High levels of cyclic‐di‐GMP in plant‐associated Pseudomonas correlate with evasion of plant immunity

The plant innate immune system employs plasma membrane‐localized receptors that specifically perceive pathogen/microbe‐associated molecular patterns (PAMPs/MAMPs). This induces a defence response called pattern‐triggered immunity (PTI) to fend off pathogen attack. Commensal bacteria are also exposed to potential immune recognition and must employ strategies to evade and/or suppress PTI to successfully colonize the plant. During plant infection, the flagellum has an ambiguous role, acting as both a virulence factor and also as a potent immunogen as a result of the recognition of its main building block, flagellin, by the plant pattern recognition receptors (PRRs), including FLAGELLIN SENSING2 (FLS2). Therefore, strict control of flagella synthesis is especially important for plant‐associated bacteria. Here, we show that cyclic‐di‐GMP [bis‐(3′‐5′)‐cyclic di‐guanosine monophosphate], a central regulator of bacterial lifestyle, is involved in the evasion of PTI. Elevated cyclic‐di‐GMP levels in the pathogen Pseudomonas syringae pv. tomato (Pto) DC3000, the opportunist P. aeruginosa PAO1 and the commensal P. protegens Pf‐5 inhibit flagellin synthesis and help the bacteria to evade FLS2‐mediated signalling in Nicotiana benthamiana and Arabidopsis thaliana. Despite this, high cellular cyclic‐di‐GMP concentrations were shown to drastically reduce the virulence of Pto DC3000 during plant infection. We propose that this is a result of reduced flagellar motility and/or additional pleiotropic effects of cyclic‐di‐GMP signalling on bacterial behaviour.     

Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase

The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.     

Climate change amplifies gross nitrogen turnover in montane grasslands of Central Europe in both summer and winter seasons

The carbon‐ and nitrogen‐rich soils of montane grasslands are exposed to above‐average warming and to altered precipitation patterns as a result of global change. To investigate the consequences of climatic change for soil nitrogen turnover, we translocated intact plant–soil mesocosms along an elevational gradient, resulting in an increase of the mean annual temperature by approx. 2 °C while decreasing precipitation from approx. 1500 to 1000 mm. Following three years of equilibration, we monitored the dynamics of gross nitrogen turnover and ammonia‐oxidizing bacteria (AOB) and archaea (AOA) in soils over an entire year. Gross nitrogen turnover and gene levels of AOB and AOA showed pronounced seasonal dynamics. Both summer and winter periods equally contributed to cumulative annual N turnover. However, highest gross N turnover and abundance of ammonia oxidizers were observed in frozen soil of the climate change site, likely due to physical liberation of organic substrates and their rapid turnover in the unfrozen soil water film. This effect was not observed at the control site, where soil freezing did not occur due to a significant insulating snowpack. Climate change conditions accelerated gross nitrogen mineralization by 250% on average. Increased N mineralization significantly stimulated gross nitrification by AOB rather than by AOA. However, climate change impacts were restricted to the 2–6 cm topsoil and rarely occurred at 12–16 cm depth, where generally much lower N turnover was observed. Our study shows that significant mineralization pulses occur under changing climate, which is likely to result in soil organic matter losses with their associated negative impacts on key soil functions. We also show that N cycling processes in frozen soil can be hot moments for N turnover and thus are of paramount importance for understanding seasonal patterns, annual sum of N turnover and possible climate change feedbacks.     

Galectin-9: From cell biology to complex disease dynamics

Galectins is a family of non-classically secreted, β-galactoside-binding proteins that has recently received considerable attention in the spatio-temporal regulation of surface ‘signal lattice’ organization, membrane dynamics, cell-adhesion and disease therapeutics. Galectin-9 is a unique member of this family, with two non-homologous carbohydrate recognition domains joined by a linker peptide sequence of variable lengths, generating isoforms with distinct properties and functions in both physiological and pathological settings, such as during development, immune reaction, neoplastic transformations and metastasis. In this review, we summarize the latest knowledge on the structure, receptors, cellular targets, trafficking pathways and functional properties of galectin-9 and discuss how galectin-9-mediated signalling cascades can be exploited in cancers and immunotherapies.     

