Biofilm induced tolerance towards antimicrobial peptides

Increased tolerance to antimicrobial agents is thought to be an important feature of microbes growing in biofilms. We address the question of how biofilm organization affects antibiotic susceptibility. We established Escherichia coli biofilms with differential structural organization due to the presence of IncF plasmids expressing altered forms of the transfer pili in two different biofilm model systems. The mature biofilms were subsequently treated with two antibiotics with different molecular targets, the peptide antibiotic colistin and the fluoroquinolone ciprofloxacin. The dynamics of microbial killing were monitored by viable count determination, and confocal laser microscopy. Strains forming structurally organized biofilms show an increased bacterial survival when challenged with colistin, compared to strains forming unstructured biofilms. The increased survival is due to genetically regulated tolerant subpopulation formation and not caused by a general biofilm property. No significant difference in survival was detected when the strains were challenged with ciprofloxacin. Our data show that biofilm formation confers increased colistin tolerance to cells within the biofilm structure, but the protection is conditional being dependent on the structural organization of the biofilm, and the induction of specific tolerance mechanisms.     

Protein Paucimannosylation Is an Enriched N‐Glycosylation Signature of Human Cancers

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics‐centric study investigates a possible link between protein paucimannosylation, an under‐studied class of human N‐glycosylation [Man_1‐3GlcNAc₂Fuc_0‐1], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non‐cancerous specimens are profiled from 467 published and unpublished PGC‐LC‐MS/MS N‐glycome datasets collected over a decade. PMGs, particularly Man_2‐3GlcNAc₂Fuc₁, are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0–50.2%). Analyses of paired (tumor/non‐tumor) and stage‐stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N‐acetyl‐β‐hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.     

NetMHCIIpan-2.0 - Improved pan-specific HLA-DR predictions using a novel concurrent alignment and weight optimization training procedure

Background

Macrophages represent the front lines of our immune system; they recognize and engulf pathogens or foreign particles thus initiating the immune response. Imaging macrophages presents unique challenges, as most optical techniques require labeling or staining of the cellular compartments in order to resolve organelles, and such stains or labels have the potential to perturb the cell, particularly in cases where incomplete information exists regarding the precise cellular reaction under observation. Label-free imaging techniques such as Raman microscopy are thus valuable tools for studying the transformations that occur in immune cells upon activation, both on the molecular and organelle levels. Due to extremely low signal levels, however, Raman microscopy requires sophisticated image processing techniques for noise reduction and signal extraction. To date, efficient, automated algorithms for resolving sub-cellular features in noisy, multi-dimensional image sets have not been explored extensively.

Results

We show that hybrid z-score normalization and standard regression (Z-LSR) can highlight the spectral differences within the cell and provide image contrast dependent on spectral content. In contrast to typical Raman imaging processing methods using multivariate analysis, such as single value decomposition (SVD), our implementation of the Z-LSR method can operate nearly in real-time. In spite of its computational simplicity, Z-LSR can automatically remove background and bias in the signal, improve the resolution of spatially distributed spectral differences and enable sub-cellular features to be resolved in Raman microscopy images of mouse macrophage cells. Significantly, the Z-LSR processed images automatically exhibited subcellular architectures whereas SVD, in general, requires human assistance in selecting the components of interest.

Conclusions

The computational efficiency of Z-LSR enables automated resolution of sub-cellular features in large Raman microscopy data sets without compromise in image quality or information loss in associated spectra. These results motivate further use of label free microscopy techniques in real-time imaging of live immune cells.     

Molecular Diversity of Rumen Methanogens from Sheep in Western Australia

The molecular diversity of rumen methanogens in sheep in Australia was investigated by using individual 16S rRNA gene libraries prepared from the rumen contents obtained from six merino sheep grazing pasture (326 clones), six sheep fed an oaten hay-based diet (275 clones), and five sheep fed a lucerne hay-based diet (132 clones). A total of 733 clones were examined, and the analysis revealed 65 phylotypes whose sequences (1,260 bp) were similar to those of cultivated methanogens belonging to the order Methanobacteriales. Pasture-grazed sheep had more methanogen diversity than sheep fed either the oaten hay or lucerne hay diet. Methanobrevibacter strains SM9, M6, and NT7 accounted for over 90% of the total number of clones identified. M6 was more prevalent in grazing sheep, and SM9, despite being found in 16 of the 17 sheep, was more prevalent in sheep fed the lucerne-based diet. Five new species were identified. Two of these species exhibited very little sequence similarity to any cultivated methanogens and were found eight times in two of the six sheep that were grazing pasture. These unique sequences appear to represent a novel group of rumen archaea that are atypical for the rumen environment.     

