ARHGEF7 (Beta-PIX) acts as guanine nucleotide exchange factor for leucine-rich repeat kinase 2

Background

Mutations within the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of familial and sporadic Parkinson''s disease. The multidomain protein LRRK2 exhibits overall low GTPase and kinase activity in vitro.

Methodology/Principal Findings

Here, we show that the rho guanine nucleotide exchange factor ARHGEF7 and the small GTPase CDC42 are interacting with LRRK2 in vitro and in vivo. GTPase activity of full-length LRRK2 increases in the presence of recombinant ARHGEF7. Interestingly, LRRK2 phosphorylates ARHGEF7 in vitro at previously unknown phosphorylation sites. We provide evidence that ARHGEF7 might act as a guanine nucleotide exchange factor for LRRK2 and that R1441C mutant LRRK2 with reduced GTP hydrolysis activity also shows reduced binding to ARHGEF7.

Conclusions/Significance

Downstream effects of phosphorylation of ARHGEF7 through LRRK2 could be (i) a feedback control mechanism for LRRK2 activity as well as (ii) an impact of LRRK2 on actin cytoskeleton regulation. A newly identified familial mutation N1437S, localized within the GTPase domain of LRRK2, further underlines the importance of the GTPase domain of LRRK2 in Parkinson''s disease pathogenesis.     

A brief history of angiogenesis assays

The major problem with angiogenesis research is the choice of an appropriate assay. Currently, many in vitro and in vivo techniques are available for research into the functions of endothelial cells during angiogenesis. In this historical review article, we describe and evaluate the methodology and specific features of some of the most frequently used of these assays.     

Cometabolic degradation of organic wastewater micropollutants by activated sludge and sludge-inherent microorganisms

Municipal wastewaters contain a multitude of organic trace pollutants. Often, their biodegradability by activated sludge microorganisms is decisive for their elimination during wastewater treatment. Since the amounts of micropollutants seem too low to serve as growth substrate, cometabolism is supposed to be the dominating biodegradation process. Nevertheless, as many biodegradation studies were performed without the intention to discriminate between metabolic and cometabolic processes, the specific contribution of the latter to substance transformations is often not clarified. This minireview summarizes current knowledge about the cometabolic degradation of organic trace pollutants by activated sludge and sludge-inherent microorganisms. Due to their relevance for communal wastewater contamination, the focus is laid on pharmaceuticals, personal care products, antibiotics, estrogens, and nonylphenols. Wherever possible, reference is made to the molecular process level, i.e., cometabolic pathways, involved enzymes, and formed transformation products. Particular cometabolic capabilities of different activated sludge consortia and various microbial species are highlighted. Process conditions favoring cometabolic activities are emphasized. Finally, knowledge gaps are identified, and research perspectives are outlined.     

Glycine inhibits angiogenic signaling in human hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a highly vascularized tumor with limited susceptibility to chemotherapy. Modern targeted therapies are aimed at specific properties of this neoplasm. Glycine is a simple non-essential amino acid with potential antiangiogenic effects. In this study, the amino acid’s effect on angiogenic signaling in an in vitro model of HCC was evaluated. HepG2 and Huh7 cells were treated with glycine-free DMEM supplemented with 0, 0.01, 0.1, 1.0, 2.0, 5.0 and 10 mM glycine. The direct effects of glycine on the viability of HCC cells were monitored using MTT assay. To detect angiogenic signaling, mRNA and protein levels of vascular endothelial growth factor (VEGF-A) were measured using RT-PCR and Western Blot assays. To determine whether or not glycine receptors (GlyR) played a significant role, the specific antagonist, strychnine, was used as a direct inhibitor. Western Blotting was performed to show the presence of GlyR. While there was no direct pro- or antiproliferative effect of either glycine or strychnine in both cell lines, glycine was shown to significantly decrease VEGF-A expression on mRNA and protein level up to 63 % in both cell lines. This effect was blunted by the presence of strychnine. GlyR was also identified in both cell lines. Glycine decreases GlyR-dependent, VEGF-A-mediated, angiogenic signaling in human HCC and thus might be a promising additive to chemotherapy treatment strategies for highly vascularized tumors.     

