information about the biochemical mechanisms of intoxication exists, research has been hampered by limited structural information, particularly of intact holotoxin. Here, we used small-angle X-ray scattering (SAXS) methods to obtain an ab initio low-resolution structure of native TcdB, which demonstrated that this molecule is monomeric in solution and possesses a highly asymmetric shape with a maximum dimension of approximately 275 A. Combining this SAXS information with crystallographic or modeled structures of individual functional domains of TcdB reveals for the first time that the three-dimensional structure of TcdB is organized into four distinct structural domains. Structures of the N-terminal glucosyltransferase, the cysteine protease, and the C-terminal repeat region can be aligned within three domains of the SAXS envelope. A fourth domain, predicted to be involved in the translocation of the glucosyltransferase, appears as a large solvent-exposed protrusion. Knowledge of the shapes and relative orientations of toxin domains provides new insight into defining functional domain boundaries and provides a framework for understanding how potential intra-domain interactions enable conformational changes to propagate between domains to facilitate intoxication processes.

The tyrosine phosphatase CD148 is an essential positive regulator of platelet activation and thrombosis.

Platelets play a fundamental role in hemostasis and thrombosis. They are also involved in pathologic conditions resulting from blocked blood vessels, including myocardial infarction and ischemic stroke. Platelet adhesion, activation, and aggregation at sites of vascular injury are regulated by a diverse repertoire of tyrosine kinase-linked and G protein-coupled receptors. Src family kinases (SFKs) play a central role in initiating and propagating signaling from several platelet surface receptors; however, the underlying mechanism of how SFK activity is regulated in platelets remains unclear. CD148 is the only receptor-like protein tyrosine phosphatase identified in platelets to date. In the present study, we show that mutant mice lacking CD148 exhibited a bleeding tendency and defective arterial thrombosis. Basal SFK activity was found to be markedly reduced in CD148-deficient platelets, resulting in a global hyporesponsiveness to agonists that signal through SFKs, including collagen and fibrinogen. G protein-coupled receptor responses to thrombin and other agonists were also marginally reduced. These results highlight CD148 as a global regulator of platelet activation and a novel antithrombotic drug target.

An essential role for talin during alpha(M)beta(2)-mediated phagocytosis.

The cytoskeletal, actin-binding protein talin has been previously implicated in phagocytosis in Dictyostelium discoideum and mammalian phagocytes. However, its mechanism of action during internalization is not understood. Our data confirm that endogenous talin can occasionally be found at phagosomes forming around IgG- and C3bi-opsonized red blood cells in macrophages. Remarkably, talin knockdown specifically abrogates uptake through complement receptor 3 (CR3, CD11b/CD18, alpha(M)beta(2) integrin) and not through the Fc gamma receptor. We show that talin physically interacts with CR3/alpha(M)beta(2) and that this interaction involves the talin head domain and residues W747 and F754 in the beta(2) integrin cytoplasmic domain. The CR3/alpha(M)beta(2)-talin head interaction controls not only talin recruitment to forming phagosomes but also CR3/alpha(M)beta(2) binding activity, both in macrophages and transfected fibroblasts. However, the talin head domain alone cannot support phagocytosis. Our results establish for the first time at least two distinct roles for talin during CR3/alpha(M)beta(2)-mediated phagocytosis, most noticeably activation of the CR3/alpha(M)beta(2) receptor and phagocytic uptake.

Two distinct cytoplasmic regions of the beta2 integrin chain regulate RhoA function during phagocytosis.

AlphaMbeta2 integrins mediate phagocytosis of opsonized particles in a process controlled by RhoA, Rho kinase, myosin II, Arp2/3, and actin polymerization. AlphaMbeta2, Rho, Arp2/3, and F-actin accumulate underneath bound particles; however, the mechanism regulating Rho function during alphaMbeta2-mediated phagocytosis is poorly understood. We report that the binding of C3bi-opsonized sheep red blood cells (RBCs) to alphaMbeta2 increases Rho-GTP, but not Rac-GTP, levels. Deletion of the cytoplasmic domain of beta2, but not of alphaM, abolished Rho recruitment and activation, as well as phagocytic uptake. Interestingly, a 16-amino acid (aa) region in the membrane-proximal half of the beta2 cytoplasmic domain was necessary for activating Rho. Three COOH-terminal residues (aa 758-760) were essential for beta2-induced accumulation of Rho at complement receptor 3 (CR3) phagosomes. Activation of Rho was necessary, but not sufficient, for its stable recruitment underneath bound particles or for uptake. However, recruitment of active Rho was sufficient for phagocytosis. Our data shed light on the mechanism of outside-in signaling, from ligated integrins to the activation of Rho GTPase signaling.