Structure of internalin,a major invasion protein of Listeria monocytogenes,in complex with its human receptor E-cadherin

Listeria monocytogenes, a food-borne bacterial pathogen, enters mammalian cells by inducing its own phagocytosis. The listerial protein internalin (InlA) mediates bacterial adhesion and invasion of epithelial cells in the human intestine through specific interaction with its host cell receptor E-cadherin. We present the crystal structures of the functional domain of InlA alone and in a complex with the extracellular, N-terminal domain of human E-cadherin (hEC1). The leucine rich repeat (LRR) domain of InlA surrounds and specifically recognizes hEC1. Individual interactions were probed by mutagenesis and analytical ultracentrifugation. These include Pro16 of hEC1, a major determinant for human susceptibility to L. monocytogenes infection that is essential for intermolecular recognition. Our studies reveal the structural basis for host tro-pism of this bacterium and the molecular deception L. monocytogenes employs to exploit the E-cadherin system.     

Complement activation,regulation, and molecular basis for complement‐related diseases

The complement system is an essential element of the innate immune response that becomes activated upon recognition of molecular patterns associated with microorganisms, abnormal host cells, and modified molecules in the extracellular environment. The resulting proteolytic cascade tags the complement activator for elimination and elicits a pro‐inflammatory response leading to recruitment and activation of immune cells from both the innate and adaptive branches of the immune system. Through these activities, complement functions in the first line of defense against pathogens but also contributes significantly to the maintenance of homeostasis and prevention of autoimmunity. Activation of complement and the subsequent biological responses occur primarily in the extracellular environment. However, recent studies have demonstrated autocrine signaling by complement activation in intracellular vesicles, while the presence of a cytoplasmic receptor serves to detect complement‐opsonized intracellular pathogens. Furthermore, breakthroughs in both functional and structural studies now make it possible to describe many of the intricate molecular mechanisms underlying complement activation and the subsequent downstream events, as well as its cross talk with, for example, signaling pathways, the coagulation system, and adaptive immunity. We present an integrated and updated view of complement based on structural and functional data and describe the new roles attributed to complement. Finally, we discuss how the structural and mechanistic understanding of the complement system rationalizes the genetic defects conferring uncontrolled activation or other undesirable effects of complement.     

Organization of excitable dynamics in hierarchical biological networks

This study investigates the contributions of network topology features to the dynamic behavior of hierarchically organized excitable networks. Representatives of different types of hierarchical networks as well as two biological neural networks are explored with a three-state model of node activation for systematically varying levels of random background network stimulation. The results demonstrate that two principal topological aspects of hierarchical networks, node centrality and network modularity, correlate with the network activity patterns at different levels of spontaneous network activation. The approach also shows that the dynamic behavior of the cerebral cortical systems network in the cat is dominated by the network's modular organization, while the activation behavior of the cellular neuronal network of Caenorhabditis elegans is strongly influenced by hub nodes. These findings indicate the interaction of multiple topological features and dynamic states in the function of complex biological networks.     

Multi‐factor climate change effects on insect herbivore performance

The impact of climate change on herbivorous insects can have far‐reaching consequences for ecosystem processes. However, experiments investigating the combined effects of multiple climate change drivers on herbivorous insects are scarce. We independently manipulated three climate change drivers (CO₂, warming, drought) in a Danish heathland ecosystem. The experiment was established in 2005 as a full factorial split‐plot with 6 blocks × 2 levels of CO₂ × 2 levels of warming × 2 levels of drought = 48 plots. In 2008, we exposed 432 larvae (n = 9 per plot) of the heather beetle (Lochmaea suturalis Thomson ), an important herbivore on heather, to ambient versus elevated drought, temperature, and CO₂ (plus all combinations) for 5 weeks. Larval weight and survival were highest under ambient conditions and decreased significantly with the number of climate change drivers. Weight was lowest under the drought treatment, and there was a three‐way interaction between time, CO₂, and drought. Survival was lowest when drought, warming, and elevated CO₂ were combined. Effects of climate change drivers depended on other co‐acting factors and were mediated by changes in plant secondary compounds, nitrogen, and water content. Overall, drought was the most important factor for this insect herbivore. Our study shows that weight and survival of insect herbivores may decline under future climate. The complexity of insect herbivore responses increases with the number of combined climate change drivers.