Characterization of Biofilm Formation by Borrelia burgdorferi In Vitro

Borrelia burgdorferi, the causative agent of Lyme disease, has long been known to be capable of forming aggregates and colonies. It was recently demonstrated that Borrelia burgdorferi aggregate formation dramatically changes the in vitro response to hostile environments by this pathogen. In this study, we investigated the hypothesis that these aggregates are indeed biofilms, structures whose resistance to unfavorable conditions are well documented. We studied Borrelia burgdorferi for several known hallmark features of biofilm, including structural rearrangements in the aggregates, variations in development on various substrate matrices and secretion of a protective extracellular polymeric substance (EPS) matrix using several modes of microscopic, cell and molecular biology techniques. The atomic force microscopic results provided evidence that multilevel rearrangements take place at different stages of aggregate development, producing a complex, continuously rearranging structure. Our results also demonstrated that Borrelia burgdorferi is capable of developing aggregates on different abiotic and biotic substrates, and is also capable of forming floating aggregates. Analyzing the extracellular substance of the aggregates for potential exopolysaccharides revealed the existence of both sulfated and non-sulfated/carboxylated substrates, predominately composed of an alginate with calcium and extracellular DNA present. In summary, we have found substantial evidence that Borrelia burgdorferi is capable of forming biofilm in vitro. Biofilm formation by Borrelia species might play an important role in their survival in diverse environmental conditions by providing refuge to individual cells.     

Systematic interpretation of cyclic nucleotide binding studies using KinetXBase

Functional proteomics aims to describe cellular protein networks in depth based on the quantification of molecular interactions. In order to study the interaction of adenosine-3',5'-cyclic monophosphate (cAMP), a general second messenger involved in several intracellular signalling networks, with one of its respective target proteins, the regulatory (R) subunit of cAMP dependent protein kinase (PKA), a number of different methods was employed. These include fluorescence polarisation (FP), isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), amplified luminescence proximity homogeneous assay (ALPHA-screen), radioligand binding or activity-based assays. Kinetic, thermodynamic and equilibrium binding data of a variety of cAMP derivatives to several cAMP binding domains were integrated in a single database system, we called KinetXBase, allowing for very distinct data formats. KinetXBase is a practical data handling system for molecular interaction data of any kind, providing a synopsis of data derived from different technologies. This supports ongoing efforts in the bioinformatics community to devise formal concepts for a unified representation of interaction data, in order to enable their exchange and easy comparison. KinetXBase was applied here to analyse complex cAMP binding data and highly site-specific cAMP analogues could be identified. The software package is free for download by academic users.     

Anthelmintic cyclcooctadepsipeptides: complex in structure and mode of action

The broad-spectrum anthelmintic cyclooctadepsipeptide PF1022A is a fungal metabolite from Rosellinia sp. PF1022, which is a Mycelia sterilia found on the leaves of Camellia japonica. A broad range of structurally related cyclooctadepsipeptides has been characterized and tested for anthelmintic activities. These metabolites have been used as starting points to generate semisynthetic derivatives with varying nematocidal capacity. Predominant among these compounds is emodepside, which exhibits a broad nematocidal potential against gastrointestinal and extraintestinal parasites. Here we review the chemical biology and mode of action of cyclooctadepsides with particular attention to PF1022A and emodepside. We illustrate how they target nematode neuromuscular function, opening up new avenues for antiparasitic treatments with potential capability for important selective toxicity.     

Serum Biomarkers in Patients with Relapsing Eosinophilic Granulomatosis with Polyangiitis (Churg-Strauss)

Introduction

Previous studies suggest a role for eotaxin-3, TARC/CCL17 and IgG4 in newly- diagnosed patients with eosinophilic granulomatosis with polyangiitis (EGPA, Churg-Strauss) with highly active disease. The role of these biomarkers in relapsing disease is unclear.

Methods

Serum levels of TARC/CCL17, eotaxin-3, IgG4, and IgG4/IgG ratio were determined in serum samples from a longitudinal cohort of patients with EGPA (105 visits of 25 patients). Epidemiological, clinical and laboratory data were available for all visits.

Results

At the first visit, 80% of patients were using glucocorticoids and 68% additional immunosuppressive drugs. Disease flares were seen at 18 visits. The median BVAS and BVAS/WG scores at time of relapse were 4 and 2, respectively. None of the biomarkers tested were useful to discriminate between active disease and remission. Patients treated with prednisone had lower eotaxin-3 and eosinophil levels compared to patients not taking glucocorticoids irrespective of disease activity. Use of immunosuppressive agents was not associated with biomarker levels.

Conclusions

Serum levels of TARC/CCL17, eotaxin-3, IgG4, and IgG4/IgG ratio do not clearly differentiate active and inactive disease in established EGPA. Defining biomarkers in EGPA remains a challenge especially during times of glucocorticoid use.     

