A synonymous mutation in Yersinia enterocolitica yopE affects the function of the YopE type III secretion signal

Yersinia spp. inject virulence proteins called Yops into the cytosol of target eukaryotic cells in an effort to evade phagocytic killing via a dedicated protein-sorting pathway termed type III secretion. Previous studies have proposed that, unlike other protein translocation mechanisms, Yops are not recognized as substrates for secretion via a solely proteinaceous signal. Rather, at least some of this information may be encoded within yop mRNA. Herein, we report that the first seven codons of yopE, when fused to the reporter protein neomycin phosphotransferase (Npt), are sufficient for the secretion of YopE1-7-Npt when type III secretion is induced in vitro. Systematic mutagenesis of yopE codons 1 to 7 reveals that, like yopQ, codons 2, 3, 5, and 7 are sensitive to mutagenesis, thereby defining the first empirical similarity between the secretion signals of two type III secreted substrates. Like that of yopQ, the secretion signal of yopE exhibits a bipartite nature. This is manifested by the ability of codons 8 to 15 to suppress point mutations in the minimal secretion signal that change the amino acid specificities of particular codons or that induce alterations in the reading frame. Further, we have identified a single nucleotide position in codon 3 that, when mutated, conserves the predicted amino acid sequence of the YopE1-7-Npt but abrogates secretion of the reporter protein. When introduced into the context of the full-length yopE gene, the single-nucleotide mutation reduces the type III injection of YopE into HeLa cells, even though the predicted amino acid sequence remains the same. Thus, yopE mRNA appears to encode a property that mediates the type III injection of YopE.     

Editorial

The inactivation of the ClC-0 chloride channel is very temperature sensitive and is greatly facilitated by the binding of a zinc ion (Zn²⁺) from the extracellular side, leading to a Zn²⁺-induced current inhibition. To further explore the relation of Zn²⁺ inhibition and the ClC-0 inactivation, we mutated all 12 cysteine amino acids in the channel and assayed the effect of Zn²⁺ on these mutants. With this approach, we found that C212 appears to be important for the sensitivity of the Zn²⁺ inhibition. Upon mutating C212 to serine or alanine, the inactivation of the channel in macroscopic current recordings disappears and the channel does not show detectable inactivation events at the single-channel level. At the same time, the channel''s sensitivity to Zn²⁺ inhibition is also greatly reduced. The other two cysteine mutants, C213G and C480S, as well as a previously identified mutant, S123T, also affect the inactivation of the channel to some degree, but the temperature-dependent inactivation process is still present, likewise the high sensitivity of the Zn²⁺ inhibition. These results further support the assertion that the inhibition of Zn²⁺ on ClC-0 is indeed due to an effect on the inactivation of the channel. The absence of inactivation in C212S mutants may provide a better defined system to study the fast gating and the ion permeation of ClC-0.     

Survival of priceless cells: active and passive protection of embryonic stem cells against immune destruction

This review focuses on our current knowledge of the mechanisms employed by embryonic stem (ES) cells to avoid destruction by cell-mediated immune responses. Recently, ES cells have been found to shield themselves against cytotoxic effector cells by expressing CD95L and serine protease inhibitor SPI-6 mediating apoptosis of the cytotoxic cells and inactivation of granzyme B, respectively. These findings are discussed in view of their implications for using ES cell-derived transplants in regenerative medicine as well as for our understanding of early embryonic stages during invasion and implantation.     

Podoplanin Immunopositive Lymphatic Vessels at the Implant Interface in a Rat Model of Osteoporotic Fractures

Insertion of bone substitution materials accelerates healing of osteoporotic fractures. Biodegradable materials are preferred for application in osteoporotic patients to avoid a second surgery for implant replacement. Degraded implant fragments are often absorbed by macrophages that are removed from the fracture side via passage through veins or lymphatic vessels. We investigated if lymphatic vessels occur in osteoporotic bone defects and whether they are regulated by the use of different materials. To address this issue osteoporosis was induced in rats using the classical method of bilateral ovariectomy and additional calcium and vitamin deficient diet. In addition, wedge-shaped defects of 3, 4, or 5 mm were generated in the distal metaphyseal area of femur via osteotomy. The 4 mm defects were subsequently used for implantation studies where bone substitution materials of calcium phosphate cement, composites of collagen and silica, and iron foams with interconnecting pores were inserted. Different materials were partly additionally functionalized by strontium or bisphosphonate whose positive effects in osteoporosis treatment are well known. The lymphatic vessels were identified by immunohistochemistry using an antibody against podoplanin. Podoplanin immunopositive lymphatic vessels were detected in the granulation tissue filling the fracture gap, surrounding the implant and growing into the iron foam through its interconnected pores. Significant more lymphatic capillaries were counted at the implant interface of composite, strontium and bisphosphonate functionalized iron foam. A significant increase was also observed in the number of lymphatics situated in the pores of strontium coated iron foam. In conclusion, our results indicate the occurrence of lymphatic vessels in osteoporotic bone. Our results show that lymphatic vessels are localized at the implant interface and in the fracture gap where they might be involved in the removal of lymphocytes, macrophages, debris and the implants degradation products. Therefore the lymphatic vessels are involved in implant integration and fracture healing.     

Yop fusions to tightly folded protein domains and their effects on Yersinia enterocolitica type III secretion

Yersinia enterocolitica organisms secrete Yop proteins via the type III pathway. Translational fusion of yop genes to ubiquitin or dihydrofolate reductase results in hybrid proteins that cannot be secreted. The folding of hybrids prevents their own transport, but it does not hinder the type III secretion of other Yops.     