Pharmacological removal of serum amyloid P component from intracerebral plaques and cerebrovascular Aβ amyloid deposits in vivo

Human amyloid deposits always contain the normal plasma protein serum amyloid P component (SAP), owing to its avid but reversible binding to all amyloid fibrils, including the amyloid β (Aβ) fibrils in the cerebral parenchyma plaques and cerebrovascular amyloid deposits of Alzheimer''s disease (AD) and cerebral amyloid angiopathy (CAA). SAP promotes amyloid fibril formation in vitro, contributes to persistence of amyloid in vivo and is also itself directly toxic to cerebral neurons. We therefore developed (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), a drug that removes SAP from the blood, and thereby also from the cerebrospinal fluid (CSF), in patients with AD. Here we report that, after introduction of transgenic human SAP expression in the TASTPM double transgenic mouse model of AD, all the amyloid deposits contained human SAP. Depletion of circulating human SAP by CPHPC administration in these mice removed all detectable human SAP from both the intracerebral and cerebrovascular amyloid. The demonstration that removal of SAP from the blood and CSF also removes it from these amyloid deposits crucially validates the strategy of the forthcoming ‘Depletion of serum amyloid P component in Alzheimer''s disease (DESPIAD)’ clinical trial of CPHPC. The results also strongly support clinical testing of CPHPC in patients with CAA.     

Acellularization-Induced Changes in Tensile Properties Are Organ Specific - An In-Vitro Mechanical and Structural Analysis of Porcine Soft Tissues

Introduction

Though xenogeneic acellular scaffolds are frequently used for surgical reconstruction, knowledge of their mechanical properties is lacking. This study compared the mechanical, histological and ultrastructural properties of various native and acellular specimens.

Materials and Methods

Porcine esophagi, ureters and skin were tested mechanically in a native or acellular condition, focusing on the elastic modulus, ultimate tensile stress and maximum strain. The testing protocol for soft tissues was standardized, including the adaption of the tissue’s water content and partial plastination to minimize material slippage as well as templates for normed sample dimensions and precise cross-section measurements. The native and acellular tissues were compared at the microscopic and ultrastructural level with a focus on type I collagens.

Results

Increased elastic modulus and ultimate tensile stress values were quantified in acellular esophagi and ureters compared to the native condition. In contrast, these values were strongly decreased in the skin after acellularization. Acellularization-related decreases in maximum strain were found in all tissues. Type I collagens were well-preserved in these samples; however, clotting and a loss of cross-linking type I collagens was observed ultrastructurally. Elastins and fibronectins were preserved in the esophagi and ureters. A loss of the epidermal layer and decreased fibronectin content was present in the skin.

Discussion

Acellularization induces changes in the tensile properties of soft tissues. Some of these changes appear to be organ specific. Loss of cross-linking type I collagen may indicate increased mechanical strength due to decreasing transverse forces acting upon the scaffolds, whereas fibronectin loss may be related to decreased load-bearing capacity. Potentially, the alterations in tissue mechanics are linked to organ function and to the interplay of cells and the extracellular matrix, which is different in hollow organs when compared to skin.     

Distinct different contributions of the alternative and classical complement activation pathway for the innate host response during sepsis

Complement activation represents a crucial innate defense mechanism to invading microorganisms, but there is an eminent lack of understanding of the separate contribution of the different complement activation pathways to the host response during sepsis. We therefore investigated different innate host immune responses during cecal ligation and puncture (CLP)-induced sepsis in mice lacking either the alternative (fD(-/-)) or classical (C1q(-/-)) complement activation pathway. Both knockout mice strains showed a significantly reduced survival and increased organ dysfunction when compared with control mice. Surprisingly, fD(-/-) mice demonstrated a compensated bacterial clearance capacity as control mice at 6 h post CLP, whereas C1q(-/-) mice were already overwhelmed by bacterial growth at this time point. Interestingly, at 24 h after CLP, fD(-/-) mice failed to clear bacteria in a way comparable to control mice. However, both knockout mice strains showed compromised C3 cleavage during sepsis. Investigating potential causes for this discrepancy, we were able to demonstrate that despite normal bacterial clearance capacity early during the onset of sepsis, fD(-/-) mice displayed increased inflammatory cytokine generation and neutrophil recruitment into lungs and blood when compared with both control- and C1q(-/-) mice, indicating a potential loss of control over these immune responses. Further in vitro experiments revealed a strongly increased Nf-κB activation capacity in isolated neutrophils from fD(-/-) mice, supporting this hypothesis. Our results provide evidence for the new concept that the alternative complement activation pathway exerts a distinctly different contribution to the innate host response during sepsis when compared with the classical pathway.     

EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy

Hepatitis C virus (HCV) is a major cause of liver disease, but therapeutic options are limited and there are no prevention strategies. Viral entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen, we identified epidermal growth factor receptor and ephrin receptor A2 as host cofactors for HCV entry. Blocking receptor kinase activity by approved inhibitors broadly impaired infection by all major HCV genotypes and viral escape variants in cell culture and in a human liver chimeric mouse model in vivo. The identified receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and viral glycoprotein-dependent membrane fusion. These results identify RTKs as previously unknown HCV entry cofactors and show that tyrosine kinase inhibitors have substantial antiviral activity. Inhibition of RTK function may constitute a new approach for prevention and treatment of HCV infection.