The Bdellovibrio bacteriovorus twin-arginine transport system has roles in predatory and prey-independent growth

Bdellovibrio bacteriovorus grows in one of two ways: either (i) predatorily [in a host-dependent (HD) manner], when it invades the periplasm of another Gram-negative bacterium, exporting into the prey co-ordinated waves of soluble enzymes using the prey cell contents for growth; or (ii) in a host-independent (HI) manner, when it grows (slowly) axenically in rich media. Periplasmic invasion potentially exposes B. bacteriovorus to extremes of pH and exposes the need to scavenge electron donors from prey electron transport components by synthesis of metalloenzymes. The twin-arginine transport system (Tat) in other bacteria transports folded metalloenzymes and the B. bacteriovorus genome encodes 21 potential Tat-transported substrates and Tat transporter proteins TatA1, TatA2 and TatBC. GFP tagging of the Tat signal peptide from Bd1802, a high-potential iron-sulfur protein (HiPIP), revealed it to be exported into the prey bacterium during predatory growth. Mutagenesis showed that the B. bacteriovorus tatA2 and tatC gene products are essential for both HI and HD growth, despite the fact that they partially complement (in SDS resistance assays) the corresponding mutations in Escherichia coli where neither TatA nor TatC are essential for life. The essentiality of B. bacteriovorus TatA2 was surprising given that the B. bacteriovorus genome encodes a second tatA homologue, tatA1. Transcription of tatA1 was found to be induced upon entry to the bdelloplast, and insertional inactivation of tatA1 showed that it significantly slowed the rates of both HI and HD growth. B. bacteriovorus is one of a few bacterial species that are reliant on a functional Tat system and where deletion of a single tatA1 gene causes a significant growth defect(s), despite the presence of its tatA2 homologue.

Evidence supporting a role for N-epsilon-(3-formyl-3,4-dehydropiperidino)lysine accumulation in Muller glia dysfunction and death in diabetic retinopathy

Purpose: Recent evidence suggests that neuroglial dysfunction and degeneration contributes to the etiology and progression of diabetic retinopathy. Advanced lipoxidation end products (ALEs) have been implicated in the pathology of various diseases, including diabetes and several neurodegenerative disorders. The purpose of the present study was to investigate the possible link between the accumulation of ALEs and neuroretinal changes in diabetic retinopathy.

Methods: Retinal sections obtained from diabetic rats and age-matched controls were processed for immunohistochemistry using antibodies against several well defined ALEs. In vitro experiments were also performed using a human Muller (Moorfields/Institute of Ophthalmology-Muller 1 [ MIO-M1]) glia cell line. Western blot analysis was used to measure the accumulation of the acrolein-derived ALE adduct N epsilon-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) in Muller cells preincubated with FDP-lysine-modified human serum albumin (FDP-lysine-HSA). Responses of Muller cells to FDP-lysine accumulation were investigated by analyzing changes in the protein expression of heme oxygenase-1 (HO-1), glial fibrillary acidic protein (GFAP), and the inwardly rectifying potassium channel Kir4.1. In addition, mRNA expression levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF alpha) were determined by reverse transcriptase PCR (RT-PCR). Apoptotic cell death was evaluated by fluorescence-activated cell sorting (FACS) analysis after staining with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide.

Results: No significant differences in the levels of malondialdehyde-, 4-hydroxy-2-nonenal-, and 4-hydroxyhexenal-derived ALEs were evident between control and diabetic retinas after 4 months of diabetes. By contrast, FDP-lysine immunoreactivity was markedly increased in the Muller glia of diabetic rats. Time-course studies revealed that FDP-lysine initially accumulated within Muller glial end feet after only a few months of diabetes and thereafter spread distally throughout their inner radial processes. Exposure of human Muller glia to FDP-lysine-HSA led to a concentration-dependent accumulation of FDP-lysine-modified proteins across a broad molecular mass range. FDP-lysine accumulation was associated with the induction of HO-1, no change in GFAP, a decrease in protein levels of the potassium channel subunit Kir4.1, and upregulation of transcripts for VEGF, IL-6, and TNF-alpha. Incubation of Muller glia with FDP-lysine-HSA also caused apoptosis at high concentrations.

Conclusions: Collectively, these data strongly suggest that FDP-lysine accumulation could be a major factor contributing to the Muller glial abnormalities occurring in the early stages of diabetic retinopathy.

