Phagocytosis Escape by a Staphylococcus aureus Protein That Connects Complement and Coagulation Proteins at the Bacterial Surface

Upon contact with human plasma, bacteria are rapidly recognized by the complement system that labels their surface for uptake and clearance by phagocytic cells. Staphylococcus aureus secretes the 16 kD Extracellular fibrinogen binding protein (Efb) that binds two different plasma proteins using separate domains: the Efb N-terminus binds to fibrinogen, while the C-terminus binds complement C3. In this study, we show that Efb blocks phagocytosis of S. aureus by human neutrophils. In vitro, we demonstrate that Efb blocks phagocytosis in plasma and in human whole blood. Using a mouse peritonitis model we show that Efb effectively blocks phagocytosis in vivo, either as a purified protein or when produced endogenously by S. aureus. Mutational analysis revealed that Efb requires both its fibrinogen and complement binding residues for phagocytic escape. Using confocal and transmission electron microscopy we show that Efb attracts fibrinogen to the surface of complement-labeled S. aureus generating a ‘capsule’-like shield. This thick layer of fibrinogen shields both surface-bound C3b and antibodies from recognition by phagocytic receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of Efb. Altogether we discover that Efb from S. aureus uniquely escapes phagocytosis by forming a bridge between a complement and coagulation protein.     

Fungi Benefit from Two Decades of Increased Nutrient Availability in Tundra Heath Soil

If microbial degradation of carbon substrates in arctic soil is stimulated by climatic warming, this would be a significant positive feedback on global change. With data from a climate change experiment in Northern Sweden we show that warming and enhanced soil nutrient availability, which is a predicted long-term consequence of climatic warming and mimicked by fertilization, both increase soil microbial biomass. However, while fertilization increased the relative abundance of fungi, warming caused only a minimal shift in the microbial community composition based on the phospholipid fatty acid (PLFA) and neutral lipid fatty acid (NLFA) profiles. The function of the microbial community was also differently affected, as indicated by stable isotope probing of PLFA and NLFA. We demonstrate that two decades of fertilization have favored fungi relative to bacteria, and increased the turnover of complex organic compounds such as vanillin, while warming has had no such effects. Furthermore, the NLFA-to-PLFA ratio for ¹³C-incorporation from acetate increased in warmed plots but not in fertilized ones. Thus, fertilization cannot be used as a proxy for effects on warming in arctic tundra soils. Furthermore, the different functional responses suggest that the biomass increase found in both fertilized and warmed plots was mediated via different mechanisms.     

An Iterative Framework for EEG-based Image Search: Robust Retrieval with Weak Classifiers

We revisit the framework for brain-coupled image search, where the Electroencephalography (EEG) channel under rapid serial visual presentation protocol is used to detect user preferences. Extending previous works on the synergy between content-based image labeling and EEG-based brain-computer interface (BCI), we propose a different perspective on iterative coupling. Previously, the iterations were used to improve the set of EEG-based image labels before propagating them to the unseen images for the final retrieval. In our approach we accumulate the evidence of the true labels for each image in the database through iterations. This is done by propagating the EEG-based labels of the presented images at each iteration to the rest of images in the database. Our results demonstrate a continuous improvement of the labeling performance across iterations despite the moderate EEG-based labeling (AUC <75%). The overall analysis is done in terms of the single-trial EEG decoding performance and the image database reorganization quality. Furthermore, we discuss the EEG-based labeling performance with respect to a search task given the same image database.     

Intracellular Nucleic Acid Delivery by the Supercharged Dengue Virus Capsid Protein

Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins.     

Experimental Diabetes Induces Structural,Inflammatory and Vascular Changes of Achilles Tendons

This study aims to demonstrate how the state of chronic hyperglycemia from experimental Diabetes Mellitus can influence the homeostatic imbalance of tendons and, consequently, lead to the characteristics of tendinopathy. Twenty animals were randomly divided into two experimental groups: control group, consisting of healthy rats and diabetic group constituted by rats induced to Diabetes Mellitus I. After twenty-four days of the induction of Diabetes type I, the Achilles tendon were removed for morphological evaluation, cellularity, number and cross-sectional area of blood vessel, immunohistochemistry for Collagen type I, VEGF and NF-κB nuclear localization sequence (NLS) and nitrate and nitrite level. The Achilles tendon thickness (µm/100g) of diabetic animals was significantly increased and, similarly, an increase was observed in the density of fibrocytes and mast cells in the tendons of the diabetic group. The average number of blood vessels per field, in peritendinous tissue, was statistically higher in the diabetic group 3.39 (2.98) vessels/field when compared to the control group 0.89 (1.68) vessels/field p = 0.001 and in the intratendinous region, it was observed that blood vessels were extremely rare in the control group 0.035 (0.18) vessels/field and were often present in the tendons of the diabetic group 0.89 (0.99) vessels/field. The immunohistochemistry analysis identified higher density of type 1 collagen and increased expression of VEGF as well as increased immunostaining for NFκB p50 NLS in the nucleus in Achilles tendon of the diabetic group when compared to the control group. Higher levels of nitrite/nitrate were observed in the experimental group induced to diabetes. We conclude that experimental DM induces notable structural, inflammatory and vascular changes in the Achilles tendon which are compatible with the process of chronic tendinopathy.     