Half of the European fruit fly species barcoded (Diptera,Tephritidae); a feasibility test for molecular?identification

A feasibility test of molecular identification of European fruit flies (Diptera: Tephritidae) based on COI barcode sequences has been executed. A dataset containing 555 sequences of 135 ingroup species from three subfamilies and 42 genera and one single outgroup species has been analysed. 73.3% of all included species could be identified based on their COI barcode gene, based on similarity and distances. The low success rate is caused by singletons as well as some problematic groups: several species groups within the genus Terellia and especially the genus Urophora. With slightly more than 100 sequences – almost 20% of the total – this genus alone constitutes the larger part of the failure for molecular identification for this dataset. Deleting the singletons and Urophora results in a success-rate of 87.1% of all queries and 93.23% of the not discarded queries as correctly identified. Urophora is of special interest due to its economic importance as beneficial species for weed control, therefore it is desirable to have alternative markers for molecular identification.We demonstrate that the success of DNA barcoding for identification purposes strongly depends on the contents of the database used to BLAST against. Especially the necessity of including multiple specimens per species of geographically distinct populations and different ecologies for the understanding of the intra- versus interspecific variation is demonstrated. Furthermore thresholds and the distinction between true and false positives and negatives should not only be used to increase the reliability of the success of molecular identification but also to point out problematic groups, which should then be flagged in the reference database suggesting alternative methods for identification.     

Grape Seed Extract Dose-Responsively Decreases Disease Severity in a Rat Model of Mucositis; Concomitantly Enhancing Chemotherapeutic Effectiveness in Colon Cancer Cells

Objective

Mucositis is a serious disorder of the gastrointestinal tract that results from cancer chemotherapy. We investigated the effects of increasing grape seed extract doses on the severity of chemotherapy in a rat model and its coincident impact on chemotherapeutic effectiveness in colon cancer cells.

Design

Female Dark Agouti rats were gavaged with grape seed extract (400–1000 mg/kg) or water (day 3–11) and were injected intraperitoneally with 5-Fluorouracil (150 mg/kg) or saline (control) on day 9 to induce mucositis. Daily metabolic data were collected and rats were sacrificed on day 12. Intestinal tissues were collected for histological and myeloperoxidase analyses. Caco-2 cell viability was examined in response to grape seed extract in combination with 5-Fluorouracil by 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide) assay.

Results

Compared with 5-Fluorouracil controls, grape seed extract (400–1000 mg/kg) significantly decreased the histological damage score (P<0.05) in the jejunum. Grape seed extract (1000 mg/kg) increased jejunal crypt depth by 25% (P<0.05) in 5-Fluorouracil treated rats compared to 5-Fluorouracil controls, and attenuated the 5-Fluorouracil -induced reduction of mucosal thickness (25%, P<0.05). Grape seed extract (600 mg/kg) decreased myeloperoxidase activity by 55% (P<0.01) compared to 5-Fluorouracil controls. Grape seed extract was more effective at ameliorating 5-Fluorouracil induced intestinal injury, with effects most pronounced in the proximal jejunum. Grape seed extract (10–25 ug/mL) significantly enhanced the growth-inhibitory effects of 5-Fluorouracil by 26% (P<0.05) in Caco-2 cells and was more potent than 5-Fluorouracil at 50–100 µg/mL.

Conclusion

Grape seed extract may represent a new therapeutic option to decrease the symptoms of intestinal mucositis while concurrently impacting on the viability of colon cancer cells.     

Pneumococci induced TLR- and Rac1-dependent NF-kappaB-recruitment to the IL-8 promoter in lung epithelial cells

Streptococcus pneumoniae is the major pathogen of community-acquired pneumonia. The respiratory epithelium constitutes the first line of defense against invading lung pathogens, including pneumococci. We analyzed the involvement of Toll-like receptors (TLR) and Rho-GTPase signaling in the activation of human lung epithelial cells by pneumococci. S. pneumoniae induced release of interleukin-8 (IL-8) by human bronchial epithelial cell line BEAS-2B. Specific inhibition of Rac1 by Nsc23766 or a dominant-negative mutant of Rac1 strongly reduced cytokine release. In addition, pneumococci-related cell activation (IL-8 release, NF-kappaB-activation) depended on MyD88, phosphatidylinositol 3-kinase, and Cdc42 but not on RhoA. Pneumococci enhanced TLR1 and TLR2 mRNA expression in BEAS-2B cells, whereas TLR4 and TLR6 expression was constitutively high. TLR1 and 2 synergistically recognized pneumococci in cotransfection experiments. TLR4, TLR6, LPS-binding protein, and CD14 seem not to be involved in pneumococci-dependent cell activation. At the IL-8 gene promoter, recruitment of phosphorylated NF-kappaB subunit p65 was blocked by inhibition of Rac1, whereas binding of the phosphorylated activator protein-1 subunit c-Jun to the promoter was not diminished. In summary, these results suggest that S. pneumoniae activate human epithelial cells by TLR1/2 and a phosphatidylinositol 3-kinase- and Rac1-dependent NF-kappaB-recruitment to the IL-8 promoter.     

No Evidence of the Ego-Depletion Effect across Task Characteristics and Individual Differences: A Pre-Registered Study

Ego-depletion, a psychological phenomenon in which participants are less able to engage in self-control after prior exertion of self-control, has become widely popular in the scientific community as well as in the media. However, considerable debate exists among researchers as to the nature of the ego-depletion effect, and growing evidence suggests the effect may not be as strong or robust as the extant literature suggests. We examined the robustness of the ego-depletion effect and aimed to maximize the likelihood of detecting the effect by using one of the most widely used depletion tasks (video-viewing attention control task) and by considering task characteristics and individual differences that potentially moderate the effect. We also sought to make our research plan transparent by pre-registering our hypotheses, procedure, and planned analyses prior to data collection. Contrary to the ego-depletion hypothesis, participants in the depletion condition did not perform worse than control participants on the subsequent self-control task, even after considering moderator variables. These findings add to a growing body of evidence suggesting ego-depletion is not a reliable phenomenon, though more research is needed that uses large sample sizes, considers moderator variables, and pre-registers prior to data collection.