A spatio-temporal contrast of the predatory impact of an invasive freshwater crustacean

Aim: Impacts of invasive species may vary across invasion gradients, owing to trait-based sorting of individuals through dispersal: those aggregating at invasion fronts may be more aggressive and voracious. We examine, in the field and laboratory, variation in the predatory impacts of an invasive Ponto-Caspian crustacean Hemimysis anomala G.O. Sars, 1907 at two sites along a spatio-temporal gradient of invasion.

Location: Republic of Ireland.

Methods: We used reciprocal transplant field-deployed mesocosms to compare predation rates of invasion front and well-established H. anomala on natural zooplankton assemblages. In the laboratory, we measured the functional response (relationship between predation rate and prey supply) of H. anomala from both sites, for a per capita mechanistic comparison of predation efficiency. We also assessed prey selectivity of H. anomala in the mesocosm experiments to further compare feeding behaviour. Finally, we used a correlative approach to assess the community impact of H. anomala across sites, including a nearby uninvaded site, by comparing zooplankton diversities and densities.

Results: Invasion front H. anomala had higher predation rates than well-established H. anomala at high in situ zooplankton densities. Invasion front H. anomala also had higher functional responses - in particular showing higher 'attack rates' - indicating a heightened ability to locate and capture prey. Prey selectivity was consistent across the spatio-temporal contrast, with positive selection for cladocerans. Zooplankton diversity and density declined with time since H. anomala invasion, both being maximal at the uninvaded site.

Main conclusions: Our study, for the first time, (1) reveals differences in predatory per capita effects and associated behavioural traits between two sites along a spatio-temporal invasion gradient and (2) shows a negative community-level impact of the invasive H. anomala in natural water bodies. Further spatio-temporal comparisons of predatory per capita effects of invaders are needed to assess the generality of these results.

Investigation of organic xenobiotic transfers, partitioning and processing in air-soil-plant systems using a microcosm apparatus. Part I:Microcosm development

A microcosm system was developed to investigate transfers of organic xenobiotics in air-soil-plant systems. This was validated using ¹⁴C labelled 1,2-dichlorobenzene (DCB) as a model compound. Trapping efficiency was 106 ± 3% for volatile compounds and 93.0 ± 2.2% for carbon dioxide in a blank microcosm arrangement. Recovery of 1,2-dichlorobenzene spiked to grassed and unplanted soils was > 90% after 1 week. The predominant DCB loss process was volatilisation with no evidence for mineralisation over 1 week and 20-30% of the added spike remained in soil. Although there was no evidence for root uptake and translocation of added label, foliar uptake of soil volatilised compound was detected. The microcosm showed good potential for study of ¹⁴C labelled and unlabelled organic xenobiotic transfers in air-soil-plant systems with single plants and also intact planted cores.

Modulation of human corticospinal excitability by paired associative stimulation

Paired Associative Stimulation (PAS) has come to prominence as a potential therapeutic intervention for the treatment of brain injury/disease, and as an experimental method with which to investigate Hebbian principles of neural plasticity in humans. Prototypically, a single electrical stimulus is directed to a peripheral nerve in advance of transcranial magnetic stimulation (TMS) delivered to the contralateral primary motor cortex (M1). Repeated pairing of the stimuli (i.e., association) over an extended period may increase or decrease the excitability of corticospinal projections from M1, in manner that depends on the interstimulus interval (ISI). It has been suggested that these effects represent a form of associative long-term potentiation (LTP) and depression (LTD) that bears resemblance to spike-timing dependent plasticity (STDP) as it has been elaborated in animal models. With a large body of empirical evidence having emerged since the cardinal features of PAS were first described, and in light of the variations from the original protocols that have been implemented, it is opportune to consider whether the phenomenology of PAS remains consistent with the characteristic features that were initially disclosed. This assessment necessarily has bearing upon interpretation of the effects of PAS in relation to the specific cellular pathways that are putatively engaged, including those that adhere to the rules of STDP. The balance of evidence suggests that the mechanisms that contribute to the LTP- and LTD-type responses to PAS differ depending on the precise nature of the induction protocol that is used. In addition to emphasizing the requirement for additional explanatory models, in the present analysis we highlight the key features of the PAS phenomenology that require interpretation.