Collagen-binding Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMM) of Gram-positive Bacteria Inhibit Complement Activation via the Classical Pathway

Members of a family of collagen-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) from Gram-positive bacteria are established virulence factors in several infectious diseases models. Here, we report that these adhesins also can bind C1q and act as inhibitors of the classical complement pathway. Molecular analyses of Cna from Staphylococcus aureus suggested that this prototype MSCRAMM bound to the collagenous domain of C1q and interfered with the interactions of C1r with C1q. As a result, C1r₂C1s₂ was displaced from C1q, and the C1 complex was deactivated. This novel function of the Cna-like MSCRAMMs represents a potential immune evasion strategy that could be used by numerous Gram-positive pathogens.     

Mechanical Resistance in Unstructured Proteins

Single-molecule pulling experiments on unstructured proteins linked to neurodegenerative diseases have measured rupture forces comparable to those for stable folded proteins. To investigate the structural mechanisms of this unexpected force resistance, we perform pulling simulations of the amyloid β-peptide (Aβ) and α-synuclein (αS), starting from simulated conformational ensembles for the free monomers. For both proteins, the simulations yield a set of rupture events that agree well with the experimental data. By analyzing the conformations occurring shortly before rupture in each event, we find that the mechanically resistant structures share a common architecture, with similarities to the folds adopted by Aβ and αS in amyloid fibrils. The disease-linked Arctic mutation of Aβ is found to increase the occurrence of highly force-resistant structures. Our study suggests that the high rupture forces observed in Aβ and αS pulling experiments are caused by structures that might have a key role in amyloid formation.     

Prevalence,quantitative load and genetic diversity of <Emphasis Type="Italic">Campylobacter</Emphasis> spp. in dairy cattle herds in Lithuania

Background

Campylobacteriosis is a zoonotic disease, and animals such as poultry, pigs and cattle may act as reservoirs for Campylobacter spp. Cattle shed Campylobacter spp. into the environment and they can act as a reservoir for human infection directly via contact with cattle or their faeces or indirectly by consumption of contaminated food. The aim of this study was to determine the prevalence, the quantitative load and the genetic strain diversity of Campylobacter spp. in dairy cattle of different age groups.

Results

Faecal samples of 200 dairy cattle from three farms in the central part of Lithuania were collected and examined for Campylobacter. Cattle herds of all three farms were Campylobacter spp. positive, with a prevalence ranging from 75% (farm I), 77.5% (farm II) to 83.3% (farm III). Overall, the highest prevalence was detected in calves (86.5%) and heifers (86.2%). In contrast, the lowest Campylobacter prevalence was detectable in dairy cows (60.6%). C. jejuni, C. coli, C. lari and C. fetus subsp. fetus were identified in faecal samples of dairy cattle. C. upsaliensis was not detectable in any sample. The high counts of Campylobacter spp. were observed in faecal material of dairy cattle (average 4.5 log₁₀ cfu/g). The highest numbers of Campylobacter spp. were found in faecal samples from calves (average 5.3 log₁₀ cfu/g), whereas, faecal samples from cows harboured the lowest number of Campylobacter spp. (average 3.7 log₁₀ cfu/g). Genotyping by fla A PCR-RFLP analysis of selected C. jejuni isolates showed that some genotypes were present in all farms and all age groups. However, farm or age specific genotypes were also identified.

Conclusions

Future studies are needed to investigate risk factors related to the degree of colonisation in cattle. Based on that, possible measures to reduce the colonisation and subsequent shedding of Campylobacter in cattle could be established. It is important to further investigate the epidemiology of Campylobacter in the cattle population in order to assess associated risks to public health.

     

MicroRNA-Mediated Suppression of Oncolytic Adenovirus Replication in Human Liver

MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3′ untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5) in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.     

Modified Lipoprotein-Derived Lipid Particles Accumulate in Human Stenotic Aortic Valves

In aortic stenosis plasma lipoprotein-derived lipids accumulate in aortic valves. Here, we first compared the lipid compositions of stenotic aortic valves and atherosclerotic plaque cores. Both pathological tissues were found to be enriched in cholesteryl linoleate, a marker of extracellularly accumulated lipoproteins. In addition, a large proportion of the phospholipids were found to contain arachidonic acid, the common precursor of a number of proinflammatory lipid mediators. Next, we isolated and characterized extracellular lipid particles from human stenotic and non-stenotic control valves, and compared them to plasma lipoproteins from the same subjects. The extracellular valvular lipid particles were isolated from 15 stenotic and 14 non-stenotic aortic valves. Significantly more apoB-100-containing lipid particles were found in the stenotic than in the non-stenotic valves. The majority of the lipid particles isolated from the non-stenotic valves had sizes (23±6.2 nm in diameter) similar to those of plasma low density lipoprotein (LDL) (22±1.5 nm), while the lipid particles from stenotic valves were not of uniform size, their sizes ranging from 18 to more than 500 nm. The lipid particles showed signs of oxidative modifications, and when compared to isolated plasma LDL particles, the lipid particles isolated from the stenotic valves had a higher sphingomyelin/phosphatidylcholine –ratio, and also higher contents of lysophosphatidylcholine and unesterified cholesterol. The findings of the present study reveal, for the first time, that in stenotic human aortic valves, infiltrated plasma lipoproteins have undergone oxidative and lipolytic modifications, and become fused and aggregated. The generated large lipid particles may contribute to the pathogenesis of human aortic stenosis.