Infection with Soil-Transmitted Helminths Is Associated with Increased Insulin Sensitivity

Objective

Given that helminth infections have been shown to improve insulin sensitivity in animal studies, which may be explained by beneficial effects on energy balance or by a shift in the immune system to an anti-inflammatory profile, we investigated whether soil-transmitted helminth (STH)-infected subjects are more insulin sensitive than STH-uninfected subjects.

Design

We performed a cross-sectional study on Flores island, Indonesia, an area with high prevalence of STH infections.

Methods

From 646 adults, stool samples were screened for Trichuris trichiura by microscopy and for Ascaris lumbricoides, Necator americanus, Ancylostoma duodenale, and Strongyloides stercoralis by qPCR. No other helminth was found. We collected data on body mass index (BMI, kg/m²), waist-to-hip ratio (WHR), fasting blood glucose (FBG, mmol/L), insulin (pmol/L), high sensitive C-reactive protein (ng/ml) and Immunoglobulin E (IU/ml). The homeostatic model assessment for insulin resistance (HOMAIR) was calculated and regression models were used to assess the association between STH infection status and insulin resistance.

Results

424 (66%) participants had at least one STH infection. STH infected participants had lower BMI (23.2 vs 22.5 kg/m², p value = 0.03) and lower HOMAIR (0.97 vs 0.81, p value = 0.05). In an age-, sex- and BMI-adjusted model a significant association was seen between the number of infections and HOMAIR: for every additional infection with STH species, the HOMAIR decreased by 0.10 (p for linear trend 0.01). This effect was mainly accounted for by a decrease in insulin of 4.9 pmol/L for every infection (p for trend = 0.07).

Conclusion

STH infections are associated with a modest improvement of insulin sensitivity, which is not accounted for by STH effects on BMI alone.     

A phosphorylation code of the Aspergillus nidulans global regulator VelvetA (VeA) determines specific functions

The velvet protein VeA is a global fungal regulator for morphogenetic pathways as well as for the control of secondary metabolism. It is found exclusively in filamentous fungi, where it fulfills conserved, but also unique functions in different species. The involvement of VeA in various morphogenetic and metabolic pathways is probably due to spatially and timely controlled specific protein–protein interactions with other regulators such as phytochrome (FphA) or velvet‐like proteins (VelB). Here we present evidence that Aspergillus nidulans VeA is a multi‐phosphorylated protein and hypothesize that at least four specific amino acids (T167, T170, S183 and Y254) undergo reversible phosphorylation to trigger development and sterigmatocystin biosynthesis. Double mutation of T167 to valine and T170 to glutamic acid exerted the largest effects with regards to sexual development and veA gene expression. In comparison with wild‐type VeA, which shuttles out of the nuclei after illumination this VeA variant showed stronger nuclear accumulation than the wild type, independent of the light conditions. The interaction between VeA and VelB or FphA, respectively, was affected in the T167V‐T170E mutant. Our results suggest complex regulation of the phosphorylation status of the VeA protein.     

Measuring Preferences on Environmental Damages in LCIA. Part 1: Cognitive Limits in Panel Surveys (9 pp)

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Part 1: Cognitive Limits in Panel Surveys · Part 2: The Question Format in Panel Surveys This series of two papers discusses the elicitation of weights for damage categories in LCIA with the aid of panel surveys. The papers focus especially on methodological aspects in panel surveys. Part 1 discusses potential cognitive limits of the panel members to understand the reference that is weighted. Part 2 focuses on the influence of the question format and compares results from two different weighting tasks: discrete choice (between alternatives) and score allocation.

Goal, Scope and Background

The weighting of environmental impacts and damages on the safeguard subjects Human Health, Ecosystems, and Resources is a significant step of full aggregated LCIA. Panel surveys have become a common approach in LCIA research to investigate the preferences of stakeholders on environmental impacts and damages. Despite the numerous studies, the knowledge on how to elicit reliable weights is still poor and inconsistent. We present a questionnaire study with 58 environmental science students to investigate so-called framing effects in panel surveys.

Main Features

The study investigates the significance of different framings, which were provided by three references. In addition, the significance of quantitative information provided in the questionnaire is tested. The references are (a1) safeguard subjects without specified additional information, (a2) damages in Europe as they are perceived by the panelist, and (a3) quantified scenarios derived from Eco-indicator99. All participants ranked and rated the importance of the safeguard subjects three times, once within each reference system. According to a test-of-scope study, quantitative information given to the panelist was varied. One level (b1) included data from the Ecoindicator99 methodology, whereas the other group (b2) received data with significantly higher Human Health damages and lower Ecosystem damages, ceteris paribus. This design allows testing the influence of quantitative data on the rating.

Results

The weighting of the safeguard subjects (a1) reveals that Human Health is considered a slightly more important safeguard subject than Ecosystems. However, both are judged to be significantly more important than Resources. This picture changes for the references (a2) and (a3) where damages were weighted. For both references, the respondents rated damages to Ecosystems as most important followed by Resources and Human Health, showing by far the lowest weights. Therefore, the framing of the reference that was weighted played a significant role. The ratings of the subgroups (b1) and (b2) did not differ with respect to the importance of damages, though substantially different quantitative information was given.

Conclusion and Outlook

The participants of the study were obviously insensitive with respect to quantitative information provided. This raises three questions, which are discussed. What is the mental model upon which respondents base their beliefs and values? Can we expect that 'more sophisticated' subjects would respond differently? Which prerequisites should an empirical weighting procedure fulfill in order to incorporate numerical data? We propose different approaches for future procedures in order to accurately analyze these questions.