How environment geometry affects grid cell symmetry and what we can learn from it

The mammalian hippocampal formation provides neuronal representations of environmental location but the underlying mechanisms are unclear. The majority of cells in medial entorhinal cortex and parasubiculum show spatially periodic firing patterns. Grid cells exhibit hexagonal symmetry and form an important subset of this more general class. Occasional changes between hexagonal and non-hexagonal firing patterns imply a common underlying mechanism. Importantly, the symmetrical properties are strongly affected by the geometry of the environment. Here, we introduce a field–boundary interaction model where we demonstrate that the grid cell pattern can be formed from competing place-like and boundary inputs. We show that the modelling results can accurately capture our current experimental observations.     

Testing the stages model in the adaptive radiation of cichlid fishes in East African Lake Tanganyika

Adaptive radiation (AR) is a key process in the origin of organismal diversity. However, the evolution of trait disparity in connection with ecological specialization is still poorly understood. Available models for vertebrate ARs predict that diversification occurs in the form of temporal stages driven by different selective forces. Here, we investigate the AR of cichlid fishes in East African Lake Tanganyika and use macroevolutionary model fitting to evaluate whether diversification happened in temporal stages. Six trait complexes, for which we also provide evidence of their adaptiveness, are analysed with comparative methods: body shape, pharyngeal jaw shape, gill raker traits, gut length, brain weight and body coloration. Overall, we do not find strong evidence for the ‘stages model’ of AR. However, our results suggest that trophic traits diversify earlier than traits implicated in macrohabitat adaptation and that sexual communication traits (i.e. coloration) diversify late in the radiation.     

Redox State of Pentraxin 3 as a Novel Biomarker for Resolution of Inflammation and Survival in Sepsis

In an endotoxaemic mouse model of sepsis, a tissue-based proteomics approach for biomarker discovery identified long pentraxin 3 (PTX3) as the lead candidate for inflamed myocardium. When the redox-sensitive oligomerization state of PTX3 was further investigated, PTX3 accumulated as an octamer as a result of disulfide-bond formation in heart, kidney, and lung—common organ dysfunctions seen in patients with sepsis. Oligomeric moieties of PTX3 were also detectable in circulation. The oligomerization state of PTX3 was quantified over the first 11 days in critically ill adult patients with sepsis. On admission day, there was no difference in the oligomerization state of PTX3 between survivors and non-survivors. From day 2 onward, the conversion of octameric to monomeric PTX3 was consistently associated with a greater survival after 28 days of follow-up. For example, by day 2 post-admission, octameric PTX3 was barely detectable in survivors, but it still constituted more than half of the total PTX3 in non-survivors (p < 0.001). Monomeric PTX3 was inversely associated with cardiac damage markers NT-proBNP and high-sensitivity troponin I and T. Relative to the conventional measurements of total PTX3 or NT-proBNP, the oligomerization of PTX3 was a superior predictor of disease outcome.Severe sepsis is a common acute illness in intensive care units (ICUs)¹ and is associated with high mortality rates and chronic morbidity. When it is associated with hypotension (termed septic shock), the mortality rate is very high (50% to 80%). Cardiovascular dysfunction during sepsis is multifactorial and often associated with minimal loss of myocardial tissue, but with the release of myocardial-specific markers such as troponins. A key unmet clinical need is the availability of a biomarker that predicts myocardial dysfunction early, monitors response to treatment, and thus identifies a cohort of patients at higher risk of septic shock to aid in targeted interventions and improve outcome (1).In the present study, we used proteomics for biomarker discovery. Over the past decade, the field of proteomics has made impressive progress. Plasma and serum, however, are the most complex proteomes of the human body (2), and less abundant proteins tend to be missed in untargeted proteomics analyses of body fluids (3). Thus, we pursued an alternative strategy: the application of proteomics to diseased tissue (4), in which the potential biomarkers are less dilute and have a less uncertain cellular origin (5–7). We employed a solubility-based protein-subfractionation methodology to analyze inflammatory proteins that are retained with sepsis tissue. This innovative proteomics approach shall reveal inflammatory molecules that reside and persist within inflamed tissue. We hypothesized that proteins that accumulate in the susceptible tissues are more likely to be biomarker candidates for organ dysfunction than proteins that just circulate in plasma or serum. We then validated our proteomics findings in the preclinical model using samples from sepsis patients admitted to ICUs.