Endosomal signaling and oncogenesis

The endosomal system provides a route whereby nutrients, viruses, and receptors are internalized. During the course of endocytosis, activated receptors can accumulate within endosomal structures and certain signal-transducing molecules can be recruited to endosomal membranes. In the context of signaling and cancer, they provide platforms within the cell from which signals can be potentiated or attenuated. Regulation of the duration of receptor signaling is a pivotal means of refining growth responses in cells. In cancers, this is often considered in terms of mutations that affect receptor tyrosine kinases and maintain them in hyperactivated states of dimerization and/or phosphorylation. However, disruption to the regulatory control exerted by the assembly of protein complexes within the endosomal network can also contribute to disease among which oncogenesis is characterized in part by dysregulated growth, enhanced cell survival, and changes in the expression of markers of differentiation. In this chapter, we will discuss the role of proteins that regulate in endocytosis as tumor suppressors or oncogenes and how changing the fate of internalized receptors and concomitant endosomal signaling can contribute to cancer.

BAR domains as sensors of membrane curvature:the amphiphysin BAR structure

The BAR (Bin/amphiphysin/Rvs) domain is the most conserved feature in amphiphysins from yeast to human and is also found in endophilins and nadrins. We solved the structure of the Drosophila amphiphysin BAR domain. It is a crescent-shaped dimer that binds preferentially to highly curved negatively charged membranes. With its N-terminal amphipathic helix and BAR domain (N-BAR), amphiphysin can drive membrane curvature in vitro and in vivo. The structure is similar to that of arfaptin2, which we find also binds and tubulates membranes. From this, we predict that BAR domains are in many protein families, including sorting nexins, centaurins, and oligophrenins. The universal and minimal BAR domain is a dimerization, membrane-binding, and curvature-sensing module.

Endosomal localization and receptor dynamics determine tyrosine phosphorylation of hepatocyte growth factor-regulated tyrosine kinase substrate

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a prominent substrate for activated tyrosine kinase receptors that has been proposed to play a role in endosomal membrane trafficking. The protein contains a FYVE domain, which specifically binds to the lipid phosphatidylinositol (PI) 3-phosphate (PI 3-P). We show that this interaction is required both for correct localization of the protein to endosomes that only partially coincides with early endosomal autoantigen 1 and for efficient tyrosine phosphorylation of the protein in response to epidermal growth factor stimulation. Treatment with wortmannin reveals that Hrs phosphorylation also requires PI 3-kinase activity, which is necessary to generate the PI 3-P required for localization. We have used both hypertonic media and expression of a dominant-negative form of dynamin (K44A) to inhibit endocytosis; under which conditions, receptor stimulation fails to elicit phosphorylation of Hrs. Our results provide a clear example of the coupling of a signal transduction pathway to endocytosis, from which we propose that activated receptor (or associated factor) must be delivered to the appropriate endocytic compartment in order for Hrs phosphorylation to occur.

Eps15 and Dap160 control synaptic vesicle membrane retrieval and synapse development

Epidermal growth factor receptor pathway substrate clone 15 (Eps15) is a protein implicated in endocytosis, endosomal protein sorting, and cytoskeletal organization. Its role is, however, still unclear, because of reasons including limitations of dominant-negative experiments and apparent redundancy with other endocytic proteins. We generated Drosophila eps15-null mutants and show that Eps15 is required for proper synaptic bouton development and normal levels of synaptic vesicle (SV) endocytosis. Consistent with a role in SV endocytosis, Eps15 moves from the center of synaptic boutons to the periphery in response to synaptic activity. The endocytic protein, Dap160/intersectin, is a major binding partner of Eps15, and eps15 mutants phenotypically resemble dap160 mutants. Analyses of eps15 dap160 double mutants suggest that Eps15 functions in concert with Dap160 during SV endocytosis. Based on these data, we hypothesize that Eps15 and Dap160 promote the efficiency of endocytosis from the plasma membrane by maintaining high concentrations of multiple endocytic proteins, including dynamin, at synapses.

Studying N-linked glycosylation of receptor tyrosine kinases

Metabolic alterations have been identified as a frequent event in cancer. This is often associated with increased flux through glycolysis, and also a secondary pathway to glycolysis, hexosamine biosynthetic pathway (HBP). HBP provides substrate for N-linked glycosylation, which occurs in the endoplasmic reticulum and the Golgi apparatus. N-linked glycosylation supports protein folding and correct sorting of proteins to plasma membrane and secretion. This process generates complex glycoforms, which can be recognized by other proteins and glycosylation of receptor tyrosine kinases (RTK) can also regulate their plasma-membrane retention time. Of special interest for experimental biologists, plants produce proteins, termed lectins, which bind with high specificity to glyco-conjugates. For the purposes of molecular biology, plant lectins can be conjugated to different moieties, such as agarose beads, which enable precipitation of specifically glycosylated proteins. In this chapter, we describe in detail how to perform pull-down experiments with commercially available lectins to identify changes in the glycosylation of RTKs.

Targeting translocator protein (18 kDa) (TSPO) dampens pro-inflammatory microglia reactivity in the retina and protects from degeneration

BACKGROUND: Reactive microglia are commonly seen in retinal degenerative diseases, and neurotoxic microglia responses can contribute to photoreceptor cell death. We and others have previously shown that translocator protein (18 kDa) (TSPO) is highly induced in retinal degenerations and that the selective TSPO ligand XBD173 (AC-5216, emapunil) exerts strong anti-inflammatory effects on microglia in vitro and ex vivo. Here, we investigated whether targeting TSPO with XBD173 has immuno-modulatory and neuroprotective functions in two mouse models of acute retinal degeneration using bright white light exposure.

METHODS: BALB/cJ and Cx3cr1 (GFP/+) mice received intraperitoneal injections of 10 mg/kg XBD173 or vehicle for five consecutive days, starting 1 day prior to exposure to either 15,000 lux white light for 1 h or 50,000 lux focal light for 10 min, respectively. The effects of XBD173 treatment on microglia and Müller cell reactivity were analyzed by immuno-stainings of retinal sections and flat mounts, fluorescence-activated cell sorting (FACS) analysis, and mRNA expression of microglia markers using quantitative real-time PCR (qRT-PCR). Optical coherence tomography (OCT), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stainings, and morphometric analyses were used to quantify the extent of retinal degeneration and photoreceptor apoptosis.

RESULTS: Four days after the mice were challenged with bright white light, a large number of amoeboid-shaped alerted microglia appeared in the degenerating outer retina, which was nearly completely prevented by treatment with XBD173. This treatment also down-regulated the expression of TSPO protein in microglia but did not change the TSPO levels in the retinal pigment epithelium (RPE). RT-PCR analysis showed that the microglia/macrophage markers Cd68 and activated microglia/macrophage whey acidic protein (Amwap) as well as the pro-inflammatory genes Ccl2 and Il6 were reduced after XBD173 treatment. Light-induced degeneration of the outer retina was nearly fully blocked by XBD173 treatment. We further confirmed these findings in an independent mouse model of focal light damage. Retinas of animals receiving XBD173 therapy displayed significantly more ramified non-reactive microglia and more viable arrestin-positive cone photoreceptors than vehicle controls.

CONCLUSIONS: Targeting TSPO with XBD173 effectively counter-regulates microgliosis and ameliorates light-induced retinal damage, highlighting a new pharmacological concept for the treatment of retinal degenerations.

Tenure-track contract helps self-selection

Tenure-track contract is criticized for curbing the incentives to expend effort after obtaining tenured status. Yet the most productive faculties seem to work on a tenure contract, and schools which aim to employ the best faculty seem to prefer to offer tenure-track contracts to their new recruits. I argue that tenure-track contracts are by construction more attractive to more able freshly minted PhDs, and therefore the observed sorting is rationalizable.

Identifying the relative importance of stock characteristics

There is no consensus in the literature as to which stock characteristic best explains returns. In this study, we employ a novel econometric approach better suited than the traditional characteristic sorting method to answer this question for the UK market. We evaluate the relative explanatory power of market, size, momentum, volatility, liquidity and book-to-market factors in a semiparametric characteristic-based factor model which does not require constructing characteristic portfolios. We find that momentum is the most important factor and liquidity is the least important based on their relative contribution to the fit of the model and the proportion of sample months for which factor returns are significant. Overall, this study provides strong evidence to support that the momentum characteristic can best explain stock returns in the UK market. The econometric approach employed in this study is a novel way to assess relevant investment risk in international financial markets outside U.S. Moreover, multinational institutions and investors can use this approach to identify regional factors in order to diversify their portfolios.